Biomedical subjects

A M Goes

Publications and source records attributed to A M Goes.

At least 19 recordsLinked to original sources

PubMed2006

Interleukin-10 and tumor necrosis factor-alpha single nucleotide gene polymorphism frequency in paracoccidioidomycosis.

Allelic variants of cytokine genes seem to be involved in mechanisms of resistance or susceptibility to several diseases. The aim of this study was to investigate the frequency of genotypes with the tumor necrosis factor-alpha TNF-alpha gene polymorphism G/A at position -308 and the IL-10 gene polymorphism G/A at position -1082, and to verify a possible association of these polymorphisms with paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. Genotyping was performed by allele-specific polymerase chain reaction (ASPCR) and restriction fragment length polymorphism (RFLP) on genomic DNA isolated of granulocytes from 54 PCM patients and 31 noninfected individuals. The analysis of SNP at position -1082 IL-10 showed a high frequency of GA genotype in both patients and controls (51% and 55%, respectively), while the allelic frequency showed 54% of G allele in the patients and 66% of A allele in the controls. The GG genotype was more frequent in patients (85%) and controls (68%) when we analyze the SNP at position -308 of TNF-alpha gene. Otherwise, 91% of PCM patients and 84% of noninfected individuals carried the G allele in -308 TNF-alpha SNP. Stimulation of cells from individuals with PCM phenotyped as A+ (GA or AA genotypes) presented elevation of TNF-alpha producing cells when compared with IL-10-producer cells. These findings reinforce the critical role of IL-10 and TNF-alpha in the paracoccidioidomycosis and can strongly suggest that the genetic screening of the -308G/A and -1082G/A polymorphisms may be a valid tool for identification of subjects needing a more appropriate therapy.

Adolescent↗

PubMed2005

Membrane and extracellular antigens of Paracoccidioides brasiliensis (Mexo): identification of a 28-kDa protein suitable for immunodiagnosis of paracoccidioidomycosis.

In this work, we analyzed serological responses of paracoccidioidomycosis (PCM) patients to membrane and extracellular antigens (Mexo) of Paracoccidioides brasiliensis by ELISA, immunoblot technique and immunofluorescence assays to identify a specific antigen profile. Among 140 PCM serum samples analyzed, a homogeneous IgG response to Mexo was observed. The specificity of this antigen was 96.6% in relation to control sera and 81.2% to sera from patients with diverse infections. Patients undergoing treatment for more than 1 year showed a reduced antibody response against Mexo. These results suggest that the presence of anti-Mexo antibodies might be an indicator of active disease. A protein from Mexo with a molecular weight of 28 kDa (Pb28) was the most specific antigen in humoral immune responses to PCM, since it reacted with 100% of patient sera and did not react with heterologous serum samples tested. This protein was purified by molecular filtration chromatography in FPLC system and, when tested by immunoblotting, it maintained its reactivity and specificity of 100% with PCM sera. The Pb28 N-terminal amino acid sequence comparison analysis in the non-redundant GenBank database at NCBI revealed no significant homology to known PCM proteins or to other fungal proteins of known function. Since the 28-kDa protein of P. brasiliensis seems to be specific for PCM, it can be used as an alternative antigen in immunoblotting diagnostic methods.

Adolescent↗

PubMed2005

Identification of immunogenic proteins from Paracoccidioides brasiliensis antigenic fractions F0, FII and FIII.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity is the principal mode of protection against this fungal infection. In this context, one of the strategies to discover proteins that are target of an effective immune response against P. brasiliensis is the partial sequencing of cDNA from an expression library previously screened with immunoglobulins (Ig) to generate antigen sequence tags (AST). In the present work, a P. brasiliensis yeast cDNA expression library was screened with affinity chromatography-purified IgG from rabbit sera immunized with P. brasiliensis antigenic fractions (F0, FII or FIII) or from paracoccidioidomycosis (PCM) patient sera by indirect ELISA. From 119 clones selected by the immunoscreening procedure, 40% were recognized by IgG from PCM patients, 25% were recognized by anti-F0, 8% were selected by anti-FII and 11% recognized by FIII specific antibodies. The remaining clones presented cross-reaction to all anti-sera tested. The AST homologies with previously reported sequences in the nonredundant GenBank at NCBI revealed high significant homology to fungal proteins of known function. One of them matched calcineurin B of Neurospora crassa with 35% identity and 55% similarity in amino acid sequence. We also identified an AST homologous to a Kinesin like protein from Ustilagus maydis and other fungi with 86% identity and 91% similarity. On the other hand, the vast majority of selected cDNA clones are new genes and represent 60% of the total. Prediction of transmembrane regions with the prediction transmembrane protein topology with a hidden markov model (TMHMM) revealed consensus sequences representing structural membrane segments in 28 encoded proteins.

Amino Acid Sequence↗

PubMed2005

Primary osteoblast cell response to sol-gel derived bioactive glass foams.

Bioactive glass macroporous structures were developed in this work to be used as scaffolds for bone tissue engineering applications. A sol-gel route was used to obtain glass foams with the introduction of a gas phase in the solution and by vigorous agitation of the sol-gel solution that contains a foam agent. Stable and homogeneous foams were formed near the gelation point, which were than dried and heat-treated. Macroporous structures with interconnected pores of up to 500 mu m, porosity as high as 88% and specific surface area of 92 m(2)/g were obtained. The porous glasses were tested in osteoblast cultures to evaluate adhesion, proliferation, collagen and alkaline phosphatase production. Osteoblast proliferation was higher in the presence of the foams as well as was the collagen secretion, when compared to control. The alkaline phosphatase production was not altered. Viable osteoblasts could be seen inside the foams, suggesting that the produced porous glass foams are a promising materials for bone repair, since it provides a good environment for the adhesion and proliferation of osteoblasts.

Alkaline Phosphatase↗

PubMed2005

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis.

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.

Acute Disease↗

PubMed2004

Schistosoma mansoni PIII antigen modulates in vitro granuloma formation by regulating CD28, CTLA-4, and CD86 expression in humans.

We investigated the in vitro responses of peripheral blood mononuclear cells (PBMC) from intestinal chronic schistosomiasis patients to PIII, a multivalent antigen prepared from Schistosoma mansoni adult worm. PIII decreased cellular proliferation and granulomatous reaction. Moreover, induced the reduction of IFN-gamma levels and increased IL-10 production. To better understand the mechanism through which the observed suppression occurs, the present study focused on the phenotypic pattern displayed by PBMC treated with PIII in an in vitro granuloma assay. Expression of the surface markers CD28, CTLA-4 and CD86 by lymphocytes and monocytes were analyzed by flow cytometry. Our results demonstrated a significant decrease of CD28+CD4+ and CD28+CD8+ T-cell percentage stimulated by PIII compared to its non-infected counterparts. This suppressive effect was related to a significant increase in the percentage of T-cells expressing CTLA-4. PIII also promoted a significant increase in the percentage of cells expressing CD86. Indeed, our results demonstrated that PIII was capable of modulating in vitro granuloma reaction, and this event was related to the balance of IL-10, IFN-gamma and CD28, CTLA-4, CD86 bringing new insight to the immunoregulation of granulomatous hypersensitivity in human schistosomiasis.

Animals↗

PubMed2004

Protective immunity induced in mice by F0 and FII antigens purified from Paracoccidioides brasiliensis.

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.

Animals↗

PubMed2004

Modulation of CD28 and CD86 expression in patients with paracoccidioidomycosis in different periods of treatment.

Paracoccidioides brasiliensis, a thermo-dimorphic fungus, is the ethiologic agent of paracoccidioidomycosis (PCM). The recidive is the greatest obstacle of this disease, because the yeast usually returns after the long treatment period. In the present work, we have investigated the cellular immune response of cells from peripheral blood drawn from patients with different duration of PCM. The classification of patients ranged from nontreated to those with long-standing disease over 5 years. Unstimulated as well as cells stimulated with phytohemaglutinin or two different antigen preparations, secreted (MEXO) or somatic (PbAg) of P. brasiliensis, were characterized. We found that cells from patients with disease proliferate considerably upon stimulation with the antigen preparations and that cells from patients with disease of long duration does not proliferate that vigorously as from patients with more recent diagnosis. Both interferon (IFN)-gamma and interleukin (IL)-4 appear to be increased in patients, but IFN-gamma tended to increase upon treatment while IL-4-secretion decreased. With respect to CD28 and CD86, we found that the subset of CD28 positive CD8 cells are decreased in all stages of the disease as compared to control individuals. A subset of CD86 positive CD19 cells appeared to be considerably increased compared to the controls. Indeed, our results demonstrated that the treatment of PCM patients promoted a regulation of IFN-gamma, IL-4 levels and CD28, CD86 expression bringing new insight to the cellular immune response in PCM.

Adolescent↗

PubMed2003

Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-gamma production, IgG avidity and antigen recognition by Western blotting.

Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Animals↗

PubMed2003

Effect of biphasic calcium phosphate on human macrophage functions in vitro.

Bioceramics may initiate several and complex biological reactions in host tissues. The cell-biomaterial interaction can determine macrophage activation that may elicit and sustain inflammatory response at the implant site. The current study describes some of the in vitro phenomena regarding the effect of surface reactivity of biphasic calcium phosphate (BCP) granules on human macrophages locomotion and secretion. X-ray diffraction analysis indicated that the synthesized ceramic presented 80% hydroxyapatite and 20% tricalcium phosphate. When BCP was put in contact with human macrophage cells, we observed that cells and BCP granules attached to each other. Cells attached to BCP presented a higher intracellular free Ca(2+) concentration compared with nonattached neighbors and secreted calcium phosphate particles into the medium. Energy dispersive X-ray analysis showed that the secreted particles presented a calcium/phosphorus ratio of 1.64 +/- 0.05, similar to hydroxyapatite. We propose that the secreted particles create a transition zone that allows further macrophage adhesion.

Biocompatible Materials↗

PubMed2003

Inhibition of the TEF/TEAD transcription factor activity by nuclear calcium and distinct kinase pathways.

Transcription enhancer factor (TEF/TEAD) is a family of four transcription factors that share a common TEA-DNA binding domain and are involved in similar cellular functions, such as cell differentiation and proliferation. All adult tissues express at least one of the four TEAD genes, so this family of transcription factors may be of widespread importance, yet little is known about their regulation. Here we examine the factors that regulate TEAD activity in CHO cells. RT-PCR indicated the presence of TEAD-1, TEAD-3, and both isoforms of TEAD-4, but not TEAD-2. Quantitative measurements showed that TEAD-4 is most abundant, followed by TEAD-3, then TEAD-1. We examined the relative effects of nuclear and cytosolic Ca(2+) on TEAD activity, since TEAD proteins are localized to the nucleus and since free Ca(2+) within the nucleus selectively regulates transcription in some systems. Chelation of nuclear but not cytosolic Ca(2+) increased TEAD activity two times above control. Inhibition of mitogen-activated protein kinase (MAPK) also increased TEAD activity, while cAMP decreased TEAD activity, and protein kinase C had no effect. Together, these results show that nuclear Ca(2+), MAPK, and cAMP each negatively regulate the activity of the TEAD transcription factor.

3T3 Cells↗

PubMed2003

Serological differentiation of acute and chronic schistosomiasis using Schistosoma mansoni recombinant protein RP26.

We obtained a recombinant protein encoded by Schistosoma mansoni gene which was able to differentiate acute from chronic schistosomiasis when applied as antigen in enzyme-linked immunosorbent assay (ELISA). A cDNA clone encoding a 26 kDa recombinant protein (RP26) was selected by screening of an adult worm S. mansoni lambdaZAP expression library with rabbit sera produced against PIII, an adult worm protein fraction already known to possess protective and immunomodulating effects. The clone cDNA presented 99% identity with S. mansoni Sm22.3 gene. We assayed IgG reactivity of sera from 18 patients with acute, 25 patients with chronic S. mansoni infection and 20 uninfected donors with RP26 in ELISA. Our results showed that 89% of sera were positive in acute schistosomiasis group, and only 26% in chronic group, without false-positive reactions in uninfected group. In mice the immune response to RP26 increased up to week 9 after infection and then diminished. We proposed that production of antibodies binding to RP26 stopped at the chronic stage of disease. The testing of sera from eight other parasitic infections with RP26 revealed no positive reactions in majority of sera. However, we observed low positive reaction in sera from 20% of leishmaniasis patients. Our results indicate that a recombinant protein RP26 can be used as immunodiagnostic reagent for detection of acute phase of schistosomiasis mansoni.

Acute Disease↗

PubMed2001

In vitro granuloma formation, NO production and cytokines profile from human mononuclear cells induced by fractionated antigens of Paracoccidioides brasiliensis.

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.

Antigens, Fungal↗

PubMed2001

Signal transduction events in human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens.

Activation of protein tyrosine kinases (PTKs) is a common step of T cell stimulation. However, the relationship between PTKs and activation of peripheral blood mononuclear cells (PBMC) from intestinal chronic schistosomiasis patients has not been explored yet. In this study, we investigated the participation of Lck and ZAP-70 protein tyrosine kinases (PTKs), as well as PLC-gamma1 and Shc proteins in PBMC activation by Schistosoma mansoni antigens. PBMC were stimulated with SEA (soluble egg antigen) or SWAP (soluble worm preparation), lysed, precipitated with specific antibodies and the level of tyrosine phosphorylation evaluated. Our results show that Lck and Shc were phosphorylated upon stimulation of the cells with SWAP, as well as with SEA. However, the phosphorylation level was more pronounced in SWAP than in SEA-stimulated cells. Phosphorylation of ZAP-70 was observed only in SWAP stimulated cells. Additionally, PLC-gamma1 phosphorylation was not observed in PBMC stimulated with SEA. Together, these results indicate that SEA and SWAP induce PBMC proliferation through distinct intracellular signaling pathways. Moreover, the weaker response of PBMC to SEA compared to SWAP stimulation suggests down-regulation of cells from intestinal chronic schistosomiasis patients to SEA, which may occur during immunomodulation to S. mansoni response.

Animals↗

PubMed2000

The role of IL-10 and IgG1 in the protection and granulomatous response in Schistosoma mansoni P24-immunized mice.

Previous work by our laboratory identified a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, designated PIII, able to elicit significant in vitro cell proliferation, and lower in vitro and in vivo granuloma formation. In the present work, we investigated some biological activities of P24, an antigenic component of PIII. Immunization of mice with this antigen induced a significant protection degree against challenge infection and significant decrease in the hepatic granuloma formation. Pre-incubation of spleen cells from P24-immunized mice with S. mansoni antigens induced a significant increase of interleukin (IL)-10 levels, but not interferon-gamma, in the cell supernatants. In addition, mice immunized with different S. mansoni antigens and P24 displayed indistinguishable levels of IgG2a in response to anti-S. mansoni antigens, while IgG1 levels were significantly increased. Collectively, our results indicate that P24 might mediate protective anti-parasite immunity and downregulate granulomatous hypersensitivity to S. mansoni eggs in part by its ability to induce a higher production of IgG1 and IL-10.

Animals↗

PubMed2000

Human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens: association between protein tyrosine kinases, mitogen-activated protein kinases and cytokine production.

Concerning schistosomiasis, little is known about the intracellular signaling response of human peripheral blood mononuclear cells (PBMC) to Schistosoma mansoni antigens. To understand the critical role of protein tyrosine kinases (PTKs) in PBMC activation by S. mansoni antigens, we investigated how inhibition of PTKs by genistein, a tyrosine kinase inhibitor, affects proliferation, cytokine production and activation of mitogen-activated protein kinases (MAPKs). Our studies showed that PTKs have an important role in proliferation of PBMC from chronic schistosomiasis patients as cells stimulated with S. mansoni soluble antigens in the presence of genistein had an impaired proliferation. In contrast, PTK inhibition failed to cause any effect on MAPKs activity. We also evaluated the cytokine production for interleukin (IL)-2, interferon gamma (IFN-gamma), and IL-10 in culture supernatants of PBMC treated with or without PTKs inhibitor. Our results show that PBMC from chronic patients produced a high amount of IL-10 when stimulated with soluble egg antigen preparation (SEA), however, the amount produced of IL-2 and IFN-gamma was not significant. In the presence of PTKs inhibitor only the production of IL-10 was decreased. The findings suggest that PTKs are involved on signal transduction pathway for PBMC activation, but may not be an absolute requirement for all signaling responses to S. mansoni antigens.

Animals↗

PubMed2000

Nitric oxide interaction with IL-10, MIP-1alpha, MCP-1 and RANTES over the in vitro granuloma formation against different Schistosoma mansoni antigenic preparations on human schistosomiasis.

Nitric oxide (NO) produced by cytokine-activated macrophages is reported to be cytotoxic against the helminth Schistosoma mansoni, although this is a controversial issue. Previous work in our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to soluble egg antigen (SEA) or to SWAP. Here we report that, in comparison to other S. mansoni antigenic preparations (SEA and SWAP), supernatants of PBMC cultivated with PIII possess higher concentrations of interleukin-10 (IL-10) and macrophage inflammatory protein (MIP-1alpha), concomitantly with lower concentrations of monocyte chemoattractant protein (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES). In the particular case of NO inhibition, supernatants of PBMC cultivated with PIII present decreased IL-10 levels. Altogether, these results indicate that IL-10, MIP-1alpha, MCP-1 and RANTES are distinctively important elements in the PIII modulating role, while NO seems to be pivotal in the regulation of granulomatous responses.

Adult↗

PubMed2000

Human IgG1 and IgG3 recognition of Schistosoma mansoni 14kDa fatty acid-binding recombinant protein.

The Schistosoma mansoni gene coding for a 14-kDa fatty acid-binding protein was amplified by PCR and subcloned into the prokaryotic expression vector pMAL-c2. Escherichia coli DH5alpha was transformed with the pMAL-Sm14 construct, and gene expression was induced hr isopropyl-beta-D-thiogalactopyranoside. The resulting recombinant (r) fusion protein was purified by affinity chromatography and confirmed by immunoblot analysis using antimaltose-binding protein or anti-Sm14 antibodies. Additionally, an antibody isotype profile was determined in sera of schistosomiasis patients to rSm14 or soluble adult worm antigen preparation. IgG1 and IgG3 subclass antibodies to rSm14 were predominant in sera of all patients studied whereas low levels of IgM, IgA or IgE were measured. Expression of a S. mansoni gene encoding a vaccine candidate is an important step to better study human immune responses to defined antigens.

Adolescent↗