Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization.

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Sm60, a mannose-binding protein from Schistosoma mansoni with inflammatory property.

We demonstrate here that a mannose-binding protein from Schistosoma mansoni, termed Sm60, was recovered in the mannose-eluted fraction (Man(+)) upon affinity chromatography on immobilised mannose of the soluble antigen fraction from adult worm tegument and cercariae. Sm60 was detected in the Man(+) fraction as a prominent doublet with an apparent molecular mass of 60-66 kDa by SDS-PAGE and appeared as a single band with a pI of approximately 6.9 by isoelectrofocusing. Sm60 was also detected in preparations of schistosomula extract and soluble egg antigens using a mouse polyclonal anti-Sm60 serum on immunoblotting assay. This antiserum demonstrated that Sm60 was localised on the tegument of S. mansoni adult worm. In order to determine the role of Sm60 in host-parasite interactions, we showed that Sm60 induced in vitro migration of human neutrophil in a dose-dependent manner and in vitro mast cell degranulation. Sm60 triggered these activities through its carbohydrate-binding site, since these activities were selectively inhibited by 0.2 M D-mannose, but not by 0.2 M D-galactose. Furthermore, Sm60 induced in vivo neutrophil migration. In contrast, mast cell-depleted rats presented a significant reduction of the neutrophil migration induced by Sm60 as compared with non-depleted controls. These data suggest that in vivo neutrophil migration induced by Sm60 is modulated by mast cell-dependent mechanisms. Sm60 might play a key role in the host-parasite interaction, and its characterization opens perspective to examine the role of this molecule in the biology of S. mansoni.

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.

KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection.

The outcome and severity of some diseases correlate with the dominance of either the T helper 1 (Th1) or Th2 immune response, which is stimulated by IL-12 or IL-4, respectively. In the present study we demonstrate that gamma interferon (IFN-gamma) secretion by murine spleen cells stimulated with KM(+), a mannose-binding lectin from Artocarpus integrifolia, is due to IL-12 induction, because (1) macrophages from several sources (including cell lines) produced IL-12 p40 in response to KM(+), and (2) lectin-free supernatants from J774 cell line cultures stimulated with KM(+) induced the secretion of IFN-gamma by spleen cell cultures, an effect blocked by the supernatant pretreatment with anti-IL-12 antibody. The known pattern of susceptibility of BALB/c mice to infection with Leishmania major, attributed to high levels of IL-4 production leading to a Th2 nonprotective immune response, was modified by administration of KM(+). Draining lymph node cells from these immunized BALB/c mice (in contrast to cells from animals immunized only with soluble leishmanial antigen [SLA]) secreted high levels of IFN-gamma and low levels of IL-4, which characterized a Th1 rather than a Th2 response pattern. The footpad thickness of BALB/c mice immunized with SLA plus KM(+) and challenged with L. major was similar to that of uninfected mice. This beneficial effect against leishmanial infection was blocked by pretreatment of these mice with anti-IL-12 antibody. These observations indicate that KM(+) induces IL-12 p40 in vivo and has a protective effect against L. major infection.

The Rubino test for leprosy is a beta2-glycoprotein 1-dependent antiphospholipid reaction.

We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red-blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae-specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The latter was recognized by the RRF IgM on immunoblot and its N-terminal sequence indicated that it was beta2-glycoprotein 1 (beta2-GP1), an anionic phospholipid-binding protein. Methanol-treated formalinized SRBC did not support the RR. Thin-layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell-surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, beta2-GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti-PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a beta2-GP1-dependent anti-PL.

Identification of IgM as the leprosy patient serum factor responsible for rapid sedimentation of formolized sheep erythrocytes.

The serum of some leprosy patients with impaired specific cellular immunity for Mycobacterium leprae causes rapid sedimentation of formolized sheep erythrocytes, a phenomenon known as the Rubino reaction. The Rubino factor was precipitated from positive sera by 5% (w/v) polyethylene glycol (PEG), bound to a concanavalin A (ConA)-Sepharose column and eluted with D-mannose, and was also eluted from a Mono Q column, pH 8.0, with 0.4 M NaCl. The Rubino factor was eluted in a volume which coincided with that of human serum IgM from a Sepharose 6 column. IgM was present in the preparation obtained by this sequence of chromatographic procedures. The correspondence of IgM with the Rubino factor was demonstrated by the following data: a) the Rubino factor was adsorbed to rabbit IgG antihuman IgM-agarose and the activity was recovered in the acid eluate of the column; b) the Rubino reaction was inhibited in the presence of rabbit antihuman IgM antibodies. This behavior was not observed when the same procedures were carried out using anti-alpha 2-macroglobulin antibodies as a control. The rapid sedimentation of formolized sheep red cells caused by the serum of lepromatous leprosy patients was not inhibited by phenolic glycolipid-I, suggesting that the IgM responsible for the Rubino reaction is not directed to this antigen which is specific for M. leprae. There was no correlation between the positivity of the Rubino reaction and the increase in total serum IgM levels observed in 42% of the lepromatous patients evaluated. The demonstration that the Rubino factor is an IgM now permits the identification of the epitope recognized by it, and this may be used as a tool to understand the specific cellular immune unresponsiveness which characterizes lepromatous leprosy.