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Paracoccidioides fungi are thermodimorphic microorganisms that cause paracoccidioidomycosis (PCM), an autochthonous disease from Latin America, with most cases in Brazil. Humans become infected by inhaling conidia or mycelial fragments that transform into yeast at body temperature. These fungi cause chronic-granulomatous inflammation, which may promote fibrosis and parenchyma destruction in the lungs. In response to stress imposed by the host, fungi Paracoccidioides spp. increase the expression of heat shock proteins (HSP), which protect them by sustaining cellular proteostasis. Our group has studied the role of HSP60 in PCM, and previous data show that the recombinant HSP60 (rHSP60) has a deleterious effect when used in a single dose as therapy for experimental PCM. Here, we investigated the mechanism by which rHSP60 could worsen the disease. We found that rHSP60 caused the viability loss of splenic or lymph node cells from both immunized and non-immunized mice, including in splenic T lymphocytes under polyclonal stimulation with concanavalin A, probably by undergoing apoptosis. Among analyzed splenic cells, lymphocytes were indeed the main cells to die. When we investigated the death mechanisms, remarkably, we found that there was no viability loss in rHSP60-stimulated splenic cells from mice deficient in Toll-like receptor 4, TRIF adapter protein, and TNF receptor 1(TNFR1), as well as rHSP60-stimulated WT cells incubated with anti-TNF antibody. Besides, caspase-8 inhibitor IETD-CHO blocked the rHSP60 effect on splenic cells, suggesting that rHSP60 induces the extrinsic apoptosis pathway dependent on signaling via TLR4/TRIF and TNFR1. [ABSTRACT FROM AUTHOR]

Abstract Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO) production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis. [ABSTRACT FROM AUTHOR]

The outcome and severity of some diseases correlatewith the dominance of either the T helper 1 (Th1) or Th2 immuneresponse, which is stimulated by IL-12 or IL-4, respectively. Inthe present study we demonstrate that gamma interferon (IFN-γ) secretion by murine spleen cells stimulatedwith KM+, a mannose-binding lectin from Artocarpusintegrifolia, is due to IL-12 induction, because (1) macrophagesfrom several sources (including cell lines) produced IL-12 p40 inresponse to KM+, and (2) lectin-free supernatantsfrom J774 cell line cultures stimulated with KM+ inducedthe secretion of IFN-γ by spleen cellcultures, an effect blocked by the supernatant pretreatment with anti-IL-12antibody. The known pattern of susceptibility of BALB/cmice to infection with Leishmania major, attributed to highlevels of IL-4 production leading to a Th2 nonprotective immuneresponse, was modified by administration of KM+. Draininglymph node cells from these immunized BALB/c mice (in contrastto cells from animals immunized only with soluble leishmanial antigen [SLA])secreted high levels of IFN-γ and lowlevels of IL-4, which characterized a Th1 rather than a Th2 responsepattern. The footpad thickness of BALB/c mice immunizedwith SLA plus KM+ and challenged with L.major was similar to that of uninfected mice. This beneficialeffect against leishmanial infection was blocked by pretreatmentof these mice with anti-IL-12 antibody. These observations indicatethat KM+ induces IL-12 p40 in vivo andhas a protective effect against L. major infection. [ABSTRACT FROM PUBLISHER]

Host cell invasion by Toxoplasma gondii isa multistep process with one of the first steps being the apicalrelease of micronemal proteins that interact with host receptors.We demonstrate here that micronemal protein 1 (MIC1) is a lactose-bindinglectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac+)fraction on affinity chromatography on immobilized lactose of thesoluble antigen fraction from tachyzoites of the virulent RH strain.MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4was also identified by sequencing cDNA clones isolated from an expressionlibrary following screening with mouse polyclonal anti-60/70kDa (Lac+ proteins) serum. This antiserum localizedthe Lac+ proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac+ fractioninduced hemagglutination (mainly type A human erythrocytes), whichwas inhibited by β-galactosides (3 mMlactose and 12 mM galactose) but not by up to 100 mM melibiose (α-galactoside), fucose, mannose, orglucose or 0.2 mg/ml heparin. The lectin activity of theLac+ preparation was attributed to MIC1, becauseblotted MIC1, but not native MIC4, bound human erythrocyte typeA and fetuin. The copurification of MIC1 and MIC4 may have beendue to their association, as reported by others. These data suggestthat MIC1 may act through its lectin activity during T.gondii infection. [ABSTRACT FROM PUBLISHER]

Paracoccidioides brasiliensis components interact with host cells and can influence the pathogenesis of paracoccidioidomycosis (PCM). Among the components released by P. brasiliensis, gp 43 and a heavily glycosylated antigen with MM>160 kDa are the most recognized by serum antibodies from patients with PCM. In order to isolate the high MM glycoconjugate, we carried out affinity chromatography of a crude exoantigen preparation on immobilized jacalin. The bound fraction (JBE, jacalin binding exoantigen) consisted of a major antigen of high MM and frequently of an additional 70-kDa minor protein. This protein, designated paracoccin, exhibited selective binding to immobilized GlcNAc, a property that was used for its purification. The structural data of paracoccin obtained by mass spectrometry of tryptic peptides did not match any known protein. Anti-paracoccin serum localized the lectin on the surface of P. brasiliensis yeasts, especially in the budding regions. Paracoccin was able to interact with laminin in a dose-dependent manner. This interaction was inhibited by GlcNAc, followed by D-glucose and D-mannose, but not by D-galactose, N-acetyl-galactosamine or L-fucose. Interestingly, paracoccin induced both resident and elicited mouse peritoneal cavity macrophages to release high and persistent levels of TNF-alpha in vitro, a fact that was associated with high nitric oxide production in elicited cells. Because binding to laminin can favor yeast adhesion and invasion of host tissues, and overproduction of NO has been associated with suppression of cell immunity, paracoccin is suggested to play an important role in PCM pathogenesis.