Antiproliferative effects of Tubi-bee propolis in glioblastoma cell lines

Propolis is a resin formed by a complex chemical composition of substances that bees collect from plants. Since ancient times, propolis has been used in folk medicine, due to its biological properties, that include antimicrobial, anti-inflammatory, antitumoral and immunomodulatory activities. Glioblastoma is the most common human brain tumor. Despite the improvements in GBM standard treatment, patients' prognosis is still very poor. The aim of this work was to evaluate in vitro the Tubi-bee propolis effects on human glioblastoma (U251 and U343) and fibroblast (MRC-5) cell lines. Proliferation, clonogenic capacity and apoptosis were analyzed after treatment with 1 mg/mL and 2 mg/mL propolis concentrations for different time periods. Additionally, glioblastoma cell lines were submitted to treatment with propolis combined with temozolomide (TMZ). Data showed an antiproliferative effect of tubi-bee propolis against glioblastoma and fibroblast cell lines. Combination of propolis with TMZ had a synergic anti-proliferative effect. Moreover, propolis caused decrease in colony formation in glioblastoma cell lines. Propolis treatment had no effects on apoptosis, demonstrating a cytostatic action. Further investigations are needed to elucidate the molecular mechanism of the antitumor effect of propolis, and the study of its individual components may reveal specific molecules with antiproliferative capacity.     

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.     

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.     

Neutrophil activation induced by the lectin KM+ involves binding to CXCR2

The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both neutrophils and the extracellular matrix depend on the lectin's ability to recognize mannose-containing glycans. In the present study, we characterized the binding of KM+ to human neutrophils and the responses stimulated by this binding. Exposure to KM+ results in cell polarization, formation of a lamellipodium, and induction of deep ruffles on the cell surface. By fluorescence microscopy, we observed that KM+ is distributed homogeneously over the cell surface. KM+/ligand complexes are rapidly internalized, reaching maximum intracellular concentrations at 120 min, and decreasing thereafter. Furthermore, KM+ binding to the surface of human neutrophils is inhibited by the specific sugars, d-mannose or mannotriose. KM+-induced neutrophil migration is inhibited by pertussis toxin as well as by inhibition of CXCR2 activity. These results suggest that the KM+ ligand on the neutrophil surface is a G protein-coupled receptor (GPCR). The results also suggest that neutrophil migration induced by KM+ involves binding to CXCR2.     

Genetic structure of drone congregation areas of Africanized honeybees in southern Brazil

As yet, certain aspects of the Africanization process are not well understood, for example, the reproductive behavior of African and European honeybees and how the first Africanized swarms were formed and spread. Drone congregation areas (DCAs) are the ideal place to study honeybee reproduction under natural conditions since hundreds of drones from various colonies gather together in the same geographical area for mating. In the present study, we assessed the genetic structure of seven drone congregations and four commercial European-derived and Africanized apiaries in southern Brazil, employing seven microsatellite loci for this purpose. We also estimated the number of mother-colonies that drones of a specific DCA originated from. Pairwise comparison failed to reveal any population sub-structuring among the DCAs, thus indicating low mutual genetic differentiation. We also observed high genetic similarity between colonies of commercial apiaries and DCAs, besides a slight contribution from a European-derived apiary to a DCA formed nearby. Africanized DCAs seem to have a somewhat different genetic structure when compared to the European.     

T helper 1-inducing adjuvant protects against experimental paracoccidioidomycosis

Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1-stimulating adjuvant in P. brasiliensis-infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.     

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.