A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.Fundacao de Amparo a Pesquisa do Estado de Minas Gerais [APQ-00621-11]Fundacao de Amparo a Pesquisa do Estado de Minas GeraisFundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao PauloCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior [23038005295/2011-40]Coordenacao de Aperfeicoamento de Pessoal de Nivel SuperiorConselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Cientifico e Tecnologic

On the solid-liquid phase diagrams of binary mixtures of even saturated fatty alcohols: systems exhibiting peritectic reaction

CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESThe solid-liquid phase diagrams of the following binary mixtures of even saturated fatty alcohols are reported in the literature for the first time: 1-octanol (C8OH) + 1-decanol (C10OH), 1-decanol + 1-dodecanol (C12OH), 1-dodecanol + 1-hexadecanol (C16OH) and 1-tetradecanol (C14OH) + 1-octadecanol (C18OH). The phase diagrams were obtained by differential scanning calorimetry (DSC) using a linear heating rate of 1 K min-1 and further investigated by using a stepscan DSC method. X-ray diffraction (XRD) and polarized light microscopy were also used to complement the characterization of the phase diagrams which have shown a complex global behavior, presenting not only peritectic and eutectic reactions, but also the metatectic reaction and partial immiscibility on solid state. © 2014 Elsevier B.V.The solid-liquid phase diagrams of the following binary mixtures of even saturated fatty alcohols are reported in the literature for the first time: 1-octanol (C8OH) + 1-decanol (C10OH), 1-decanol + 1-dodecanol (C12OH), 1-dodecanol + 1-hexadecanol (C16OH) and 1-tetradecanol (C14OH) + 1-octadecanol (C18OH). The phase diagrams were obtained by differential scanning calorimetry (DSC) using a linear heating rate of 1 K min-1 and further investigated by using a stepscan DSC method. X-ray diffraction (XRD) and polarized light microscopy were also used to complement the characterization of the phase diagrams which have shown a complex global behavior, presenting not only peritectic and eutectic reactions, but also the metatectic reaction and partial immiscibility on solid state.589137147CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES142193/2010-0; 304495/2010-7; 552280/2010-0; 483340/2012-0; 479533/2013-02007/06162-1; 2008/09502-0; 2008/56258-8; 2012/05027-1Sem informaçãoFisk, P.R., Wildey, R.J., Girling, A.E., Sanderson, H., Belanger, S.E., Veenstra, G., Nielsen, A., Stanton, K., Environmental properties of long chain alcohols. 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Acta, 91, pp. 1286-1298The authors are grateful to CNPq (483340/2012-0, 304495/2010-7, 552280/2010-0, 142193/2010-0, 479533/2013-0), FAPESP (2007/06162-1, 2008/09502-0, 2008/56258-8 and 2012/05027-1), CAPES and FAEPEX/UNICAMP for their financial support and assistantships

P21-His₆ binds to CXCR4 chemokine receptor.

<p>CCR4 knockout did not affect P21-His₆ pro-phagocytic activity (A). CXCR4 inhibitor abolished the pro-phagocytic P21-His₆ activity (B). Peritoneal macrophages treated with P21-His₆ showed an increased expression of CXCR4 revealed by Western blotting (C). *P<0.5, **P<0.01, ***P<0.001.</p

P21-His₆ augments phagocytosis of different intracellular parasites.

<p>Peritoneal macrophages were incubated with <i>Trypanosoma cruzi</i>, <i>Leishmania amazonensis</i> or <i>Toxoplasma gondii</i> and treated or not with P21-His₆. The number of internalized parasites in treated cells was higher than in control cells, indicating that P21-His₆ increases parasite internalization. *P<0.5, **P<0.01, ***P<0.001.</p

P21-His₆ activity depends on PI3-kinase pathway.

<p>Peritoneal macrophages were incubated with zymosan, treated or not with P21-His₆, and also treated or not with inhibitors to PI3-kinase (A), AKT (B), n-Ras (C), ERK 2 (D), MEK 1 (E), MEK1/2 (F), m-Tor (G). P21-His₆ activity was not detected in the presence of PI3-K inhibitor. *P<0.5, **P<0.01, ***P<0.001.</p

Peritoneal macrophages treated with P21-His₆ show increased actin polymerization.

<p>Peritoneal macrophages were treated or not with P21-His₆ for 0.5, 1.0 and 3.0 hours in the presence or not of anti-P21-His₆ polyclonal antibody and FcR blocking. The actin cytoskeleton was stained with TRITC-phalloidin. Flow cytometry showed that the cells treated with the recombinant protein had a higher fluorescence intensity than the control cells following 0.5 (A) and 1.0 hour (B) of incubation. After 3.0 hours of incubation, the protein lost its activity (C). Representative histograms are also shown. Macrophages not treated with P21-His₆ (D); macrophages treated with P21-His₆ (E); macrophages with FcR-blocking and treated with P21-His₆ previously incubated with anti-P21-His₆ polyclonal antibody (F).*P<0.5, **P<0.01, ***P<0.001.</p

P21-His₆ enhances phagocytosis of zymosan particles.

<p>Phagocytosis assay using zymosan particles treated or not with P21-His₆. (A) peritoneal macrophages, treated in different periods,1, 3, 6, 12, 24 and 48 hours. P21-His₆ enhanced zymosan internalization, in all treatment periods. (B) Peritoneal macrophages (grey bar), treated with BSA (white bars) or P21-His6 (black bars) at different concentrations: 1, 20, 40 and 80 µg/ml. P21-His6, but not BSA, enhanced zymosan internalization at all concentrations tested. (C) Peritoneal macrophages obtained from wild-type (WT) or <i>toll4^−/−</i> animals, treated or not with P21-His₆, and those also treated with polymyxin B. P21-His₆ treatment increased zymosan phagocytosis in both, the WT and <i>toll4^−/−</i> macrophages. Also, treatment with polymyxin B did not inhibit P21-His₆ activity. (D) Phagocytosis assay using folded and denatured P21-His₆ (dP21-His₆) at 100°C. Note that only folded protein was able to upregulate phagocytosis. (E) Peritoneal macrophages with (white bars) or without (black bars) FcR blocked were incubated with P21-His₆ (40 µg/ml) previously opsonized in different concentrations of serum. Peritoneal macrophages with FcR did not show an enhanced zymosan internalization as those without FcR blocked. *P<0.05 **P<0.01 ***P<0.001.</p

Galectin-3: A Friend but Not a Foe during Trypanosoma cruzi Experimental Infection

Trypanosoma cruzi interacts with host cells, including cardiomyocytes, and induces the production of cytokines, chemokines, metalloproteinases, and glycan-binding proteins. Among the glycan-binding proteins is Galectin-3 (Gal-3), which is upregulated after T. cruzi infection. Gal-3 is a member of the lectin family with affinity for β-galactose containing molecules; it can be found in both the nucleus and the cytoplasm and can be either membrane-associated or secreted. This lectin is involved in several immunoregulatory and parasite infection process. Here, we explored the consequences of Gal-3 deficiency during acute and chronic T. cruzi experimental infection. Our results demonstrated that lack of Gal-3 enhanced in vitro replication of intracellular parasites, increased in vivo systemic parasitaemia, and reduced leukocyte recruitment. Moreover, we observed decreased secretion of pro-inflammatory cytokines in spleen and heart of infected Gal-3 knockout mice. Lack of Gal-3 also led to elevated mast cell recruitment and fibrosis of heart tissue. In conclusion, galectin-3 expression plays a pivotal role in controlling T. cruzi infection, preventing heart damage and fibrosis