Leonardo Marques da Fonseca

The role of KATP channels on propofol preconditioning in a cellular model of renal ischemia-reperfusion.

BACKGROUND: Propofol (2,6-diisopropylphenol) has been shown to protect several organs, including the kidneys, from ischemia-reperfusion (I-R)-induced injury. Although propofol affects adenosine triphosphate-sensitive potassium (KATP) channels in nonrenal tissues, it is still not clear by which mechanisms propofol protects renal cells from such damage. In this study, we investigated whether propofol induces renal preconditioning through renal KATP channels. METHODS: A reversible ATP depletion (antimycin A) followed by restoration of substrate supply in LLC-PK1 cells was used as an in vitro model of renal I-R. Cell viability was assessed by dimethylthiazol-diphenyltetrazol bromide and trypan blue dye exclusion test assays. Apoptosis was evaluated by annexin V–fluorescein isothiocyanate staining by flow cytometry and immunofluorescence. Propofol treatments were initiated at various time intervals: 1 or 24 h before ischemia, only during ischemia, or only during reperfusion. To evaluate the mechanisms of propofol protection, specific KATP channel inhibitors or activators were used in some experiments during propofol pretreatment. RESULTS: Propofol attenuated I-R injury on LLC-PK1 cells when present either 1 or 24 h before initiated I-R, and also during the recovery period, but not when added only during ischemia. Propofol pretreatment significantly protected LLC-PK1 from I-R-induced apoptosis. The protective effect of propofol was prevented by glibenclamide (a sarcolemmal ATP-dependent K+ channel blocker) and decreased by 5-hydroxidecanoic acid (a mitochondrial ATP-dependent K+ channel blocker), but it was not modified by diazoxide (a selective opener of ATP-sensitive K+ channel). CONCLUSION: Propofol protected cells against apoptosis induced by I-R. This protection was probably due to a preconditioning effect of propofol and was, at least in part, mediated by KATP channels.

Glycosylation in Cancer: Interplay between Multidrug Resistance and Epithelial-to-Mesenchymal Transition?

The expression of unusual glycan structures is a hallmark of cancer progression, and their functional roles in cancer biology have been extensively investigated in epithelial-to-mesenchymal transition (EMT) models. EMT is a physiological process involved in embryonic development and wound healing. It is characterized by loss of epithelial cell polarity and cell adhesion, permitting cell migration, and thus formation of new epithelia. However, this process is unwanted when occurring outside their physiological limit, resulting in fibrosis of organs and progression of cancer and metastasis. Several studies observed that EMT is related to the acquisition of multidrug resistance (MDR) phenotype, a condition in which cancer cells acquire resistance to multiple different drugs, which has virtually nothing in common. However, although some studies suggested interplay between these two apparently distinct phenomena, almost nothing is known about this possible relationship. A common pathway to them is the need for glycosylation, a post-translational modification that can alter biological function. Thus, this review intends to compile the main facts obtained until now in these two areas, as an effort to unravel the relationship between EMT and MDR.

Preliminary Anticancer Potency Evaluation and Phytochemical investigation of Methanol Extract of Piper claussenianum (Miq.) C. DC.

Andre M. Marques*, Renan A. de Paiva, Leonardo M. da Fonseca, Marcia A. M. Capella, Elsie F. Guimaraes and Maria Auxiliadora C. Kaplan Nucleo de Pesquisas de Produtos Naturais (NPPN), Centro de Ciencias da Saude, Bloco H, Universidade Federal do Rio de Janeiro (UFRJ). CEP: 21941590 Rio de Janeiro, RJ, Brazil. Instituto de Biofisica Carlos Chagas Filho, Programa de Oncobiologia, Universidade Federal do Rio de Janeiro (UFRJ). CEP: 21941-590 Rio de Janeiro, RJ, Brazil. Instituto de Pesquisa Jardim Botânico do Rio de Janeiro. CEP: 22.460-030 Rio de Janeiro, RJ, Brazil.

Modulation of Cell Sialoglycophenotype: A Stylish Mechanism Adopted by Trypanosoma cruzi to Ensure Its Persistence in the Infected Host

Trypanosoma cruzi, the etiological agent of Chagas disease exhibits multiple mechanisms to guarantee its establishment and persistence in the infected host. It has been well demonstrated that T. cruzi is not able to synthesize sialic acids (Sia). To acquire the monosaccharide, the parasite makes use of a multifunctional enzyme called trans-sialidase (Tc-TS). Since this enzyme has no analogous in the vertebrate host, it has been used as a target in drug therapy development. Tc-TS preferentially catalyzes the transfer of Sia from the host glycoconjugates to the terminal β-galactopyranosyl residues of mucin-like molecules present on the parasite’s cell surface. Alternatively, the enzyme can sialylate/re-sialylate glycoconjugates expressed on the surface of host cells. Since its discovery, several studies have shown that T. cruzi employs the Tc-TS activity to modulate the host cell sialoglycophenotype, thus favoring its perpetuation in the infected vertebrate. In this review, we summarize the dynamic of host/parasite sialoglycophenotype modulation, highlighting its role in the subversion of host immune response in order to promote the establishment of persistent chronic infection.

Distribution of theO-acetyl groups and β-galactofuranose units in galactoxylomannans of the opportunistic fungusCryptococcus neoformans

Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that β-galactofuranose (β-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

Metabolic Symbiosis and Immunomodulation: How Tumor Cell-Derived Lactate May Disturb Innate and Adaptive Immune Responses

The tumor microenvironment (TME) is composed by cellular and non-cellular components. Examples include the following: (i) bone marrow-derived inflammatory cells, (ii) fibroblasts, (iii) blood vessels, (iv) immune cells, and (v) extracellular matrix components. In most cases, this combination of components may result in an inhospitable environment, in which a significant retrenchment in nutrients and oxygen considerably disturbs cell metabolism. Cancer cells are characterized by an enhanced uptake and utilization of glucose, a phenomenon described by Otto Warburg over 90 years ago. One of the main products of this reprogrammed cell metabolism is lactate. “Lactagenic” or lactate-producing cancer cells are characterized by their immunomodulatory properties, since lactate, the end product of the aerobic glycolysis, besides acting as an inducer of cellular signaling phenomena to influence cellular fate, might also play a role as an immunosuppressive metabolite. Over the last 10 years, it has been well accepted that in the TME, the lactate secreted by transformed cells is able to compromise the function and/or assembly of an effective immune response against tumors. Herein, we will discuss recent advances regarding the deleterious effect of high concentrations of lactate on the tumor-infiltrating immune cells, which might characterize an innovative way of understanding the tumor-immune privilege.

Multiple Myeloma Cells Express Key Immunoregulatory Cytokines and Modulate the Monocyte Migratory Response

Multiple myeloma (MM) is a plasma cell disorder that still remains incurable. The immune dysfunction of the host is a striking characteristic of MM, leading to tumor growth and reducing the survival rate of patients. Monocytes are precursors of conventional dendritic cells (DCs), a major player in the immunity mechanisms driving protective T cell responses against tumor. Herein, we report that human MM RPMI 8226 cell line shows a pronounced chemoattractant activity for monocytes and also expresses enhanced levels of the leukocyte chemotactic cytokines CXCL12, CCL5, MIP-1β, and CXCL10 in association with elevated levels of both key immunoregulatory interleukins such as IL-4 and IL-10. This cytokine profile was observed together with reduced expression of IFN-γ by MM RPMI 8226 cell line, a determinant interleukin involved in the acquisition of cellular-mediated protective responses against tumor cells. We further demonstrate that MM RPMI 8226 cell line expresses elevated levels of soluble form of the intercellular adhesion molecule-1 known to inhibit antitumoral T cell responses. This attractive modulation of immune responses by MM cells might provide a means to impair early antitumor responses during the establishment of cytokine-mediated immunosuppressive tumor niche.

Role of Inactive and Active Trypanosoma cruzi Trans-sialidases on T Cell Homing and Secretion of Inflammatory Cytokines

Trans-sialidase from Trypanosoma cruzi (Tc-TS) belongs to a superfamily of proteins that may have enzymatic activity. While enzymatically active members (Tc-aTS) are able to transfer sialic acid from the host cell sialyl-glycoconjugates onto the parasite or to other molecules on the host cell surface, the inactive members (Tc-iTS) are characterized by their lectinic properties. Over the last 10 years, several papers demonstrated that, individually, Tc-aTS or Tc-iTS is able to modulate several biological events. Since the genes encoding Tc-iTS and Tc-aTS are present in the same copy number, and both proteins portray similar substrate-specificities as well, it would be plausible to speculate that such molecules may compete for the same sialyl-glycan structures and govern numerous immunobiological phenomena. However, their combined effect has never been evaluated in the course of an acute infection. In this study, we investigated the ability of both proteins to modulate the production of inflammatory signals, as well as the homing of T cells to the cardiac tissue of infected mice, events that usually occur during the acute phase of T. cruzi infection. The results showed that the intravenous administration of Tc-iTS, but not Tc-aTS protected the cardiac tissue from injury caused by reduced traffic of inflammatory cells. In addition, the ability of Tc-aTS to modulate the production of inflammatory cytokines was attenuated and/or compromised when Tc-iTS was co-injected in the same proportions. These results suggest that although both proteins present structural similarities and compete for the same sialyl-glycan epitopes, they might present distinct immunomodulatory properties on T cells following T. cruzi infection.

Expanding the knowledge of the chemical structure of glycoconjugates from Trypanosoma cruzi TcI genotype. Contribution to taxonomic studies

One of the main obstacles to the treatment of Chagas disease is the genetic and phenotypical variance displayed by T. cruzi strains, resulting in differences in morphology, virulence, pathogenicity and drug susceptibility. To better understand the role of glycoconjungates in Chagas disease, we performed the molecular characterization of the O-linked chains from mucins and glycoinositolphospholipids (GIPLs) of the Silvio X10 clone 1 strain. We demonstrated the presence of a β-galactofuranose (β-Galf) unity linked to the O-4 position of the α-N-acetylglucosamine (α-GlcNAc)O-4 in Tc-mucins. GIPLs analysis showed that the lipidic portion is exclusively composed of ceramide and the PI-oligossacharidic portion contains the Man4(AEP)GlcN-Ins-PO4 core, substituted by ethanolamine-phosphate (EtNP) on the third distal mannose from inositol, which may or may not have a terminal β Galf unity. These results confirm the classification of the Silvio X10/1 strain in group T. cruzi I. Again, it is noted that the study of T. cruzi surface glycoconjugates confirm the molecular results and the hypothesis that surface glycoconjugates may be interesting biomarker for the differentiation of trypanosomatid strains.

ABCC1 is related to the protection of the distal nephron against hyperosmolality and high sodium environment: possible implications for cancer chemotherapy.

Aims Glutathione (GSH) plays an important role in protecting cells against oxidative damage. ABCC1 protein transports GSH. Although this protein is largely studied in cancer, due to multidrug resistance phenotype, its role in the tubular cells of the kidney is unknown. The goal of this study was to find out whether ABCC1 has a role in protecting cells from the distal nephron against the stress caused by high medullar osmolality. Main Methods MA104 cells were treated with high concentrations of sodium chloride, urea, or both to raise the osmolality of the culture medium. Cell viability was accessed by MTT and trypan blue assays. ABCC1 expression and extrusion of carboxi-fluorescein (CF), a fluorescent ABCC1 substrate, were measured by flow cytometry. Key Findings Incubation of MA104 cells in a high sodium concentration medium resulted in changes in cell granularity and altered expression and activity of ABCC1. Urea did not alter ABCC1 expression or activity, but reversed the observed NaCl effects. High sodium concentrations also had a negative effect on cell viability and urea also protected cells against this effect. Significance Our findings demonstrate that ABCC1 plays a significant role in the protection of kidney epithelial cells against the stress caused by high sodium environment present in renal medulla.