Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites.

Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles

African Trypanosomes undermine humoral responses and vaccine development : link with inflammatory responses?

African trypanosomosis is a debilitating disease of great medical and socioeconomical importance. It is caused by strictly extracellular protozoan parasites capable of infecting all vertebrate classes including human, livestock, and game animals. To survive within their mammalian host, trypanosomes have evolved efficient immune escape mechanisms and manipulate the entire host immune response, including the humoral response. This report provides an overview of how trypanosomes initially trigger and subsequently undermine the development of an effective host antibody response. Indeed, results available to date obtained in both natural and experimental infection models show that trypanosomes impair homeostatic B-cell lymphopoiesis, B-cell maturation and survival and B-cell memory development. Data on B-cell dysfunctioning in correlation with parasite virulence and trypanosome-mediated inflammation will be discussed, as well as the impact of trypanosomosis on heterologous vaccine efficacy and diagnosis. Therefore, new strategies aiming at enhancing vaccination efficacy could benefit from a combination of (i) early parasite diagnosis, (ii) anti-trypanosome (drugs) treatment, and (iii) anti-inflammatory treatment that collectively might allow B-cell recovery and improve vaccination

Microvesicles: ROS scavengers and ROS producers

This review analyzes the relationship between microvesicles and reactive oxygen species (ROS). This relationship is bidirectional; on the one hand, the number and content of microvesicles produced by the cells are affected by oxidative stress conditions; on the other hand, microvesicles can directly and/or indirectly modify the ROS content in the extra- as well as the intracellular compartments. In this regard, microvesicles contain a pro-oxidant or antioxidant machinery that may produce or scavenge ROS: direct effect. This mechanism is especially suitable for eliminating ROS in the extracellular compartment. Endothelial microvesicles, in particular, contain a specific and well-developed antioxidant machinery. On the other hand, the molecules included in microvesicles can modify (activate or inhibit) ROS metabolism in their target cells: indirect effect. This can be achieved by the incorporation into the cells of ROS metabolic enzymes included in the microvesicles, or by the regulation of signaling pathways involved in ROS metabolism. Proteins, as well as miRNAs, are involved in this last effect

MicroRNA-126 regulates Hypoxia-Inducible Factor-1alfa which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence

Whereas a healthy endothelium maintains physiological vascular functions, endothelial damage contributes to the development of cardiovascular diseases. Endothelial senescence is the main determinant of endothelial dysfunction and thus of age-related cardiovascular disease. The objective of this study is to test the involvement of microRNA-126 and HIF-1 alpha in a model of replicative endothelial senescence and the interrelationship between both molecules in this in vitro model. We demonstrated that senescent endothelial cells experience impaired tube formation and delayed wound healing. Senescent endothelial cells failed to express HIF-1 alpha, and the microvesicles released by these cells failed to carry HIF-1 alpha. Of note, HIF-1 alpha protein levels were restored in HIF-1 alpha stabilizer-treated senescent endothelial cells. Finally, we show that microRNA-126 was downregulated in senescent endothelial cells and microvesicles. With regard to the interplay between microRNA-126 and HIF-1 alpha, transfection with a microRNA-126 inhibitor downregulated HIF-1 alpha expression in early passage endothelial cells. Moreover, while HIF-1 alpha inhibition reduced tube formation and wound healing closure, microRNA-126 levels remained unchanged. These data indicate that HIF-1 alpha is a target of miRNA-126 in protective and reparative functions, and suggest that their therapeutic modulation could benefit age-related vascular disease

Endothelial Extracellular Vesicles Produced by Senescent Cells: Pathophysiological Role in the Cardiovascular Disease Associated with all Types of Diabetes Mellitus

Endothelial senescence-associated with aging or induced prematurely in pathological situations, such as diabetes, is a first step in the development of Cardiovascular Disease (CVDs) and particularly inflammatory cardiovascular diseases. The main mechanism that links endothelial senescence and the progression of CVDs is the production of altered Extracellular Vesicles (EVs) by senescent endothelial cells among them, Microvesicles (MVs). MVs are recognized as intercellular signaling elements that play a key role in regulating tissue homeostasis. However, MVs produced by damage cell conveyed epigenetic signals, mainly involving microRNAs, which induce many of the injured responses in other vascular cells leading to the development of CVDs. Many studies strongly support that the quantification and characterization of the MVs released by senescent endothelial cells may be useful diagnostic tools in patients with CVDs, as well as a future therapeutic target for these diseases. In this review, we summarize the current knowledge linking senescence-associated MVs to the development of CVDs and discuss the roles of these MVs, in particular, in diabetic-associated increases the risk of CVDs

Chagas' disease: an update on immune mechanisms and therapeutic strategies

The final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order. Among the pathogenic trypanosomatids, some species are of particular interest due to their medical importance. These species include the agent responsible for Chagas' disease, Trypanosoma cruzi. Approximately 8 to 10 million people are infected in the Americas, and approximately 40 million are at risk. in the present review, we discuss in detail the immune mechanisms elicited during infection by T. cruzi and the effects of chemotherapy in controlling parasite proliferation and on the host immune system.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Biologia Estrutural e QuImica Medicinal em Doencas Infecciosas (INBEQMeDI)Univ São Paulo, Inst Biomed Sci, Dept Parasitol, São Paulo, BrazilMackenzie Presbeterian Univ, Ctr Biomol Sci & Hlth, São Paulo, BrazilNatl Univ Rosario, Sch Med Sci, Inst Immunol, Rosario, Santa Fe, ArgentinaCSIC, Inst Parasitol & Biomed Lopez Neyra, Granada, SpainUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPESP: 08/57596-4FAPESP: 07/08648-9CNPq: 473906/2008-2Web of Scienc

Endothelial Senescence and the Chronic Vascular Diseases: Challenges and Therapeutic Opportunities in Atherosclerosis

Atherosclerosis is probably one of the paradigms of disease linked to aging. Underlying the physiopathology of atherosclerosis are cellular senescence, oxidative stress, and inflammation. These factors are increased in the elderly and from chronic disease patients. Elevated levels of oxidative stress affect cellular function and metabolism, inducing senescence. This senescence modifies the cell phenotype into a senescent secretory phenotype. This phenotype activates immune cells, leading to chronic systemic inflammation. Moreover, due to their secretory phenotype, senescence cells present an increased release of highlighted extracellular vesicles that will change nearby/neighborhood cells and paracrine signaling. For this reason, searching for specific senescent cell biomarkers and therapies against the development/killing of senescent cells has become relevant. Recently, senomorphic and senolityc drugs have become relevant in slowing down or eliminating senescence cells. However, even though they have shown promising results in experimental studies, their clinical use is still yet to be determinedInstituto de Salud Carlos IIISociedad Española de NefrologíaComunidad de MadridFondo Europeo de Desarrollo Regiona

Molecular and Structural Discrimination of Proline Racemase and Hydroxyproline-2-Epimerase from Nosocomial and Bacterial Pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for ‘proline racemase’ virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not ‘one way’ directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on contraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host

Endothelial Cell Senescence in the Pathogenesis of Endothelial Dysfunction

Aging is the main risk factor for cardiovascular diseases (CVD), and senescence in endothelial cells seems to be an initial step in the cascade of events that will culminate with the development of these pathologies. In this chapter, we examine the pathophysiological mechanism(s) involved in endothelial senescence, leading to CVD as well as the biochemical and cellular pathways that may explain the activation and development of the process of endothelial senescence, and we discuss new hypotheses supported by experimental results which suggest that the senescent endothelial cell may induce a general process of vascular senescence. This process is probably induced either by soluble molecules secreted by these senescent cells and/or by intercellular signals transported in cellular vesicles that may be useful as biomarkers and as potential therapeutic targets in endothelial senescence

Increasing the Magnesium Concentration in Various Dialysate Solutions Differentially Modulates Oxidative Stress in a Human Monocyte Cell Line

Oxidative stress is exacerbated in hemodialysis patients by several factors, including the uremic environment and the use of dialysis fluids (DFs). Since magnesium (Mg) plays a key role in modulating immune function and in reducing oxidative stress, we aimed to evaluate whether increasing the Mg concentration in different DFs could protect against oxidative stress in immunocompetent cells in vitro. Effect of ADF (acetate 3 mM), CDF (citrate 1 mM), and ACDF (citrate 0.8 mM + acetate 0.3 mM) dialysates with Mg at standard (0.5 mM) or higher (1, 1.25, and 2 mM) concentrations were assessed in THP-1 monocyte cultures. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were quantified under basal and uremic conditions (indoxyl sulfate (IS) treatment). Under uremic conditions, the three DFs with 0.5 mM Mg promoted higher ROS production and lipid damage than the control solution. However, CDF and ACDF induced lower levels of ROS and MDA, compared to that induced by ADF. High Mg concentration (1.25 and/or 2 mM) in CDF and ACDF protected against oxidative stress, indicated by reduced ROS and MDA levels compared to respective DFs with standard concentration of Mg. Increasing Mg concentrations in ADF promoted high ROS production and MDA content. Thus, an increase in Mg content in DFs has differential effects on the oxidative stress in IS-treated THP-1 cells depending on the dialysate used.Instituto de Salud Carlos IIISociedad Española de Nefrologí