Suramin attenuates dystrophin‐deficient cardiomyopathy in the mdx mouse model of duchenne muscular dystrophy

Introduction: The purpose of this study was to determine the effects of suramin, an antifibrotic agent, on cardiac function and remodeling in mdx mice. Methods: mdx mice (8 months old) received intraperitoneal injections of suramin twice a week for 3 months. Control mdx mice (8 months old) were injected with saline. Results: Suramin improved the electrocardiography profile with the main corrections seen in S‐ to R‐wave ratio, PR interval, and Q amplitude, and a significant decrease in the cardiomyopathy index. Suramin decreased myocardial fibrosis, inflammation, and myonecrosis. Conclusions: These findings suggest that suramin may be a new adjunctive therapy to help improve cardiomyopathy in DMD. Muscle Nerve 48 : 911–919, 2013     

Spatial analyzes of HLA data in Rio Grande do Sul,south Brazil: genetic structure and possible correlation with autoimmune diseases

Background

HLA genes are the most polymorphic of the human genome and have distinct allelic frequencies in populations of different geographical regions of the world, serving as genetic markers in ancestry studies. In addition, specific HLA alleles may be associated with various autoimmune and infectious diseases. The bone marrow donor registry in Brazil is the third largest in the world, and it counts with genetic typing of HLA-A, -B, and -DRB1. Since 1991 Brazil has maintained the DATASUS database, a system fed with epidemiological and health data from compulsory registration throughout the country.

Methods

In this work, we perform spatial analysis and georeferencing of HLA genetic data from more than 86,000 bone marrow donors from Rio Grande do Sul (RS) and data of hospitalization for rheumatoid arthritis, multiple sclerosis and Crohn’s disease in RS, comprising the period from 1995 to 2016 obtained through the DATASUS system. The allele frequencies were georeferenced using Empirical Bayesian Kriging; the diseases prevalence were georeferenced using Inverse Distance Weighted and cluster analysis for both allele and disease were performed using Getis-Ord Gi* method. Spearman’s test was used to test the correlation between each allele and disease.

Results

The results indicate a HLA genetic structure compatible with the history of RS colonization, where it is possible to observe differentiation between regions that underwent different colonization processes. Spatial analyzes of autoimmune disease hospitalization data were performed revealing clusters for different regions of the state for each disease analyzed. The correlation test between allelic frequency and the occurrence of autoimmune diseases indicated a significant correlation between the HLA-B*08 allele and rheumatoid arthritis.

Conclusions

Genetic mapping of populations and the spatial analyzes such as those performed in this work have great economic relevance and can be very useful in the formulation of public health campaigns and policies, contributing to the planning and adjustment of clinical actions, as well as informing and educating professionals and the population.

     

Fluoride intake from regular and low fluoride dentifrices by 2-3-year-old children: influence of the dentifrice flavor

This study evaluated the fluoride intake from dentifrices with different fluoride concentrations ([F]) by children aged 24-36 months, as well as the influence of the dentifrice flavor in the amount of fluoride ingested during toothbrushing. Thirty-three children were randomly divided into 3 groups, according to the [F] in the dentifrices: G-A (523 microgF/g), G-B (1,062 microgF/g) and G-C (1,373 microgF/g). Dentifrices A and B are marketed for children, while dentifrice C is a regular product. The amount of F ingested was indirectly obtained, subtracting the amount expelled and the amount left on the toothbrush from the amount initially loaded onto the brush. The results were analyzed by ANOVA, Tukey's test and linear regression analysis (p < 0.05). Children ingested around 60% of the dentifrice loaded onto the brush, but no significant differences were seen among the groups (p > 0.05). Mean daily fluoride intake from dentifrice for G-A, G-B and G-C was 0.022(a) feminine, 0.032(a) feminine and 0.061(b) mg F/kg body weight, respectively (p < 0.01). There was a strong positive correlation (r = 0.86, p < 0.0001) between the amount of dentifrice used and the amount of fluoride ingested during toothbrushing. The results indicate the need for instructing children's parents and care givers to use a small amount of dentifrice (< 0.3 g) to avoid excessive ingestion of fluoride. The use of low-[F] dentifrices by children younger than 6 years also seems to be a good alternative to minimize fluoride intake. Dentifrice flavor did not influence the percentage of fluoride intake.     

Composite depth of cure obtained with QTH and LED units assessed by microhardness and micro-Raman spectroscopy

This study analyzed the depth of cure of a composite assessed by microhardness and the degree of conversion as a function of the light cure unit (LCU) used. Two light cure units, one LED (Ultraled-Dabi Atlante) and one quartz-tungsten-halogen (QTH, Optilux 401-Demetron) unit were used to cure 4.0 x 4.0 mm and 5.0 mm deep composite specimens (Filtek Z250, 3M ESPE). After 24 hours storage at 37 degrees C, Knoop microhardness and degree of conversion were measured on the irradiated surface and at each millimeter of the sample's depth. The degree of conversion was determined by using micro-Raman spectroscopy. The specimens cured with the QTH unit presented uniform decay in microhardness up to 4 mm in depth. Beyond 4 mm, the drop was abrupt. With LED photoactivation, uniform decay was observed only up to 2 mm. At higher depths, the decay in microhardness increased rapidly, especially beyond 3 mm. Depth of cure assessed by micro-Raman revealed that the degree of conversion behaved similarly to microhardness for both LCUs. A strong linear regression between microhardness and the degree of conversion, including both LCUs, was established with R2 = 0.980.     

A comparison of histamine effects on the sympathetic neurotransmission of testicular capsule and rat vas deferens

Histamine is an important modulatory agent of the sympathetic neurotransmission, but its exact action on the testicular capsule or rat vas deferens is not fully understood. The present study sought to further investigate the functional effects of histamine on the neuronal and exogenous noradrenaline-induced contraction of the testicular capsule and rat vas deferens as well as to evaluate the contractile properties of this drug. The testicular capsule or vas deferens from Wistar rats, 3–4 months old, weighing 300–400 g, was isolated and mounted in organ baths for functional experiments. The results indicated that the neuronally evoked contraction of the testicular capsule was affected by histamine (10^?10 to 10^?8 M) with participation of inhibitory (H₃ receptors) and excitatory (H₁ receptors) receptors. Histamine (10^?7 to 10^?4 M) modulated the field-stimulated vas deferens by excitatory (H₂ receptors) and inhibitory (H₁ receptors) receptors. Histamine was able to decrease the tonic response for noradrenaline-induced contractions with participation of H₁ receptors (testicular capsule) and H₃ receptors (vas deferens) followed by nitric oxide generation. At high concentration, histamine exerts contractile effects in both tissues. In the testicular capsule, the histamine-induced contractions were related to H₁ receptor activation followed by release of prostaglandins. In contrast, the contractile effects of histamine in the vas deferens were related to H₂ receptor activation followed by release of catecholamines from sympathetic nerve endings. Therefore, our results indicate that histamine induced several effects on the sympathetic neurotransmission of rat testicular capsule and vas deferens. These effects are dependent on the concentration used and with participation of multiple histamine receptors.     

Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4⁺ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4⁺ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.     

Association between Genetic Variants in NOS2 and TNF Genes with Congenital Zika Syndrome and Severe Microcephaly

Zika virus (ZIKV) causes Congenital Zika Syndrome (CZS) in individuals exposed prenatally. Here, we investigated polymorphisms in VEGFA, PTGS2, NOS3, TNF, and NOS2 genes as risk factors to CZS. Forty children with CZS and forty-eight children who were in utero exposed to ZIKV infection, but born without congenital anomalies, were evaluated. Children with CZS were predominantly infected by ZIKV in the first trimester (p < 0.001) and had mothers with lower educational level (p < 0.001) and family income (p < 0.001). We found higher risk of CZS due the allele rs2297518[A] of NOS2 (OR = 2.28, CI 95% 1.17–4.50, p = 0.015). T allele and TT/CT genotypes of the TNF rs1799724 and haplotypes associated with higher expression of TNF were more prevalent in children with CZS and severe microcephaly (p = 0.029, p = 0.041 and p = 0.030, respectively). Our findings showed higher risk of CZS due ZIKV infection in the first trimester and suggested that polymorphisms in NOS2 and TNF genes affect the risk of CZS and severe microcephaly.     

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H₂O₂) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins—including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein—was blocked by preincubation with PEG-catalase, which degrades H₂O₂. ADP treatment promoted the H₂O₂-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H₂O₂. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl–interacting protein CrkII are mediated by H₂O₂. We transfected endothelial cells with differentially targeted HyPer2 H₂O₂ biosensors and found that ADP promoted a marked increase in H₂O₂ levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H₂O₂ in control of ADP-mediated signaling responses in the vascular wall.Beyond their established roles in intracellular energy flux and nucleic acid metabolism, purine nucleotides also serve as intercellular messenger molecules that regulate signal transduction pathways in a broad range of cells and tissues (1–3). The purine nucleotide ADP binds to G protein-coupled P2Y purinergic cell surface receptors, which are expressed in diverse mammalian cells, including blood platelets and vascular endothelial cells (reviewed in refs. 2 and 4). ADP is a critical determinant of platelet aggregation, blood vessel tone, and vascular wall integrity. Platelet granules contain high concentrations of ADP, which is released during platelet aggregation. The released ADP binds to P2Y12 and P2Y1 cell surface receptors for ADP on platelets and further potentiates platelet aggregation. P2Y receptor antagonists play a central role in cardiovascular therapeutics (2, 4): The P2Y12 blocker clopidogrel is one of the most commonly prescribed drugs in the United States, and other P2Y1 and P2Y12 blockers are being actively developed and tested for treatment of cardiovascular and cerebrovascular disease states. ADP also binds to P2Y1 receptors in vascular endothelial cells and rapidly activates endothelial nitric oxide synthase (eNOS) (5). Endothelium-generated nitric oxide (NO) inhibits platelet aggregation (3, 6) and provides an important feedback loop between endothelial cells and platelets that serves to attenuate the direct proaggregatory effects of ADP on platelets. ADP may also be released from the vascular endothelium and act in an autocrine or paracrine fashion to exert longer-term effects on vascular cell migration and barrier function (1, 3, 7–9). Clearly, a deeper understanding of P2Y receptor pharmacodynamics could inform current efforts in the development of novel purinergic antagonist drugs.Purinergic receptors for ADP can be classified by their structure and mode of action into two distinct receptor families, P2X and P2Y. P2X receptors are ligand-gated ion channels, whereas members of the P2Y receptor family are G protein-coupled receptors. ADP signaling pathways in platelets have been extensively characterized, yet the roles of ADP in the modulation of endothelial responses are less well understood. The current studies have focused on exploring the signaling pathways activated by P2Y1 receptors in vascular endothelial cells. We have shown (6) that ADP acts via P2Y1 receptors to activate the endothelial isoform of nitric oxide synthase (eNOS) in cultured endothelial cells and also modulates the activation of key signaling protein kinases including the AMP-activated protein kinase (AMPK). We also found that ADP promotes the P2Y1 ADP receptor-dependent endothelial cell migration through activation of the small GTPase Rac1 (6, 10). Discovering the involvement of Rac1 provided an important clue to the mechanisms whereby ADP exerts its influence on endothelial cell responses.Rac1 is an actin-binding cytoskeletal regulatory protein and is a member of the Rho GTPase protein family. The activation of eNOS by P2Y1 receptors for ADP depends on Rac1 (1, 6). Rac1 has been identified as a critical determinant of endothelial cell migration and barrier function, at least in part by modulating the levels of intracellular NO and hydrogen peroxide (H₂O₂) (11–15). H₂O₂ is a stable reactive oxygen species (ROS) that has been identified in recent years as a physiologically important intracellular messenger molecule (11–14), belying the classical concept of ROS functioning solely as deleterious molecules responsible for pathological states such as aging and neurodegeneration (14, 16). We reported (17, 18) that endogenous H₂O₂ regulates endothelial cell migration via dynamic signaling pathways involving the MARCKS protein, a ubiquitous phosphoprotein that translocates from the cell membrane to the actin cytoskeleton. The MARCKS protein also reversibly sequesters the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate (PIP₂). PIP₂ is an important activator of proteins that initiate actin nucleation, including the phosphoprotein c-Abl, a nonreceptor tyrosine kinase that has been implicated in the dynamic cytoskeletal rearrangements that modulate endothelial barrier function. Endogenous H₂O₂ induces changes in cellular phospholipid metabolism via the phosphorylation and translocation of MARCKS in endothelial cells, yet the connections between receptor activation and intracellular modulation of H₂O₂ levels are incompletely understood.The roles of H₂O₂ as a physiological intracellular messenger molecule were initially discovered through studies of growth factor-dependent activation of their cognate receptor tyrosine kinases (19, 20), which then signal to redox-regulated phosphoprotein phosphatases via H₂O₂ (20). In contrast to the widespread involvement of H₂O₂ in receptor tyrosine kinase signaling, only a handful of G protein-coupled receptors have been shown to directly modulate H₂O₂ levels (11, 16, 21, 22). Indeed, the roles of H₂O₂ in modulation of physiological responses have not yet been clearly defined for G protein-coupled receptors. In these studies, we present observations that establish that the G protein-coupled P2Y1 receptor for ADP modulates key H₂O₂-dependent signaling responses in the vascular endothelium via transactivation of the receptor tyrosine kinase Flt3.