Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Lymphocyte Activation In Silica-exposed Workers.

Exposure to silica dust has been examined as a possible risk factor for autoimmune diseases, including systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus and ANCA-associated vasculitis. However, the underlying cellular and molecular mechanisms resulting in the increased prevalence of autoimmunity remain elusive. To clarify these mechanisms, we studied various markers of immune activation in individuals occupationally exposed to silica dust, i.e., serum levels of soluble IL-2 receptor (sIL-2R), levels of IL-2, other pro- and anti-inflammatory cytokines and lymphoproliferation. Our results demonstrate that silica-exposed individuals present important alterations in their immune response when compared to controls, as shown by increased serum sIL-2R levels, decreased production of IL-2 and increased levels of the pro-inflammatory (IFN-γ, IL-1α, TNF-α, IL-6) as well as anti-inflammatory (IL-10 and TGF-β) cytokines. Furthermore, silica-exposed individuals presented enhanced lymphoproliferative responses. Our findings provide evidence that the maintenance of immune homeostasis may be disturbed in silica-exposed individuals, possibly resulting in autoimmune disorders.

Analysis Of The Contribution Of Immunologically-detectable Her2, Steroid Receptors And Of The Triple-negative" Tumor Status To Disease-free And Overall Survival Of Women With Epithelial Ovarian Cancer."

We assessed associations between steroid receptors including: estrogen-alpha, estrogen-beta, androgen receptor, progesterone receptor, the HER2 status and triple-negative epithelial ovarian cancer (ERα-/PR-/HER2-; TNEOC) status and survival in women with epithelial ovarian cancer. The study included 152 women with primary epithelial ovarian cancer. The status of steroid receptor and HER2 was determined by immunohistochemistry. Disease-free and overall survival were calculated and compared with steroid receptor and HER2 status as well as clinicopathological features using the Cox Proportional Hazards model. A mean follow-up period of 43.6 months (interquartile range=41.4 months) was achieved where 44% of patients had serous tumor, followed by mucinous (23%), endometrioid (9%), mixed (9%), undifferentiated (8.5%) and clear cell tumors (5.3%). ER-alpha staining was associated with grade II-III tumors. Progesterone receptor staining was positively associated with a Body Mass Index≥25. Androgen receptor positivity was higher in serous tumors. In stand-alone analysis of receptor contribution to survival, estrogen-alpha positivity was associated with greater disease-free survival. However, there was no significant association between steroid receptor expression, HER2 status, or TNEOC status, and overall survival. Although estrogen-alpha, androgen receptor, progesterone receptor and the HER2 status were associated with key clinical features of the women and pathological characteristics of the tumors, these associations were not implicated in survival. Interestingly, women with TNEOC seem to fare the same way as their counterparts with non-TNEOC.

Indole-3-carbinol Attenuates The Deleterious Gestational Effects Of Bisphenol A Exposure On The Prostate Gland Of Male F1 Rats.

Bisphenol A (BPA) is a chemical that has been investigated for it potential to cause prostate diseases. In this study, pregnant Sprague-Dawley rats were treated with 25 or 250 μg/kg BPA from gestational day (GD) 10 to GD21 with or without concurrent indole-3-carbinol (I3C) feeding. I3C is a phytochemical, and it affords chemoprotection against many types of neoplasia. Male F1 rats from different litters were euthanized on post-natal day (PND) 21 and PND180. BPA-treated groups showed a significant increase in histopathological lesions, but I3C feeding reversed many of these changes, mainly at PND180. Maternal I3C feeding increased prostate epithelial apoptosis in the BPA-treated groups and across age groups. Furthermore, I3C induced partial normalization of the prostate histoarchitecture. The results pointed to a protective effect of maternal I3C feeding during pregnancy in the BPA-exposed male offspring, thereby indicating reduction in the harmful effects of gestational BPA imprinting on the prostate.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Monitoramento de parâmetros imunológicos em atletas adolescentes de basquetebol durante e após a temporada esportiva= : Monitoring of immunological parameters in adolescent basketball athletes during and after a sports season

Universidade Estadual de Campinas . Faculdade de Educação Física

Macrophage Cell Activation With Acute Apical Abscess Contents Determined By Interleukin-1 Beta And Tumor Necrosis Factor Alpha Production.

This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Frequency Of The Allelic Variant C.1150t > C In Exon 10 Of The Fibroblast Growth Factor Receptor 3 (fgfr3) Gene Is Not Increased In Patients With Pathogenic Mutations And Related Chondrodysplasia Phenotypes.

Mutations in the FGFR3 gene cause the phenotypic spectrum of FGFR3 chondrodysplasias ranging from lethal forms to the milder phenotype seen in hypochondroplasia (Hch). The p.N540K mutation in the FGFR3 gene occurs in ∼70% of individuals with Hch, and nearly 30% of individuals with the Hch phenotype have no mutations in the FGFR3, which suggests genetic heterogeneity. The identification of a severe case of Hch associated with the typical mutation c.1620C > A and the occurrence of a c.1150T > C change that resulted in a p.F384L in exon 10, together with the suspicion that this second change could be a modulator of the phenotype, prompted us to investigate this hypothesis in a cohort of patients. An analysis of 48 patients with FGFR3 chondrodysplasia phenotypes and 330 healthy (control) individuals revealed no significant difference in the frequency of the C allele at the c.1150 position (p = 0.34). One patient carrying the combination `pathogenic mutation plus the allelic variant c.1150T > C' had a typical achondroplasia (Ach) phenotype. In addition, three other patients with atypical phenotypes showed no association with the allelic variant. Together, these results do not support the hypothesis of a modulatory role for the c.1150T > C change in the FGFR3 gene.