Acute Administration of n-3 Rich Triglyceride Emulsions Provides Cardioprotection in Murine Models after Ischemia-Reperfusion

Dietary n-3 fatty acids (FAs) may reduce cardiovascular disease risk. We questioned whether acute administration of n-3 rich triglyceride (TG) emulsions could preserve cardiac function and decrease injury after ischemia/reperfusion (I/R) insult. We used two different experimental models: in vivo, C57BL/6 mice were exposed to acute occlusion of the left anterior descending coronary artery (LAD), and ex-vivo, C57BL/6 murine hearts were perfused using Langendorff technique (LT). In the LAD model, mice treated with n-3 TG emulsion (1.5g/kg body weight), immediately after ischemia and 1h later during reperfusion, significantly reduced infarct size and maintained cardiac function (p<0.05). In the LT model, administration of n-3 TG emulsion (300mgTG/100ml) during reperfusion significantly improved functional recovery (p<0.05). In both models, lactate dehydrogenase (LDH) levels, as a marker of injury, were significantly reduced by n-3 TG emulsion. To investigate the mechanisms by which n-3 FAs protects hearts from I/R injury, we investigated changes in key pathways linked to cardioprotection. In the ex-vivo model, we showed that n-3 FAs increased phosphorylation of AKT and GSK3β proteins (p<0.05). Acute n-3 TG emulsion treatment also increased Bcl-2 protein level and reduced an autophagy marker, Beclin-1 (p<0.05). Additionally, cardioprotection by n-3 TG emulsion was linked to changes in PPARγ protein expression (p<0.05). Rosiglitazone and p-AKT inhibitor counteracted the positive effect of n-3 TG; GSK3β inhibitor plus n-3 TG significantly inhibited LDH release. We conclude that acute n-3 TG injection during reperfusion provides cardioprotection. This may prove to be a novel acute adjunctive reperfusion therapy after treating patients with myocardial infarction.     

Hormonal regulatory role of eyestalk factors on growth of heart in mud crab,Scylla serrata

The present study was attempted to know the growth regulation of eyestalk factors on the growth of heart in Scylla serrata using eyestalk extractions and bilateral eyestalk ablations. The bilateral eyestalk ablation led to the maximum growth indices of the heart ((H) indices) to 0.162 and 0.158 in ablated male and female, respectively, in comparison to 0.153 and 0.167 in the control male and female and 0.147 and 0.157 in injected male and female, respectively. The data have shown that the heart of male crabs grows faster than female crabs. The study has also shown that bilateral eyestalk ablation resulted in a significant increase in the heart indices in males and has least effect on the growth of the female heart. The results presented strongly support a potential role of the eyestalk factors and molting hormone regulating the growth of the heart in S. serrata.     

Phosphatidylserine receptors: what is the new RAGE?

The identification of RAGE as a phophatidylserine receptor—in this issue of EMBO reports by He et al—adds to the range of molecules that can sense this ‘eat-me'' signal, and suggests new potential therapeutic opportunities.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.28The recognition of apoptotic cells by phagocytes is a complex, yet highly orchestrated event. Many receptors have been identified that recognize phosphatidylserine (PS; Fig 1)—which is exposed on early apoptotic cells—leading to downstream signalling and apoptotic cell engulfment. In a paper published this month in EMBO reports, the receptor for advanced glycation end-products (RAGE) is described as a new PS receptor on alveolar macrophages that participates in the clearance of apoptotic cells (He et al, 2011).…[RAGE] is described as a new phosphatidylserine receptor on alveolar macrophages that participates in the clearance of apoptotic cellsOpen in a separate windowFigure 1Phosphatidylserine-dependent apoptotic cell recognition.Schematic of the known PS receptors and downstream signalling to Rac. Dashed lines indicate unknown signalling mechanisms. PS, phosphatidylserine; RAGE, receptor for advanced glycation end-products; sRAGE, soluble RAGE.More than 200 billion cells undergo apoptosis every day in a human body, yet few apoptotic cells are detected in healthy tissue (Ravichandran, 2010). Apoptotic cells are generated during development, as part of normal homeostatic turnover and in disease states. The efficient clearance of apoptotic cells is crucial to prevent them from becoming secondarily necrotic, thereby limiting the immune response to apoptotic cell-derived self-antigens (Green et al, 2009). Disruptions to the clearance of apoptotic cells are linked to several diseases including atherosclerosis, chronic inflammation and autoimmunity (Elliott & Ravichandran, 2010).More than 200 billion cells undergo apoptosis every day in a human body, yet few apoptotic cells are detected in healthy tissueApoptotic cell engulfment can be divided into several steps. The first is the release of ‘find-me'' signals—such as triphosphate nucleotides (ATP and UTP), sphingosine-1-phosphate (S1P), lysophosphatidylcholine (LPC) and the chemokine CX₃CL1—by apoptotic cells (Ravichandran, 2010). Then, phagocytes sense the find-me signals and migrate toward the apoptotic cell. When they are in close proximity, recognition is mediated by the interaction between engulfment receptors on phagocytes and ligands, known as ‘eat-me'' signals, that are expressed on the dying cells (Ravichandran, 2010). The best-studied eat-me signal is PS, which is flipped from the inner leaflet to the outer leaflet of the plasma membrane during early apoptosis. Many receptors have been linked to the recognition of the exposed PS on apoptotic cells, and they are discussed below. The recognition of an apoptotic cell results in a downstream signalling cascade that leads to cytoskeletal rearrangement of the phagocytic membrane and subsequent engulfment of the apoptotic cell. Once the corpse is internalized, the phagocyte must process and digest the cellular contents.The exposure of PS on the outer leaflet of the membrane is the most-characteristic marker of an apoptotic cell. Phagocytes can recognize PS directly through receptors such as Bai1, TIM-4 and stabilin 2, or through soluble bridging molecules that bind to both PS and specific phagocyte receptors. For example, bridging molecules MFG-E8 and Gas6 interact with α_Vβ_3/5 and MER on the phagocytic membrane, respectively. Other eat-me signals and the molecules that bind to them have been characterized: thrombospondin is recognized by the vitronectin receptor, calreticulin by LRP1, oxidized LDL by scavenger receptors, ICAM3 might bind to CD14 and altered sugars bind to lectins (Lauber et al, 2004). Not all receptors need to be engaged for engulfment to occur, and different cell types have different receptor-expression levels.In a paper published this month in EMBO reports, the Yamamoto team identify RAGE as a new type of PS receptor on macrophages (He et al, 2011). There are two functional forms of RAGE, an abundant full-length transmembrane form that can initiate signalling through its intracellular tail, and a soluble isoform (sRAGE) that acts as a decoy receptor. RAGE is characteristically regarded as a pro-inflammatory receptor and has a variety of ligands, including advanced glycation end-products (AGEs) and many other damage-associated molecular patterns (DAMPs; Sims et al, 2010). One ligand in particular—high-mobility group protein B1 (HMGB1)—is released by cells undergoing necrosis and has been shown to bind to RAGE and induce inflammation (Sims et al, 2010). Therefore, RAGE might function during pro-inflammatory conditions and—as proposed by He and colleagues—during the anti-inflammatory process of apoptotic cell clearance. RAGE is mainly expressed in the lungs, but levels of it quickly increase at sites of inflammation, mostly on inflammatory and epithelial cells. Given the multitude of RAGE ligands and its inducible expression levels, RAGE is implicated in a variety of inflammation-related pathological states such as neurological and pulmonary disorders, vascular disease, cancer and diabetes (Sims et al, 2010).He and colleagues suggest that RAGE is a PS receptor during apoptotic cell engulfment in alveolar macrophages (He et al, 2011). Furthermore, sRAGE—which can bind to PS and apoptotic thymocytes—acts as a decoy and inhibits RAGE recognition of PS. By using PS liposomes as an artificial apoptotic target, the authors find RAGE in areas of the membrane in which a pseudopod forms to engulf a PS liposome. Additionally, sRAGE can compete with transmembrane RAGE to block the recognition of PS by the phagocyte and subsequently decrease the engulfment of apoptotic cells. Under homeostatic conditions, alveolar macrophages isolated from RAGE-deficient mice have defects in phagocytosis of apoptotic thymocytes. In a model of lung injury induced by lipopolysaccharide administration, RAGE-deficient mice accumulate neutrophils in the alveolar space and RAGE-deficient macrophages have defects in neutrophil engulfment. Previous works have implicated RAGE expression and/or upregulation in inflammatory conditions. In fact, genetic deletion of RAGE in mice can result in attenuated atherosclerosis, resistance to septic shock and reduced diabetic kidney disease (Ramasamy et al, 2010). Apoptotic cell clearance is generally an immunologically silent process and, therefore, if RAGE significantly contributes to engulfment, RAGE-deficient mice would be expected to have defects in cell clearance, leading to enhanced inflammation and disease. However, this does not seem to be the case. Thus, future studies should examine cell-type specific deletions of RAGE to clarify its apparently contradictory role in cell clearance and inflammation in these diseases.Given that several modes of PS recognition have been identified (Ravichandran, 2010), there must be some redundancy. The way in which RAGE contributes to this scenario remains to be investigated. Analysis of the expression levels of each PS receptor on different cell types will also help to define their relative importance in individual cells. As RAGE is highly expressed in the lung, it would be interesting to analyse its contribution to apoptotic cell engulfment in this tissue, in comparison with the other PS receptors. Furthermore, RAGE is induced by inflammation, suggesting that it is probably important during disease states to facilitate engulfment and reduce inflammation in the microenvironment.Another interesting question that remains is how RAGE signals to the phagocyte for engulfment. RAGE signalling results in pro-inflammatory cytokine production through activation of NF-κB (Yan et al, 1994), which seems to be different from the production of anti-inflammatory cytokines—such as IL-10 and TGFβ—by phagocytes during cell engulfment. However, as several RAGE ligands exist, the way in which they bind to RAGE could result in differential signalling. RAGE has also been shown to interact with mouse Dia1, leading to downstream activation of Rac1 and Cdc42, and cell migration (Hudson et al, 2008). Now, He and colleagues suggest that RAGE signals to Rac1 through Dia1 in the context of apoptotic cell clearance, as RAGE-deficient macrophages have decreased Rac1 activity in response to PS-liposome engulfment. Two evolutionarily conserved Rac-dependent pathways have been identified to mediate corpse internalization. Engagement of some engulfment receptors such as Bai1, results in Rac activation through the ELMO–Dock180–CrkII complex. ELMO and Dock180 mediate the exchange of GDP to GTP on Rac, whereas CrkII has been proposed to function as an adaptor protein. Another pathway involves signalling from the engulfment receptor LRP1 or stabilin 2, leading to Rac activation through the engulfment adaptor protein (GULP). Additional work is necessary to determine whether RAGE–mDia1 signalling constitutes a third intracellular signalling pathway for cell engulfment.Another interesting question that remains is how RAGE signals to the phagocyte for engulfmentThe study from the Yamamoto team identifies RAGE as a new PS-recognition molecule implicated in apoptotic cell-clearance in the lung. As each new receptor is identified, we are reminded of the redundancy and cell-type-specific expression of PS receptors. Defects in apoptotic cell-clearance lead to a variety of inflammatory diseases, including cardiovascular and autoimmune diseases. This study could also open an interesting therapeutic avenue; if sRAGE blocks the recognition of PS by RAGE and other PS receptors, it might be beneficial as a therapy by enhancing cell clearance and decreasing the severity of cell-clearance-associated diseases.     

Differential roles for the interferon-inducible IFI16 and AIM2 innate immune sensors for cytosolic DNA in cellular senescence of human fibroblasts

The IFN-inducible IFI16 and AIM2 proteins act as innate immune sensors for cytosolic double-stranded DNA (dsDNA). On sensing dsDNA, the IFI16 protein induces the expression of IFN-β whereas the AIM2 protein forms an inflammasome, which promotes the secretion of IL-1β. Given that the knockdown of IFI16 expression in human diploid fibroblasts (HDF) delays the onset of cellular senescence, we investigated the potential roles for the IFI16 and AIM2 proteins in cellular senescence. We found that increased IFI16 protein levels in old (vs. young) HDFs were associated with the induction of IFN-β. In contrast, increased levels of the AIM2 protein in the senescent (vs. old) HDFs were associated with increased production of IL-1β. The knockdown of type I IFN-α receptor subunit, which reduced the basal levels of the IFI16 but not of the AIM2, protein delayed the onset of cellular senescence. Accordingly, increased constitutive levels of IFI16 and AIM2 proteins in ataxia telangiectasia mutated (ATM) HDFs were associated with the activation of the IFN signaling and increased levels of IL-1β. The IFN-β treatment of the young HDFs, which induced the expression of IFI16 and AIM2 proteins, activated a DNA damage response and also increased basal levels of IL-1β. Interestingly, the knockdown of AIM2 expression in HDFs increased the basal levels of IFI16 protein and activated the IFN signaling. In contrast, the knockdown of the IFI16 expression in HDFs decreased the basal and dsDNA-induced activation of the IFN signaling. Collectively, our observations show differential roles for the IFI16 and AIM2 proteins in cellular senescence and associated secretory phenotype.     

A 32 kb critical region excluding Y402H in CFH mediates risk for age-related macular degeneration

Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10⁻¹⁰⁹); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10⁻⁹) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.     

Immune Biomarker Differences and Changes Comparing HCV Mono-Infected,HIV/HCV Co-Infected,and HCV Spontaneously Cleared Patients

Background

Immune biomarkers are implicated in HCV treatment response, fibrosis, and accelerated pathogenesis of comorbidities, though only D-dimer and C-reactive protein have been consistently studied. Few studies have evaluated HIV/HCV co-infection, and little longitudinal data exists describing a broader antiviral cytokine response

Methods

Fifty immune biomarkers were analyzed at baseline(BL) and HCV end of treatment follow-up(FU) time point using the Luminex 50-plex assay in plasma samples from 15 HCV-cleared, 24 HCV mono- and 49 HIV/HCV co-infected patients receiving antiretroviral treatment, who either did or did not receive pegylated-interferon/ribavirin HCV treatment. Biomarker levels were compared among spontaneous clearance patients, mono- and co-infected, untreated and HCV-treated, and sustained virologic responders (SVR) and non-responders (NR) at BL and FU using nonparametric analyses. A Bonferroni correction, adjusting for tests of 50 biomarkers, was used to reduce Type I error

Results

Compared to HCV patients at BL, HIV/HCV patients had 22 significantly higher and 4 significantly lower biomarker levels, following correction for multiple testing. There were no significantly different BL levels when comparing SVR and NR in mono- or co-infected patients; however, FU levels changed considerably in co-infected patients, with seven becoming significantly higher and eight becoming significantly lower in SVR patients. Longitudinally between BL and FU, 13 markers significantly changed in co-infected SVR patients, while none significantly changed in co-infected NR patients. There were also no significant changes in longitudinal analyses of mono-infected patients achieving SVR or mono-infected and co-infected groups deferring treatment

Conclusions

Clear differences exist in pattern and quantity of plasma immune biomarkers among HCV mono-infected, HIV/HCV co-infected, and HCV-cleared patients; and with SVR in co-infected patients treated for HCV. Though >90% of patients were male and co-infected had a larger percentage of African American patients, our findings may have implications for better understanding HCV pathogenesis, treatment outcomes, and future therapeutic targets     

Novel limonene and citral based 2,5-disubstituted-1,3,4-oxadiazoles: A natural product coupled approach to semicarbazones for antiepileptic activity

Two novel series of N⁴-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N¹-(2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enylidene)semicarbazide and N⁴-(5-(2/3/4-substituted-phenyl)-1,3,4-oxadiazol-2-yl)-N¹-(3,7-dimethylocta-3,6-dienylidene)-semicarbazide were synthesized to meet structural prerequisite indispensable for anticonvulsant activity. The anticonvulsant activities of the compounds were investigated using maximal electroshock seizure (MES), subcutaneous pentylenetrtrazole (scPTZ) and subcutaneous strychnine (scSTY) models. The rotorod test was conducted to evaluate neurotoxicity. Some of the selected active compounds were subjected to GABA assay to confirm their mode of action. The outcome of the present investigations proved that the four binding sites pharmacophore model is vital for anticonvulsant activity. The efforts were also made to establish structure–activity relationships among test compounds.