APOE gene is one of currently only two genes that have consistently been associated with longevity. Apolipoprotein E (APOE) is a plasma protein which plays an important role in lipid and lipoprotein metabolism. In humans, there are three major APOE isoforms, designated APOE2, APOE3, and APOE4. Of these three isoforms, APOE3 is most common while APOE4 was shown to be associated with age-related diseases, including cardiovascular and Alzheimer’s disease, and therefore an increased mortality risk with advanced age. Evidence accumulates, showing that oxidative stress and, correspondingly, mitochondrial function is affected in an APOE isoform-dependent manner. Accordingly, several stress response pathways implicated in the aging process, including the endoplasmic reticulum stress response and immune function, appear to be influenced by the APOE genotype. The investigation and development of treatment strategies targeting APOE4 have not resolved any therapeutic yet that could be entirely recommended. This mini-review provides an overview on the state of research concerning the impact of the APOE genotype on stress response-related processes, emphasizing the strong interconnection between mitochondrial function, endoplasmic reticulum stress and the immune response. Furthermore, this review addresses potential treatment strategies and associated pitfalls as well as lifestyle interventions that could benefit people with an at risk APOE4 genotype.The  FOXO3) and apolipoprotein E (APOE) to be consistently associated with human longevity [loci, APOE was the first to be identified [APOE has since then been extensively studied in the context of aging [Aging is characterized by both a proceeding decline in biological functions and a decreased stress resistance . This raongevity –14. Of tentified . Not surof aging .In the 1970s, APOE was discovered as a constituent of lipoproteins and potent modulator of plasma lipoprotein and cholesterol concentrations. Up to 75 % of the plasma APOE is synthesized by liver parenchymal cells ; howeverApoe knockout (KO) mice and in vitro studies revealed functions of APOE which can not entirely be attributed to its role in lipid metabolism and strongly suggest APOE to be involved in the aging process. Apoe KO mice lack endogenous Apoe expression due to gene targeting-induced inactivation of the murine Apoe gene [Apoe KO mice show an altered lipoprotein profile compared with wild type mice and spontaneously develop atherosclerosis [Apoe KO mice suffer from neurological disorders [Apoe KO mice showed elevated markers of oxidative stress [APOE gene affects multiple phenotypic traits simultaneously depending on the site of APOE protein synthesis. The multiple functions of the APOE protein make it a potent modulator of cellular stress response processes and the aging process.Over the course of time, studies on poe gene . These mpoe gene  and alsopoe gene , 25. Apoclerosis , which mclerosis . Moreoveisorders ; they deisorders , 29 and isorders . Furthere stress  and in ve stress . Althouge stress . Oxidatie stress . By its APOE gene is located on the long arm of chromosome 19, in close proximity to the genes of apolipoprotein C-I and C-II. The low density lipoprotein receptor (LDLR) is located on the short arm of the same chromosome, suggesting chromosome 19 to play an important role in lipoprotein metabolism [APOE gene is composed of four exons, separated by three introns [APOE gene product comprises 317 amino acids with an N-terminal 18 amino acids long signaling peptide being removed co-translationally. Prior to secretion, the APOE protein undergoes O-linked glycosylation and sialylation in the Golgi apparatus; but most plasma APOE is post-secretory de-sialylated [APOE gene also inherits the two non-synonymous single nucleotide polymorphisms, which gave rise to the three major APOE alleles ε2, ε3, and ε4 . These alleles encode six major APOE genotypes (three homozygous and three heterozygous). Basis of the APOE protein isoforms are single amino acid interchanges at positions 112 and 158 . Woectively .The amino acid interchanges influence both the structure and the function of the resulting isoproteins. Lipid-free APOE consists of two independently folded domains, which are separated by a hinge region. The N-terminal LDL-receptor binding domain is arranged in a four-helix bundle. The major C-terminal domain contains amphipathic α-helices, which mediate the high-affinity binding of APOE to lipoproteins , 41. APOAPOE gene. Interestingly, APOE isoforms confer different disease susceptibilities. APOE2 is associated with increased plasma levels of cholesterol and triglycerides, which makes it a risk factor for type III hyperlipoproteinemia [APOE ε4 carriers [ε4 allele increases the risk and decreases the age of onset of late-onset Alzheimer’s disease (AD) in a dose-dependent manner [APOE ε4 carriers decreases with age [APOE a “frailty gene” rather than referring to it as a determinant of longevity.Later, the NMR structure of full-length APOE3 revealed several interactions between the two domains to also occur in APOE3 , 46. Mizteinemia . APOE4 icarriers . More obt manner . Not unewith age . Therefowith age  suggest APOE genotype to the cellular stress response and to the aging process. Overall, APOE4 appears rather defective in its response to stressors as it will be discussed below.Increasing evidence links the Apoe KO mice had revealed antioxidative properties of APOE [APOE genotype and oxidative stress  mice; [APOE genotype and coronary heart disease (CHD) risk, there is no clear evidence of smoking being in fact a modifier of the APOE genotype/CHD risk-association [APOE genotype on antioxidant enzyme activities in APOE TR mice and brains of AD patients yielded conflicting results [Shortly after studies on  of APOE , in vitr of APOE . Later,  of APOE . Since tiewed in ). Consisiewed in , 56 and iewed in . Accordiiewed in . Compareviduals; ), other R) mice; ). It appR) mice; , like toR) mice; , 62 or eR) mice; . Howeverociation . Studies results , 58, 65. results , and lik results . Interes results , and low results . However results .APOE genotype-dependent regulation of antioxidant defense mechanisms, increasing evidence suggests the APOE genotype to influence mitochondrial and endoplasmic reticulum (ER) stress-related processes in an APOE isoform-specific manner. Mitochondrial dysfunction and disturbances in the ubiquitin proteasome system which both may evoke by oxidative stress are considered hallmarks of the aging process. Both systems have been implicated in the pathogenesis of various age-related diseases, particularly neurodegenerative disorders, such as AD and Parkinson’s disease [2+ exchange via the calcium transfer channels voltage-dependent anion channel 1 (VDAC1) and inositol 1,4,5-triphosphate receptor Ca2+ channel (IP3R), located on both the mitochondrial and ER site of the MAM [vice versa. The two organelles both significantly contribute to the endogenous production of reactive oxygen species (ROS) [Besides the differential association of the APOE isoforms with oxidative stress and the potential  disease . The ER  disease . Structu the MAM , 72. Thees (ROS) .The ER mediates the synthesis, posttranslational modification and folding of almost all secretory and membrane proteins and is in these processes highly dependent on intra-compartmental calcium levels, an oxidative environment and the availability of specialized proteins, so-called chaperones, which facilitate proper protein folding. Changes in ER calcium levels, redox state but also metabolic or inflammatory changes may either lead to compromised ER function or an increased demand for (folded) proteins. Exceedance of the ER folding capacity causes an accumulation of unfolded or misfolded proteins in the ER lumen, referred to as ER stress . In turnAPOE genotype with ER stress. Interestingly, there is evidence to suggest that ER stress is indeed an early feature of APOE4 pathogenicity. APOE4 compared with APOE3 TR mice showed enhanced phosphorylation of EIF2A in the brain as early as 4 months of age and already displayed mild cognitive impairment [APOE genotype with ER stress in tissues other than the brain is rather limited. Two studies on the influence of the APOE genotype on ER stress parameters in peritoneal macrophages report conflicting results. Eberlé et al. [APOE genotype-dependent differential expression of markers of ER stress  [It has been hypothesized that APOE4, due to its structural characteristics, might be recognized by the ER as misfolded, subsequently leading to the activation of the ER stress response . Furtherpairment . Moreovepairment  reportedpairment , which wpairment , 45, as pairment . Data oné et al.  found ERé et al. . By conté et al. . A reducé et al. . Human a., ATF4) .Table 1S2+ homeostasis, processes that depend on proper OXPHOS function [One way through which protein homeostasis may be impaired is oxidative damage which has been shown to be often tightly linked to mitochondrial dysfunction . The prifunction .2+ overload and/or oxidative stress initiate steps of the apoptotic cascade, including cytochrome c release by mitochondria. This is usually based on the coordinated action of the pro- and anti-apoptotic proteins of the B cell lymphoma 2 family members (BCL2), opening of the mitochondrial permeability transition pore and organelle fragmentation [In situations of increased ATP demands, mitochondrial mass is increased by mitochondrial biogenesis. The key players in this process are peroxisome proliferator-activated receptors (PPARs), PPAR co-activators  and nuclear respiratory factors (NRFs), which are induced by the energy sensors adenosine monophosphate (AMP)-activated protein kinase (AMPK) and sirtuin 1 (SIRT1). Architecture and function of mitochondria are tightly linked and depend on the coordinated actions of fission-  and fusion-  promoting proteins. Fission and fusion enable exchange of proteins and mitochondrial DNA between mitochondria and the production of proper sized organelles , 83. Mitentation , 85. Damentation .APOE ε4 with AD, much of the data concerning mitochondrial function in relation to the APOE genotype was obtained in models of AD. Mitochondrial dysfunction has been reported to occur early in the pathogenesis of AD [APOE ε4 carriers with [ε4 allele [APOE genotype-specific effects, substantiating detrimental effects of APOE4 to be rather neuron-specific [post mortem human hippocampus [APOE ε4 to improvements of mitochondrial function by dietary interventions, at least as far as curcumin is concerned [APOE ε4 to dietary factors may depend on the origin of the substances. Furthermore, differential modulation of mitochondrial functions seems to depend on the cell types investigated; an observation that may not be limited to the brain but may also be applicable to other organs and tissues. Table APOE genotype with mitochondrial function.Due to the strong association of is of AD  and alsois of AD . Positroers with –90 and wers with –95 diagners with . In the ers with . Cell cuers with –100. Inters with  and, cor4 allele , 102. Sp4 allele , 99. Con4 allele . The APO4 allele , 104. Mo4 allele . In the specific . Down-repocampus . James epocampus  performepocampus . More repocampus . Pathwaypocampus . In contpocampus  observedpocampus . Consistpocampus . Interesoncerned . In contoncerned . It shouAPOE genotype and mitochondrial function is further substantiated by linkage disequilibrium (LD) structure analysis of the APOE gene and its neighboring regions which revealed high LD levels between three SNPs in the TOMM40 gene and the APOE ε4 allele [An association between the 4 allele .ε4 allele [TREM2 (triggering receptor expressed on myeloid cells 2) was suppressed in presence of APOE4 [APOE ε4 may be deleterious to the organism with advanced age, it may confer greater protection from pathogens early in life  and bacteria  [As stated earlier, all three pathways of the UPR may trigger the inflammatory response by activating the pro-inflammatory transcription factor NF-kB . Further4 allele . The mai4 allele , 122. Veof APOE4 . However theory, ). Indeedo 40.7 % . Intereso 40.7 %  and stilo 40.7 %  were foug embryo , 127. Fug embryo  reporteds common –130. Mormalaria) –133, thomalaria) , 135  ).APOE gene is gaining more and more public attention. The role of APOE4 as a mortality factor in the elderly is undisputed. Because of the magnitude of impact of APOE4 on various age-related diseases and the relatively high frequency of the ε4 allele in the population, prevention strategies targeting APOE4 gain in importance, particularly in view of the ongoing “population aging” [Nowadays, several companies offer genetic testing to the public and provide their customers with information about ancestry, disease susceptibility genes, and about effects of dietary habits, drug use and other environmental factors on the individuals’ health. Websites offering information and support for people genotyped positive for certain gene variants arise quickly. The n aging” . SeveralAPOE ε4 carriers have been shown to display lower APOE levels in blood [ε2 allele, whose carriers seem more likely to reach centenarian age, is associated with the highest blood APOE levels among the APOE isoforms [ε4 allele.One target of interest in pharmacoprevention research is the discovery of drugs that raise APOE levels. in blood –141 and in blood . Interesisoforms . Few canisoforms . Probucoisoforms . Agonistisoforms , 149. Fuisoforms . Indeed,isoforms , 152. Thisoforms . Next toisoforms , 154. Itisoforms . The reaAnother pharmacotherapeutic approach, though still in its infancy, is the search for so-called small-molecule structure correctors, i.e., substances that induce correct APOE4 folding to restore proper protein function . As mentAPOE genotype yielded inconsistent results. However, APOE variation may influence the effects of fish oil supplementation on blood LDL levels. In a group of individuals with an atherogenic lipoprotein phenotype, APOE ε4 carriers showed a greater raise in LDL cholesterol levels after fish oil supplementation (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), 3 g/d) compared with carriers of the other APOE isoforms [APOE ε4 appeared rather insensitive to other dietary interventions. Quercetin supplementation, for example, exerted beneficial effects  reduction) only in presence of APOE ε3, but not APOE ε4 in humans and APOE TR mice [APOE ε4 carriers may have a slightly increased demand for dietary vitamin E, since vitamin E uptake into extrahepatic tissues was shown to be reduced and vitamin E degradation enhanced in APOE4 compared with APOE3 mice [APOE ε4 carriers were shown to exhibit higher intestinal absorption of dietary vitamin D and calcium [APOE ε4 carriers can also benefit from increasing their physical activity. It was shown that physical activity reduced the risk of CVD and protected cognitive functions especially in carriers of the ε4 allele [Pharmacoprevention may be the most effective strategy to ameliorate detrimental outcomes associated with APOE4; however, the discovery of new drugs is time-consuming and cost-intensive. Furthermore, drug discovery trials often come to nothing, when safety concerns are encountered. Therefore, alternative strategies become more and more important, including dietary and lifestyle interventions . Studiesisoforms , though isoforms . As afor TR mice , 161. Si TR mice . However TR mice , as statOE3 mice –164. On  calcium . APOE ε44 allele , 167. Ho4 allele .APOE ε4 into APOE ε3 were based on homologous recombination [APOE ε4 [New therapeutic possibilities may arise from the use of ‘genome editing’, i.e., the direct manipulation of the DNA sequence. Initial methods aiming at a conversion of bination . First ibination . Improvebination . Substanbination . It rema[APOE ε4 .APOE genotype on the modulation of stress response processes. However, the findings reviewed within this work support tissue-specific pathological patterns of APOE4. In the light of the pleiotropic nature of the APOE protein, therapeutic interventions targeting APOE4 should be chosen carefully not to abolish beneficial features of APOE4. As mentioned earlier, APOE4 may be beneficial early in life when infectious diseases are of greater importance to health than chronic disorders. This may especially apply to populations under high infectious pressure and a high prevalence of malnutrition. Furthermore, we previously reported associations of APOE ε4 with higher levels of both vitamin D [APOE ε4 being protective in age-related macular degeneration, a classical age-related disease [Taken together, the current review highlights the impact of the itamin D  and adipitamin D . Strikin disease . Neverth disease  opens upAD, Alzheimer’s disease; ADAS-Cog, Alzheimer’s Disease Assessment Scale-Cognitive subscale; AMP, adenosine monophosphate; AMPK, adenosine monophosphate-activated protein kinase; APOE, apolipoprotein E; Aß, amyloid beta; ATF4, activating transcription factor 4, ATF6, activating transcription factor 6; ATG7, autophagy related 7; ATP, adenosine triphosphate; BCL2, B cell lymphoma 2 family members; BIP, binding immunoglobulin protein; CGAS, candidate gene association study; CHD, coronary heart disease; CHOP, c/EBP-homologous protein 10; CI-V, complexes I-V of the mitochondrial respiratory chain; CM, chylomicron; CMR, chylomicron remnant; CRISPR/Cas9, Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 nuclease; CSF, cerebrospinal fluid; CVD, cardiovascular disease; DDIT3, DNA damage-inducible transcript 3; DNM1L or DRP1, dynamin 1-like protein 1; EGFP, enhanced green fluorescent protein; EIF2A, eukaryotic translation initiation factor 2A; ER, endoplasmic reticulum; ETC, electron transport chain; FFA, free fatty acids; FOXN3, forkhead box N3; FOXO3, forkhead box O3; FRET, fluorescence resonance energy transfer; Gabpa, GA-binding protein alpha chain; GADD34, protein phosphatase 1, regulatory subunit 15A; GCN, eukaryotic translation initiation factor 2 alpha kinase 4; GRP78, 78 kDa glucose-regulated protein; GRP94, heat shock protein 90, beta; GSH, glutathione; GSSG, glutathione disulfide; GWAS, genome-wide association study; HCV, hepatitis C virus; HDL, high density lipoprotein; HERP, homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1; HIV, human immunodeficiency virus; HL, hepatic lipase; HSPG, heparan sulfate proteoglycans; HSV, herpes simplex virus; IP3R, inositol 1,4,5-triphosphate receptor; IRE1α, serine/threonine-protein kinase/endoribonuclease; JNK, mitogen-activated protein kinase 8; KO, knockout; LD, linkage disequilibrium; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; LPL, lipoprotein lipase; LRP, low density lipoprotein receptor-related protein; LXR, liver X receptor; MAM, mitochondria-associated endoplasmic reticulum membranes; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; MFN1/2, mitofusin 1/2; mt-CO1 or CIV, mitochondrial cytochrome oxidase I; NRF, nuclear respiratory factors; OASIS, old astrocyte specifically induced substance; OPA1, optic atrophy 1 protein; OXPHOS, oxidative phosphorylation; PDI, protein disulfide isomerase associated 3; PERK, eukaryotic translation initiation factor 2-alpha kinase 3; PET, positron-emission tomography; PGE2, prostaglandin E2; PKR, eukaryotic translation initiation factor 2-alpha kinase 2; PON1, paraoxonase 1; PPAR, peroxisome proliferator-activated receptors; Ppargc1a or PGC-1α, peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; RCT, reverse cholesterol transport; ROS, reactive oxygen species; RXR, retinoid X receptor; SIRT1, sirtuin 1; TALEN, transcription activator-like effector nucleases; TCA, tricarboxylic acid; TNF-α, tumor necrosis factor alpha; TOMM40, translocase of the outer mitochondrial membrane; TR, targeted replacement; TRB3, tribbles homolog 3; TREM2, triggering receptor expressed on myeloid cells 2; UBA5, ubiquitin like modifier activating enzyme 5; UPR, unfolded protein response; VDAC1, voltage-dependent anion channel 1; VLDL, very low density lipoprotein; XBP1(S), X-box binding protein 1, spliced; XBP1, X-box binding protein 1; ZFN, zinc-finger nucleases


Firmicutes and unclassified bacteria dominated across all sites, Proteobacteria, especially Gammaproteobacteria, declined, and Epsilonproteobacteria increased in the vent with an increase in Bacteroidetes at both the vent and transition sites.Bacterial communities at a CO CO2 concentration gradients from where the samples were collected ranged from modern levels to projected late-21st-century conditions (pH ~7.6); thus, reference samples had mean pH 8.0, transition pH 7.6, and vent pH 6.7, respectively. Samples were filtered through 0.22-µm pore-size filters and shipped overnight to the Chauhan laboratory, where metagenomic DNA was extracted from the filters with a PowerWater DNA isolation kit , and pyrotag sequencing of the 16S rRNA gene V1-V3 region was performed using standard protocols , which is rather unusual for marine waters. However, previous studies have, in fact, identified bacteria, such as novel Bacillus vulcani and Bacillus aeolius  on which Bacteroidetes are known to thrive (2 potentially stimulates DOM production and consumption, as demonstrated previously in mesocosms held at different CO2 levels (2-low pH “natural laboratory” may strengthen our ability to predict the impacts of ocean acidification on marine microorganisms.Of particular interest was the decline of athogens , 17. Theo thrive , 19. Beco thrive , elevate2 levels . FurtherSRP050984.The DNA sequences from this metagenomic project were deposited in the Sequence Read Archive under the accession no.


Innate lymphoid cells (ILCs), a newly identified member of the lymphoid population, play a critical role in the transition from innate to adaptive immunity in host defense. ILCs are important in mucosal barrier immunity, tissue homeostasis, and immune regulation throughout the body. Significant alterations in ILC responses in lung diseases have been observed and reported. Emerging evidence has shown that ILCs are importantly involved in the pathogenesis and development of a variety of lung diseases, i.e., helminth infections, allergic airway inflammation, and airway hyper-responsiveness. However, as a tissue-resident cell population, the role of ILCs in the lung remains poorly characterized. In this review, we discuss the role of ILCs in lung diseases, the mechanisms underlying the ILC-mediated regulation of immunity, and the therapeutic potential of modulating ILC responses. Innate lymphoid cells (ILCs) are emerging as an important cell population of innate immunity and play complex roles in lymphoid tissue formation, tissue remodeling, tissue stromal cell homeostasis, and regulation of host responses to infection and inflammation. Compared to adaptive lymphocytes, ILCs are relatively rare in lymphoid tissue, but they populate barrier surfaces, such as the skin, intestine, and lung, as well as in adipose and some mucosal-associated lymphoid tissues –3. CompaThe molecular and cellular components of the innate and adaptive immune systems influence and regulate both lung homeostasis and the development of lung diseases. The emerging ILC family has been shown to have critical roles in the initiation, modulation, and resolution of lung diseases. Studies using mouse models indicated that ILCs play an important role in the remodeling of damaged lung tissue following influenza virus infection, contribute to the exacerbated allergic asthma induced by viruses –10, prom+ hematopoietic progenitor cells (HPCs) in human bone marrow and peripheral blood that express ROR-γt and selectively differentiate toward ILC3s. HPCs are located in the tonsils and intestinal lamina propria (LP) and are able to differentiate into either NK cells or LTi-like cells through a process influenced by aryl hydrocarbon receptor (Ahr) ligands, stem cell factor (SCF), and IL-15 [+ progenitors differentiate into three groups of ILCs, termed Group 1 ILCs (ILC1s), Group 2 ILCs (ILC2s) and Group 3 ILCs (ILC3s)  differentiate into cells of the adaptive immune system, i.e., T cells and B cells; however, a subset of CLPs gives rise to ILCs , 14. ILCnd IL-15 . Under t+ intraepithelial ILC1s, develop from an as yet unknown precursor in a process that requires T-bet, NFIL3, and Id2, but not IL-15; ILC1s express Eomes [ILC1s respond to IL-12, constitutively express T-bet, and produce effector cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF) , 32, 33.ss Eomes , 32.ILC2s respond to the cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP); they constitutively express high levels of GATA3; and they produce the effector cytokines IL-4, IL-5, IL-9, IL-13, and amphiregulin , 34, 35.- ILC3s and NCR+ ILC3s, according to the expression of natural cytotoxicity receptors . These cells can express IFN-γ and IL-22, and their development requires T-bet and Ahr [+ ILC3s are nearly exclusively located in the skin and intestinal lamina propria, whereas LTi-like ILC3s are positioned in the intestine and lymphoid tissues. However, ILC3s with a unique phenotype, which express Thy1, stem cell antigen 1 (SCA-1), ROR-γt and IL-23R, can develop in some inflammatory contexts in the liver and intestine [ILC3s respond to IL-1β, IL-6, and IL-23; they constitutively express ROR-γt, and they produce the effector cytokines IL-17 and/or IL-22 –53. LTi  and Ahr –58. T-bentestine , 60.-/- mice, the ILC2s down-regulated ILC2s genes, such as Gata3 and Il1rl1 (encoding IL-33 receptor), and up-regulated Rorc and the ILC3 genes. Recent studies reported plasticity between different ILC groups. ILC3 subsets could down-regulate ROR-γt expression in humans and mice, leading to dominant T-bet expression and sustained IFN-γ expression, both of which are known characteristics of ILC1s [Inter-transfer between different types of ILCs has been reported. The ILC2 population can differentiate into IL-17-producing ILC3-like cells . Many faof ILC1s , 54, 55.of ILC1s , 54, 55.ILCs in both lymphoid and non-lymphoid organs are tissue-resident cells that are locally renewed and expanded in response to acute environmental challenges. The maintenance of ILCs depends on their self-renewal ability in different microenvironments and physiological conditions .4 cells in the lung of a naïve mouse and representing 0.4-1 % of the total live cells in the lung [- ILC3s. In the contrast, the percentages of ILC1s and ILC2s are 10 % and 30 %, respectively [ILCs have been identified in the human and mouse lungs and airways . In the the lung . In humaectively . ILC2s, ectively .Studies have shown that more than 95 % of the lung-resident ILCs are of host origin and are maintained and expanded locally under physiologic or pathological conditions . During Maintenance of the epithelial barrier function in respiratory tract mucosal sites is critical to limit exposure to physiological and immunological stimuli , 65. RecIn the lung, ILCs interact with other cells, including local epithelial cells, NKT cells, myeloid cells and granulocytes, to form an immune system network.Studies have suggested that epithelial cell-derived mediators are important for ILC regulation. For example, epithelial cell- or myeloid cell-derived IL-25, IL-33, and TSLP can elicit ILC2 responses after allergen, chemical, or helminth parasite exposure or influenza virus infection , 71–73. −/− mice, which lack B and T cells, infected with helminths or challenged with the protease papain [−/− ILC2s into Il17br−/− mice, in which the secretion of T-cell-derived IL-13 was delayed, restored the antigen-specific T cell production of IL-13. The results suggest that ILC2s regulate antigen-specific T cell responses and enhance T cell cytokine production in an IL-13-independent manner [In vitro co-culture of lung ILC2s with CD4+ T cells induced the enhanced proliferation of the CD4+ T cells and the production of Th2 cytokines [h2) responses, ILC2s can promote chronic inflammation in mice. This occurs either by migration of activated DCs to the lung draining lymph node and subsequent Th2 cell priming in response to IL-13 [+ T cells in a major histocompatibility complex class II (MHCII)-dependent manner [Moreover, T cells have been shown to interact with ILCs in the lung. ILCs were unable to induce worm expulsion in Rag2e papain . In micet manner . In vitrytokines . In papaytokines . Furtherto IL-13  or by tht manner , 87.h2 cytokines, including IL-5, IL-6 and IL-13. IL-5 and IL-6 regulate B cell antibody production and self-renewal of B1 cells [Crosstalk between B cell and ILCs in the lung has also been reported. The fat-associated lymphoid clusters (FALC)-derived ILC2s proliferate in response to IL-2 and produce large amounts of TB1 cells –90. OtheB1 cells , 72, 91.Studies have shown that IL-27 and IFN-γ, which can be released by ILC1s, antagonize the function of ILC2s and type 2 innate immune responses; in ILC2s lacking the IFN-γ receptor, ILC2-mediated lung inflammation was enhanced. The transcription factor STAT1 seems important in mediating the suppressive effects of IL-27 and IFN-γ on ILC2 functions , 93. HowN. brasiliensis infection in mouse lungs, autocrine IL-9 production contributes to the survival of activated ILC2s, amplifies ILC2-mediated amphiregulin production, and promotes tissue repair [The recovery of lung tissue following injury is critical for restoring lung homeostasis and is a complex process involving multiple cellular and molecular regulators, such as interleukins , chemokines (MCP-1), growth factors , and extracellular matrix proteins  –96. TissCurrent studies have recently emphasized the complex role of ILCs and their alterations in disease-association studies of patients and experimental models, such as lung transplantation, influenza virus infection, allergic asthma, chronic rhinosinusitis and chronic obstructive pulmonary disease (COPD).In influenza virus infection, a population of ILCs that express CD90, CD25, CD127, and T1-ST2 was identified in the mouse and human lungs . A mouseAsthma is a prevalent disease of chronic inflammation in which the Th2 cytokines IL-4, IL-5, and IL-13 are all tightly linked to the pathogenesis of asthma. IL-4 induces IgE production by B cells , IL-5 ac+ ILCs were observed in the nasal polyps of patients with chronic rhinosinusitis compared to control subjects [Chronic rhinosinusitis is a typical type 2 inflammatory disease associated with IL-13 release from ILC2s. Human ILC2s express a prostaglandin D2 receptor named chemoattractant receptor expressed on Th2 cells (CRTH2), and elevated numbers of CRTH2subjects , 77. Thesubjects . Furthersubjects .- ILC3 was observed in the lungs of patients with COPD compared with the controls [- ILC3s, contribute to the formation of lymphoid follicles [COPD is characterized by a chronic inflammation of the airways. The formation of lymphoid follicles and neutrophilic airway inflammation are two characteristics of COPD , 105. Incontrols . Additioollicles , 108. Neollicles . IL-13 mollicles . Importaollicles . AlthougBased on the findings showing that ILCs play an important role in lung homeostasis and diseases, there is an urgent need to investigate whether the modulation of ILC responses is a practical therapeutic strategy that could provide a clinical benefit. Indeed, various therapeutic strategies have been developed that aim to modify ILC development, cell-cell crosstalk, effector molecules, cytokine-receptor pathways, and tissue repair functions and maintenance. On the other hand, ILCs can be a biomarker for several diseases. Because many therapies were used to treat patients with multiple sclerosis or Crohn's disease, options are available for use to determine whether these strategies also work in patients with lung disease. A therapeutic example is the treatment of patients with multiple sclerosis with daclizumab, which is a humanized monoclonal antibody against α chain of the interleukin-2 receptor . Daclizumab modulates ILC responses by decreasing the number of ILCs by modifying the cells’ phenotypes and shifting the cells toward a NK cell lineage .However, it is important to consider how the therapeutic strategies may affect the pathologic versus protective functions of ILCs. Some methods include targeting molecules that are critical for the development and migration of ILCs, e.g. α4β7 and MAdCAM-1. A clinical study revealed that Vitamin D can inhibit cytokine production and integrin α4β7 expression in human ILCs . HoweverDespite these advances, further studies are required to reveal the ILC responses in defined patient populations and to fully reveal how ILC function can be modulated to limit human disease.Our knowledge of ILC development and regulation and their roles in lung diseases has been greatly advanced by recent research. However, a better definition of innate immune cells, i.e., a universal consensus on the markers for ILCs in humans and mice, is needed. At the cellular and molecular level, the interactions between ILCs and other immune and tissue cells, as well as the mechanisms involved in these interactions, need to be addressed. A critical appraisal of translational studies is also expected, including the determination of the expression profiles of these cells in patients and the ILC responses to currently used medications. Furthermore, several questions regarding the potential plasticity of ILC populations, ILCs’ novel functions, and the regulatory pathways affecting ILC responses should be addressed using animal approaches. Knowledge of ILC biology and their roles in resting conditions and disease states will be necessary for us to better understand the mechanisms of lung diseases and to develop novel therapeutic options for these diseases.Ahr, Aryl hydrocarbon receptor; CHILP, Common helper innate lymphoid precursor; CLPs, Common lymphoid progenitors; COPD, Chronic obstructive pulmonary disease; CRTH2, Chemoattractant receptor expressed on Th2 cells; Eomes, Eomesodermin; FALC, Fat-associated lymphoid clusters; GATA3, GATA binding protein 3; HPCs, Hematopoietic progenitor cells; Id2, Inhibitor of DNA binding 2; IFN-γ, Interferon-γ; ILC1s, Group 1 ILCs; ILC2s, Group 2 ILCs; ILC3s, Group 3 ILCs; ILCps, ILC progenitors; ILCs, Innate lymphoid cells; IRF4, Interferon regulatory factor 4; LP, Lamina propria; LT, Lymphotoxin; LTi, Lymphoid tissue-inducer cells; NCR, Natural cytotoxicity receptors; NFIL3, Nuclear factor interleukin-3; NKps, NK cell precursors; NKs, Natural killer cells; PGD2, Prostaglandin D2; PLZF, Promyelocytic leukemia zinc finger; RAG, Recombination activating gene; ROR-α, Retinoic acid receptor-related orphan receptor-α; ROR-γt, Retinoic acid receptor-related orphan receptor-γt; SCA-1, Stem cell antigen 1; SCF, Stem cell factor; TCF1, T cell factor 1; Th2, T helper 2 cell; TNF, Tumor necrosis factor; TOX, Thymocyte selection–associated high-mobility group box protein; TSLP, Thymic stromal lymphopoietin


Alphaherpesvirinae that causes equine coital exanthema. Here, we report the first complete genome sequence of EHV-3. The 151,601-nt genome encodes 76 distinct genes like other equine alphaherpesviruses, but genetically, EHV-3 is significantly more divergent.Equid herpesvirus 3 (EHV-3) is a member of the subfamily  Varicellovirus of the subfamily Alphaherpesvirinae. EHV-3 is antigenically, genetically, and pathogenically distinct from the other equine herpesviruses and has a worldwide distribution. EHV-3 is the causative agent of equine coital exanthema (ECE). ECE is an infectious, venereally transmitted disease with characteristic papules, vesicles, pustules, and ulcers on the external genitalia of mares and stallions. Although EHV-3 is highly contagious, the virus is non-invasive, and ECE is a fairly benign disease. ECE, however, has a significant negative economic impact on the equine breeding industry due to forced temporary disruption of mating activities (––Equid herpesvirus 3 (EHV-3) belongs to the genus tivities . Althougtivities –5, the sivities ––8. We th50 105) was clarified , concentrated , and purified with a 30% sucrose gradient ultracentrifugation . Subsequently, residual equine DNA was degraded through nuclease digestion, after which viral DNA was extracted as previously described  for two passages and plaque purified three times. Clone 3A was passaged seven additional times in the same cell line, and 600 mL of culture supernatant , both flanked by inverted repeats (TRL/IRL and IRS/TRS). It has a total size of 151,601 nt and a G+C content of 68.1%. EHV-3 open reading frames (ORFs) are arranged in the same order as in other equine alphaherpesviruses with no proof of additional ORFs. Due to a different position of the junction between US and IRS/TRS, ORF 76 is positioned inside the IRS/TRS and is duplicated. Consequently, EHV-3 is predicted to encode 76 distinct genes, 4 of which are duplicated , resulting in a total of 80 genes. Genetically, EHV-3 is the most divergent of the equine alphaherpesviruses with overall nucleotide identities with the other viruses ranging from 62.1% to 64.9%, whereas EHV-1, EHV-4, EHV-8, and EHV-9 have identities of at least 78.2%.KM051845.The genome sequence of EHV-3 has been deposited in GenBank under the accession number


Igf2, Igfb2, Igf1r, Fgf1, Mdk, Ntf3, Bdnf), transcription factors , potassium channels (Kcne2), neuropeptides , peptide receptors , neurotransmitter receptors , and vesicular neurotransmitter transporters . Protein-protein interaction analysis revealed networking of clusters associated with the regulation of growth/troph factor signaling, transcription, translation, neurotransmitter and neurohormone signaling mechanisms and potassium channels. Collectively, the results reveal the contribution of ERβ-mediated processes to the regulation of transcription, translation, neurogenesis, neuromodulation, and neuroprotection in the hippocampal formation of ovariectomized, middle-aged rats and elucidate regulatory channels responsible for DPN-altered functional patterns. These findings support the notion that selective activation of ERβ may be a viable approach for treating the neural symptoms of E2 deficiency in menopause.Estradiol (E2) robustly activates transcription of a broad array of genes in the hippocampal formation of middle-aged ovariectomized rats via estrogen receptors . Selective ERβ agonists also influence hippocampal functions, although their downstream molecular targets and mechanisms are not known. In this study, we explored the effects of long-term treatment with ERβ agonist diarylpropionitrile  on the hippocampal transcriptome in ovariectomized, middle-aged (13 month) rats. Isolated hippocampal formations were analyzed by Affymetrix oligonucleotide microarray and quantitative real-time PCR. Four hundred ninety-seven genes fulfilled the absolute fold change higher than 2 (FC > 2) selection criterion. Among them 370 genes were activated. Pathway analysis identified terms including glutamatergic and cholinergic synapse, RNA transport, endocytosis, thyroid hormone signaling, RNA degradation, retrograde endocannabinoid signaling, and mRNA surveillance. PCR studies showed transcriptional regulation of 58 genes encoding growth factors ( This treatment lasted for 29 days providing a dosage of 0.02 mg DPN/animal/day. The same animals and hippocampal samples have been used in a recent PCR study  and in accordance with regulations of the European Community (Decree 86/609/EEC). Female Harlan-Wistar rats were purchased from Toxicoop  and housed on a 12 h light/12 h dark cycle in the animal care facility of IEM. The rats were used as breeders and retired at their age of 8 months, and then housed individually for the subsequent months. At their age of 13 months, rats were deeply anesthetized and ovariectomized bilaterally .Hippocampal formations from 16 animals were prepared and total RNA was isolated and analyzed as described previously  and hypoxanthine guanine phosphoribosyl-transferase (Hprt) were used as housekeeping genes. Expression of these genes did not vary among experimental groups. A computed internal control corresponding to the geometric mean of cycle threshold (Ct) values of Gapdh and Hprt was used for Ct calculation. PCR data evaluation were performed as described previously  were designed to study mRNA expression by real-time PCR. Sixteen samples were examined. One microgram of total RNA was used for reverse transcription. Reverse transcription and PCR were carried out by using Applied Biosystems' High Capacity cDNA Reverse Transcription Kit and TaqMan Universal PCR Master Mix II, respectively. The ViiA7 RUO 1.2.1 (Applied Biosystems) software and relative quantification against calibrator samples (ΔΔCt) were used for data evaluation. Glyceraldehyde-3-phosphate dehydrogenase , exceeded 2. Symbols and description of regulated genes are listed in Supplementary Table Ttr), claudin 2 (Cldn2), klotho (Kl), insulin-like growth factor-binding protein 2 (Igfbp2), secretoglobin (Scgb1c1), folate (Folr1), and prolactin (Prlr) receptors, sclerostin domain containing (Sostdc1), small nuclear ribonucleoprotein polypeptide G (Snrpg), prostaglandin D2 synthase (Ptgds), insulin-like growth factor 2 (Igf2), malectin (Mlec), SEC63 homolog (Sec63), membrane frizzled-related protein (Mfrp), spectrin beta, non erythrocytic 4 (Sptbn4), midkine (Mdk), angiotensin I converting enzyme (Ace), and poliovirus related receptor 1 (Pvrl). The single robustly suppressed target gene (FC < 0.25) was stem-loop binding protein (Slbp).The top 59 upregulated and 11 downregulated genes, which satisfied the mFC > 2.7 criterion, were listed in Table The heat map represents mRNA expression of the top 70 differentially expressed genes in the hippocampal formation of vehicle treated (M-OVX) and DPN-treated (M-OVX+DPN) animals Figure . The heaGng12, Dlgap1, Cacna1a, Shank2, Gls, Gnbr, Kcnj3, Grm1, Grm8), cholinergic synapse , RNA transport , endocytosis, thyroid hormone signaling pathway , RNA degradation , retrograde endocannabinoid signaling , and mRNA surveillance pathway . By the use of STRING program, putative interactions of certain proteins encoded by the DPN-regulated genes, obtained from the KEGG pathway analysis, were revealed and plotted  identified 12 clusters with high enrichment score (ES), such as membrane-enclosed lumen ES = 4.39), mRNA metabolic process (3.16), protein transport (2.24), nuclear mRNA splicing (2.18), regulation of transcription (1.92), fatty acid transport (1.47), regulation of carbohydrate metabolism (1.45), nucleotide binding (1.44), potassium channel complex (1.41), macromolecular complex assembly (1.41), vesicle coating (1.39), and DNA-dependent transcription (1.3) clusters  contained Cdkn1c, Kcne2, Cox8b, Fgf1, Mdk, Sfrp1, Msx1, Anxa2, Ptgds, A2m, Aldh1a2, Mrc1, Slc12a2, Cdkn1a, Htr2a, Gpx1, Gria2, Oprm1, Igf1r, Inpp5f, Ntf3, Gnrhr, Chrm5, Nr3c1, Grm5, Gria3, Gphn, Gabra1. The group of downregulated genes coded for neuropeptides , neuropeptide receptors , neurotransmitter receptors , vesicular transporters , neurotrophic factor (Bdnf), and others . Importantly, DPN-dependent regulation of the 21 selected genes including Sostdc1, Ttr, Kl, Prlr, Slco1a5, Mfrp, Aqp1, Otx2, Cldn2, Igfbp2, Folr1, Igf2, Ace, Htr2c, Slc13a4, Enpp2, Kcne2, Mdk, Ptgds, Inpp5f, A2m was confirmed by the PCR study.We applied quantitative real-time PCR to confirm DPN-dependent regulation of selected genes and to get further insight to the impact of DPN on neurotransmission. We selected 21 genes from the microarray gene list to confirm regulation and 37 additional genes which are associated with neurotransmitter and neuropeptide signaling. Selected genes and changes in their mRNA expression are summarized in Table Altogether, we identified 534 DPN-regulated genes by expression profiling. We searched for interactions among proteins encoded by these genes using the STRING 10 platform. At high confidence level 0.72), STRING predicted a large number of putative interactions and clusters composed of more than four elements indicating that DPN may modulate multiple regulatory mechanisms involved in hippocampal functions  take part in the formation of GABAA complexes. α1, α2, and α4 are found in all sectors of the hippocampus  fill synaptic vesicles with GABA. The transporter protein is highly expressed in the pyramidal and granular layers of the hippocampus  in the hippocampal formation  is mainly expressed in granule cells and CA3 pyramidal neurons while mGluR5 is highly expressed in all subfields of the rat hippocampus  are activated in response to DPN. Activation of HT2A receptors has been shown to decrease the expression of BDNF in the granule cell layer without affecting the CA subfields  from angiotensin I. The rat hippocampus contains high level of angiotensin II  subtypes show wide, distinct but partially overlapping expression in the rat brain including the hippocampus  is expressed throughout the rat brain  receptor is expressed in the rat hippocampus  subtypes mediate the protective and trophic effects of galanin in the hippocampus. GalR1 and GalR2 are predominantly expressed in the CA1 subfield and the dentate gyrus, respectively  receptors are found in the rat hippocampus , is expressed in the hippocampus  and Kcnc2 (Kv3.2), and A-type Kcnd3 (Kv4.3) family members. The single modulatory beta subunit is Kcne2. DPN strongly activates transcription of Kcne2 which is in line with the presence of estrogen response elements in the promoter region of the gene , inwardly rectifying channel (Kcnj3), and tetramerization domain-containing 1 (Kctd1). With the exception of Kcnk2 and Kctd4, the DPN-controlled potassium channel coding genes were upregulated. Increased expression of Kcne2 may result in modulation of neuronal excitability via KCNQ2-KCNQ3 channel function in the hippocampus  mRNA is localized in neurons of the pyramidal cell layer, the granular layer and the hilus of the dentate gyrus  survival. Deletion of Cdkn1c initially causes increased neurogenesis, while on the long run leads to impaired neurogenesis in the hippocampus , spliceosome components (Snrpg) and ribonucleoproteins . These findings suggest that DPN widely alters the transcriptional machinery in the hippocampus.Cyclin-dependent kinases (Cdk) form complexes with cyclins and operate as transcription factors. Depletion of In females, structure and function of the tri-synaptic circuit of the hippocampal formation is responsive to changes of gonadal hormone levels. The neuronal response to E2, which takes place within 15 min, includes dendritic remodeling and synaptic turnover. The role of fast, nongenomic effects has been explored and recognized, while the role of genomic effects has remained unexplored. This study demonstrates that selective activation of ERβ leads to robust alteration of mRNA expression of growth/troph factor genes and modest changes of neurotransmitter and peptide receptor genes.Gria2) and GluR3 (Gria3) may increase constitutive insertion of GluR2/3 heterodimers shifting the composition of synaptic AMPA receptors. The DPN-evoked increase of GluR2 content may reduce Ca++ influx and decrease the potential of excitatory events  are composed of GluR1/2 and GluR2/3 heterodimers. As AMPAR mediates the majority of fast synaptic transmission, the modulation of its composition and phosphorylation control synaptic strength and cell firing , Fgf1, Mdk, Prlr, Cdk10, and Cdkn1c. Insulin-like growth factor 2 is the master regulator of adult neurogenesis in the subgranular zone  and migrating (DC+) cells of the dentate gyrus express ERβ and ERα mRNA  which is downregulated by DPN. Trpm7 in hippocampal neurons detects extracellular divalent cations , cytochrome c oxidase VIII (Cox8b) and some mitochondrial ribosomal protein genes  which may reflect elevated mitochondrial ATP synthesis supporting hippocampal functions.It has been demonstrated that some E2 binding sites were associated with mitochondria in dendritic shafts and spines of the rat hippocampus  in the hippocampus (Sarvari et al., Ttr. It is in accord with the presence of a putative estrogen-response element within the 5′ flanking region of TTR (Martinho et al., Ttr in the hippocampus (Wang et al., Concerning limitations, first we note that pharmacological approaches in ERβ research are hindered by the moderate selectivity of ERβ agonists. In a chronic paradigm it seems reasonable to assume that nonselective actions on ERα may occur. However, the lack of the hallmark of ERα, namely its proliferating effect in the uterus, does not support this notion. In the hippocampus, DPN in part exerts overlapping transcriptional effects with E2, but there are clear differences (Sarvari et al., Summing up, long-term ERβ agonist treatment evokes a broad array of changes in the hippocampal transcriptome of middle-aged, ovariectomized rats Figure . A signiZL, MS conceived and designed experiments. MS, IK, EH, AR performed experiments. MS, NS, ZL analyzed data. MS, ZL wrote the paper.This work was supported by the Hungarian Scientific Research Fund Grant OTKA K100722 for ZL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The goal of this review is to discuss how the TLRs and NRLs signaling pathways collaborate in a cooperative or synergistic manner to counteract the infectious agents. A deep knowledge of the biochemical events initiated by each of these receptors will undoubtedly have a high impact in the design of more effective strategies to control inflammation.Early sensing of pathogenic bacteria by the host immune system is important to develop effective mechanisms to kill the invader. Microbial recognition, activation of signaling pathways, and effector mechanisms are sequential events that must be highly controlled to successfully eliminate the pathogen. Host recognizes pathogens through pattern-recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs). Some of these PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene-I- (RIG-I-) like receptors (RLRs), and C-type lectin receptors (CLRs). TLRs and NLRs are PRRs that play a key role in recognition of extracellular and intracellular bacteria and control the inflammatory response. The activation of TLRs and NLRs by their respective ligands activates downstream signaling pathways that converge on activation of transcription factors, such as nuclear factor- All living organisms are constantly challenged by microorganisms and a variety of particles (from air pollution and cellular stresses) that represent a health threat. To counteract this burden, the innate immune system needs to react promptly and adequately to eliminate them, while at the same time to preserve tissue normal function. In the last decade there has been an enormous progress in the study of the molecular mechanisms that allow the host to fight against any antigenic stimuli and to keep internal homeostasis. In general, the innate immune host defense includes three essential sequentially events: (1) microbial recognition, (2) activation of signaling pathways, and (3) effector mechanisms.κB, AP-1 or IRFs. Activation of these transcription factors regulates the inflammatory and innate immune response through the expression of proinflammatory mediators and antimicrobial effectors. Since some pathogenic bacteria possess PAMPs that can be simultaneously recognized by several PRRs and this leads to the activation of common transcription factors, it is likely that a collaborative response among different signaling molecules may exert regulatory functions after recognition of pathogenic bacteria. Therefore, the aim of this review is to discuss recent findings on the collaborative activity of TLRs and NLRs in the modulation of the inflammatory response induced by virulence factors of pathogenic bacteria.Hosts are able to recognize distinct PAMPs present in microorganisms through a wide variety of PRRs. To date, a broad range of PRRs have been reported that include TLRs, NLRs, RLRs, and CLRs . The di Trypanosoma cruzi, zymosan isolated from yeast, profilin from Toxoplasma gondii, and DNA from bacteria and mycobacteria [Animals, including humans, respond to a wide range of antigenic stimuli in order to preserve their homeostatic conditions . Professbacteria –8. To daTLRs constitute a family of receptors, with different specificities, 10 in humans and 12 in murines. They are able to recognize a structural diversity of PAMPs like glycans, lipids, proteins, lipoproteins, and nucleic acids  and are Monocytes, mature macrophages, and dendritic cells virtually express all TLRs, while TLR4 and TLR5 are also expressed in intestinal epithelium. Moreover, it has been reported that plasmacytoid dendritic cells and mast cells express TLR7 and TLR8, respectively , 23–25. TLRs also play a major role in cutaneous host defense against microorganisms , 38. NorNLRs constitute a family of intracellular receptors that detects PAMPs and endogenous molecules. This family contains 16 members that have been categorized into five structurally different subfamilies: NLRA, with an acidic transactivation domain; NLRB, with a baculovirus inhibitor of apoptosis protein repeat; NLRC, with a CARD domain; NLRP, with a Pyrin domain; and NLRX that contains an uncharacterized domain . Probabl Shigella flexneri [ Shigella, Salmonella, Listeria, Yersinia, pathogenic Escherichia coli strains, and Mycobacterium species [The expression of NLRs has been described in a variety of cellular types , for exaflexneri , 56, othflexneri –60. The flexneri . NOD1 an species . The exp species . Chlamydophila pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa [ Streptococcus pneumoniae, Staphylococcus aureus, E. coli, C. pneumoniae, and M. tuberculosis [In contrast to NOD1 that is expressed in a wide range of cells and tissues, the expression of NOD2 seems to be restricted to macrophages, neutrophils, dendritic cells, and lung epithelium , 65. Speruginosa , 70–73. ruginosa –76 and srculosis –79.β and pro-IL-18 to IL-1β and IL-18 and are involved in a form of cell death termed pyroptosis [NLRPs are a subgroup of NLRs constituted by proteins such as NLRP1, NLRP3, NLRP4, NLRP6, NLRP7, and NLRP12 that are involved in the formation of multiprotein complexes termed inflammasomes . These croptosis . K. pneumoniae, Listeria monocytogenes [ S. pneumoniae, S. aureus [ C. pneumoniae [ M. tuberculosis [ L. pneumophila [ Porphyromonas gingivalis [ Aspergillus fumigatus [ Aeromonas veronii [ Citrobacter rodentium and Clostridium difficile in mice [NLRPs respond to a broad variety of bacteria and it has been shown that NLRP3 is activated by the lung pathogenic microorganismsytogenes , 85, S. . aureus , C. pneueumoniae , M. tuberculosis , L. pneuumophila , influenumophila , 91, Porngivalis , Aspergiumigatus , and Aer veronii . NLRP3 s in mice ; however Bacillus anthracis [ T. gondii in mice and rats infection models [β stimulation [ Mycoplasma as well as S. aureus infection, leading to formation of an inflammasome [Although NLRP1 was the first NLR described as a part of an inflammasome, its mechanism of activation is not well studied. It is abundantly expressed in lymphocytes, respiratory epithelial cells, and myeloid cells , 96. Thenthracis ; LT actinthracis . A recenn models . NLRP7 imulation . Despiteammasome .κB activation and IFNβ production [κB signaling downstream of TLRs in macrophages in vitro and mouse in vivo [ Nlrp6 confers resistance to L. monocytogenes and Salmonella typhimurium infections [ Nlrp6 gene could be beneficial to control infection caused by these pathogens, it must be studied how its deficiency might affect the gut homeostasis. Another member of the NLRs family NLRP12 functions as a negative regulator of inflammation. It is expressed in myeloid cells and its expression is reduced by TNFα and TLR stimulation [ in vivo host innate immune response to LPS stimulation, K. pneumoniae infection, or M. tuberculosis [NLRPs also negatively control the inflammatory response by lowering the NF-oduction , 102. Thoduction . On the  in vivo , which s in vivo . Also itfections . Althougmulation , 107. Horculosis .β/IL-18 secretion in macrophages infected with Salmonella enterica serovar Typhimurium, L. pneumophila, and P. aeruginosa. [ S. flexneri, a pathogen bacterium lacking flagellum, also induces the activation of the NLRC4 inflammasome through PrgJ, a protein that forms the basal body rod of the type three secretion system [ Salmonella beyond the gut [Early experiments revealed that flagellin delivered into the cytosol is an important bacterial component for the activation of the NLRC4 inflammasome independently of TLR5 activation . Besidesuginosa. , 111. S.n system –114. Fur the gut .β maturation and pyroptotic cell death [ Francisella tularensis in macrophages [ Francisella novicida in dendritic cells [ L. monocytogenes in macrophages [ Mycobacterium sp. in macrophages [ S. pneumoniae in macrophages [ P. gingivalis in gingival epithelial cells [β release and survival during acute central nervous system (CNS) S. aureus infection [On the other hand, the absent in melanoma-2 (AIM2) protein is a member of the IFI20X/IFI16 (PYHIN) protein family that binds DNA from virus and bacterial pathogens. Upon DNA sensing, AIM2 triggers the assembly of the inflammasome, leading to caspase-1 activation, IL-1ll death , 117. Serophages , Francisic cells , L. monorophages , 120–122rophages , 124, S.rophages , and P. al cells . Even AInfection .κB, AP-1 or IRFs transcription factors, which driving the expression of genes that mediate several processes such as cell proliferation and release of antimicrobial molecules and cytokines that regulate the immune response [Initially, sensing of pathogenic bacteria by host activate signaling pathways that turn on mechanisms to kill the microorganism . Howeverresponse . In the β- (TRIF-) dependent manner, with MyD88 signaling predominantly leading to the activation of NF-κB, while TRIF signaling leading to both interferon regulatory factor 3 (IRF3) and, to a lesser extent, NF-κB activation  , 130. TLtivation  [131, 13 T. gondii, and diacyl-lipopeptides from mycoplasma [β-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and TAB2 at the plasma membrane, promoting the phosphorylation of TAB2 and TAK1. IRAK1 is degraded at the plasma membrane and the remaining complex, consisting of TRAF6, TAK1, TAB1, and TAB2, associates with the ubiquitin ligases, ubiquitin-conjugating enzyme 13 (UBC13) and ubiquitin-conjugating enzyme E2 variant 1 (UEV1A) in the cytosol. Ubiquitination of TRAF6 induces the activation and phosphorylation of mitogen-activated protein kinases (MAPKs), Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK), and the inhibitor of nuclear factor-κB- (IκB-) kinase (IKK) complex, which consists of IKK-α, IKK-β, and IKK-γ  [κB (IκB), which leads to its ubiquitination and subsequent degradation by the proteasome 26S, allowing the translocation of NF-κB to the nucleus and expression of inflammatory cytokines, chemokines, costimulatory molecules, and other effectors necessary to build up the host cell “weapons” against the invading pathogen [TLRs have an extracellular leucine-rich repeat (LRR) domain, a transmembrane domain and a cytoplasmic Toll/IL-1 receptor (TIR) domain. The LRR domain of TLRs is involved in the recognition of proteins (e.g. flagellin and porin from bacteria), carbohydrates (e.g. zymosan from fungi), lipids (LPS), lipid A, and lipoteichoic acid (LTA from bacteria), nucleic acids , protein or peptide derivatives (lipoprotein and lipopeptides from various pathogens), lipid derivatives (LAM from mycobacteria), profilin fromcoplasma , 134. Oncoplasma . In the , resp.) , 135. Thpathogen , 137. Thpathogen .κB and MAP kinases, the TRIF-dependent signaling pathway also induces the activation of IFNβ. TRIF contains a Rip homotypic interaction motif (RHIM) in its C-terminal region that mediates the interaction with members of the receptor-interacting protein (RIP) family. It was observed that TRIF activates NF-κB either by direct interaction with TRAF6 or through RIP-1. Both TRIF/RIP-1 and TRIF/TRAF6 pathways converge at the IKK complex to achieve maximum activation of NF-κB-dependent gene expression. Expression of the IFNβ gene is controlled by cooperative activation of NF-κB, ATF2/c-Jun, IRF3, and IRF7. Activation of TRAF3 by TRIF is important to generate a link between TRIF and TANK-binding kinase 1 , and this in turn activates TBK1 and IKKε. TBK1 and IKKε then activate these two molecules that are responsible for the activation of TRAF family member-associated NF-κB activator (TANK), which phosphorylates IRF3 and IRF7. Phosphorylated IRF3 and IRF7 form homodimers and move to the nucleus where it binds to IFN-stimulated response elements (ISRE), resulting in the production of type I IFNs and IFN stimulatory genes (ISGs) [α response [α production [ Candida albicans [Apart from the activation of NF-s (ISGs) , 139–142response , IRF5 aloduction , 145. Moalbicans , 147. N-acetylglucosamine-N-acetylmuramic acid tripeptide motif with diaminopimelic acid (DAP). NOD2 detects muramyl dipeptide , which is a motif common of PGN from both Gram-negative and Gram-positive bacteria [κB and AP-1 (κB-inducing kinase (NIK) that triggers the p100/p52-dependent induction of the noncanonical NF-κB pathway was demonstrated by Pan et al. [κB activation through phosphorylation and ubiquitination of IκBα, inducing proinflammatory cytokines production [κB pathways, leading to cytokine, chemokine, and antimicrobial peptide production [As stated above, the NLRs NOD1 and NOD2 regulate proinflammatory cytokine expression induced by intracellular bacterial ligands. NOD1 recognizes mainly Gram-positive PGN fragments containing thebacteria –152. In bacteria . Once babacteria , 154, aland AP-1  [153, 15n et al. . Althougn et al. , 159–161oduction , 163. Mooduction , and NODoduction . Cellulaoduction , 167.κB activation was suppressed in NOD1 and NOD2 genes silenced monocytes [β-defensin 2 in human oral epithelial cells via NF-κB [α, IL-1β, IL-6, and IL-8 [−/− macrophages, via NF-κB, p38 and ERK signaling pathways [γ production [α, IL-1β, IL-4, IL-6, GM-CSF, IL-10, and TNF-α. In the same study carried out by van Heel et al. [β production was observed for TLR1/2, TLR2/6, TLR4, TLR5, and TLR7/8 ligands  combined with M-TriDAP. Assays performed with homozygotic macrophages for the 3020insC mutation and/or TLR2−/− mice demonstrated that the NOD2 ligand MDP has a synergistic effect on the induction of TNF-α, IL-1β, and IL-10 upon costimulation with specific TLR2 agonists Pam3Cys and MALP2 [As it was explained earlier TLRs and NLRs regulate the cytokine and chemokine expression in response to bacterial ligands through their respective signaling pathways , 169. Itonocytes . This syia NF-κB . Interesand IL-8 . The synpathways . Additiooduction . Anotherl et al.  a strongnd MALP2 .γ, and TNF-α that was related to increased activation of NF-κB, JNK, and p38 signaling pathways [β and IL-23 and expression of the inhibitor suppressor of cytokine signaling 2 (SOCS2), where the NOD1/TLR agonists combination was more relevant for the synergistic activity observed [ M. tuberculosis. They found a synergistic action between these two receptors for cytokine expression that was lost in cells from individuals homozygous for NOD2 3020insC mutation or macrophages harvested from TLR2−/− mice  fragments [ L. monocytogenes caused an increased production of CXCL1 and CCL2 chemokines, which was notably decreased in MC deficient in NOD1 and RICK [In a more recent work, it was shown that NOD1 and TLR2 cooperate to enhance human CD8 T cells proliferation and expansion, and this cooperating action caused an enhanced secretion of IL-2, IFN-pathways . Simultaobserved . Altogetobserved  demonstr−/− mice . The samnfection . Evidencragments . Interesand RICK .C. pneumoniae, which is a common pathogen that causes pneumonia in humans can be recognized by TLR2 and TLR4 [ C. pneumoniae by these receptors induced a reduction in the expression of IL-6, IL-12p40, and IFN-γ in RIP-2−/− mice at day 3 after infection compared with wild-type mice. However, at days 5 and 14 after infection, the production of these cytokines was significantly increased in wild-type mice, indicating an initial impaired and delayed kinetics of cytokine production in C. pneumonia-infected RIP-2−/− mice. Thus, the collaborative activity of both NLRs and TLRs is fundamental for efficient pathogen bacterial clearance. This idea has been supported by later experiments in which NLRC4 was necessary to eliminate L. pneumophila, while TLR5 was necessary to recruit neutrophils [ L. pneumophila [ P. aeruginosa macrophage infection, since NLRC4 and caspase-1 activation attenuated the autophagy activated by TLR5 and reduced the type I interferon production [and TLR4 , as welland TLR4 . Sensingtrophils . Furtherumophila . Howeveroduction , besidesoduction . Helicobacter pylori, the cooperative interaction between TLR2 and NOD2 showed to be important for IL-1β production and NLRP3 activation. This cooperative interaction in H. pylori infection was confirmed in IL-1β- and IL-1 receptor-deficient mice in which the clearance of bacteria from the stomach was impaired compared with wild-type mice [ S. enterica clearance and improved mice survival. This effect was accompanied with an increase in IL-5, IL-6, IL-13, IL-21, IL-22, TNF-α, and β-defensin 3 in small intestine [ S. aureus [β production, revealed an additive effect of both receptors in keratinocytes from murine oral epithelium, since in TLR2- or NOD2-deficient keratinocytes the cytokine release was decreased by approximately 50% compared to wild-type cells [ Vibrio vulnificus and Vibrio cholerae revealed the activation of caspase-1 via the NLRP3 inflammasome [Myd−/−Trif−/−), demonstrated that NLRP3 activation required NF-κB-dependent TLR stimulation. Altogether, these results indicate that several sensors are necessary to fight against pathogenic bacteria. Moreover, the specific combination of PRRs seems to be coordinated according to the bacteria or PAMPs involved, which subsequently affect the host response by driving collaborative/synergistic activity.On the other hand, in DCs infected withype mice . Costimuntestine . More ev. aureus . Analysipe cells . Infectiammasome . In thisκB, MAPKs, AP-1, or IRFs signaling pathways; however, the molecules that are involved in the synergistic activation of these pathways have not been identified. Then, for a better understanding of the molecular mechanisms by which NLRs and TLRs collaborate, this synergistic activity will have to be further analyzed. Since TRLs and NLRs play a fundamental role in the eradication of invading pathogen microorganisms through the induction of inflammatory and antimicrobial peptides, this collaborative activity could be exploited to modulate or improve the host response against pathogenic bacteria that causes an exacerbated inflammatory response.Most studies have focused on the characterization of the inflammatory response triggered by several virulence factors alone. However, it is important to take into account that in physiological conditions the participation of several PRRs that respond to different PAMPs could be more effective for the host to combat infections. Regarding this issue, the experimental evidence accumulated so far has pointed out that the host response against pathogenic bacteria may be the sum of several pathways induced by the recognition of different PAMPs by different PRRs, which in turn trigger and shape the subsequent innate and adaptive immune responses. Although synergistic activity among NLRs and TLRs has been demonstrated, the subjacent mechanisms are not clear. Both receptors are able to activate NF-Finally, there is a growing interest in targeting these PRRs for the treatment of sepsis, but also to fight against inflammatory diseases such as cancer, rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus , 192. Se


The present review will focus on n-3 PUFAs and bioactive peptides effects on cardiovascular disease risk factors. Moreover, since considerable controversy exists regarding the association between n-3 PUFAs and major cardiovascular endpoints, we have also reviewed the main clinical trials supporting or not this association.Cardiovascular disease remains the most common health problem in developed countries, and residual risk after implementing all current therapies is still high. Permanent changes in lifestyle may be hard to achieve and people may not always be motivated enough to make the recommended modifications. Emerging research has explored the application of natural food-based strategies in disease management. In recent years, much focus has been placed on the beneficial effects of fish consumption. Many of the positive effects of fish consumption on dyslipidemia and heart diseases have been attributed to  Cardiovascular disease (CVD) is the most common cause of human morbidity and mortality in the world, and elevated blood lipids have been strongly associated with increased incidence of CVD. Expenses for the treatment of CVD to the European health care system are presumed to be as high as 200 billion Euros each year . In the Atherosclerosis is the dominant cause of CVD including myocardial infarction, heart failure, stroke and claudication. Atherosclerosis is mainly located in the intima of middle sized and large arteries, especially where the vessels divide ,5. It isLowering low density lipoprotein-cholesterol (LDL-C) is a central target in the prevention of CVD, especially coronary heart disease. It is estimated that for every 1% reduction in LDL-cholesterol concentration, there is a corresponding 1% to 2% decrease in the risk of CVD ,8,9,10.Although genetic factors and aging are important in determining the overall risk, a substantial proportion of CVD occurs in conjunction with a series of modifiable risk factors, such as hyperlipidemia, hypertension, obesity, insulin resistance and diabetes, susceptible to lifestyle modifications, which include diet and physical exercise ,12. PermWith marine species comprising approximately one-half of the global biodiversity, the ocean offers a wonderful resource for novel compounds, which may serve in improving health of the worldwide population.n-3) polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid  and docosahexaenoic acid  [n-3 PUFAs intake were challenged by recent clinical trials that failed to replicate protective effects of EPA and DHA on CVD [n-3 PUFAs, be attributed to other nutrients, such as minerals, vitamins and proteins [Observational studies first reported in the 1970s showed that the Greenland Inuit populations had a low incidence of coronary artery disease that was related to their traditional lifestyle and in particular their distinctive dietary habits, characterized by the consumption of cold-water marine fish and artic mammals, rich in Ω-3 (or 2:6 n-3) ,15,16. S2:6 n-3) ,18. Howecid EPA, :5 n-3 anproteins .n-3 PUFAs and bioactive peptides, and their effects on risk factors for CVD and on specific clinical endpoints.This review examines current recommendations for fish intake, as source of n-3 PUFAs are able to affect: lipid profile [Several data highlighted that  profile ,23,24, a profile , platele profile ,27, endo profile ,29, infl profile , and blo profile .n-3 PUFAs have well known and long-appreciated TG-lowering properties [Fasting and nonfasting triglycerides (TG) have long been associated with CVD . Fish oioperties . Intake operties , a drug operties ,35,36.i.e., 2, 3, and 4 g/day [n-3 PUFA, the free fatty acid form does not require pancreatic lipase hydrolysis with carboxyl ester lipase, an enzyme which activity is highly dependent on fat meal content [Recently, the EpanoVa fOr Lowering Very high triglyceridEs (EVOLVE) trial, a double-blind, randomized, controlled investigation of a lipid-altering drug in patients with severe hypertriglyceridemia, demonstrated that Epanova , a novel 4 g/day . Specifi 4 g/day . Since i 4 g/day ,39. The n-3 PUFAs accomplish this hypotriglyceridemic effect have been explored in kinetic studies in humans [in vitro and ex vivo experiments [de novo lipogenesis, decrease of serum levels of nonesterified fatty acids and increase in fatty acid beta-oxidation); (2) reduced hepatic enzyme activity for TG synthesis; (3) increased phospholipid synthesis and apoB degradation [n-3 PUFAs exert their effect on TG by inhibiting hepatic very low density lipoprotein (VLDL) secretion rate and by stimulating serum TG clearance mechanisms [The mechanisms by which n humans ,41 and an humans ,43, as weriments . The majradation ,45,46. Hchanisms ,50,51,52n-3 PUFAs have little or no effect on TC, but affect LDL-C and HDL-C concentrations as well as HDL and LDL particle size; these effects are variable and depend on dose and population studied [With regard to total cholesterol (TC), HDL-C and LDL-C levels,  studied ,54,55,56 studied . DHA sup studied , a prepa studied . Similar studied .n-3 PUFA formulations are approved in the United States to treat adult people with very high levels of TG (>5.6 mmolL) with or without concomitant elevation of other atherogenic parameters: omega-3-acid ethyl esters , omega-3-carboxylic acids (Epanova), which contain both EPA and DHA, and icosapent ethyl (Vascepa), which is an EPA-only formulation [Currently, six mulation .In vitro and animal studies showed that n-3 PUFAs directly affect cardiac ion channels [n-3 PUFAs exert a marked inhibitory effect on sodium channels, thus reducing excitability, and a wide range of effects on potassium channels, L-type calcium channels and sodium-calcium exchanger [n-3 PUFAs have been shown to alter membrane fluidity that could contribute to effects on ion transport [n-3 PUFAs reduces ischaemia-induced ventricular fibrillation, by acting on potassium channels [n-3 PUFAs may be mediated in part by their actions on autonomic control, especially by an increased vagal tone [About 80%–90% of sudden cardiac deaths in the early stages after a myocardial infarction are linked to ventricular arrhythmias, and arrhythmias are associated with electrophysiological mechanisms controlling muscle contraction . In vitrchannels ,63. Dataxchanger ,64,65,66ransport ,68. Studchannels ,70. Antigal tone . Altogetgal tone ,73,74,75n-3 PUFAs are considered to have anti-thrombotic effects due to their ability to inhibit platelet thromboxane A2 synthesis and to act as antagonists of the pro-aggregant thromboxane A2/prostaglandin H2 receptor [receptor . Howeverreceptor .n-3 PUFAs influence endothelial function is mediated by their incorporation into biological membrane phospholipids; this allows modulation of membrane composition and fluidity [n-3 PUFAs may exert their beneficial effects, which include increased NO production and reduced production of pro-inflammatory mediators. Molecular evidence of enhanced endothelial nitric oxide synthase activity/expression following administration of n-3 PUFAs derives from in vitro and in vivo experimental studies [n-3 PUFAs attenuate NF-κB activation, resulting in reduced vascular cell adhesion molecule-1 expression [n-3 PUFAs exert systemic anti-inflammatory effects by raising the plasma levels of adiponectin [n-3 PUFAs are precursors of a novel series of lipid mediators  with potent anti-inflammatory and pro-resolving properties [The mechanism by which fluidity . Endothefluidity . Caveolafluidity ,81. By mfluidity , n-3 PUF studies ,84. In epression ,86. Addiponectin  and suppponectin . Moreoveoperties .n-3 PUFAs are able to reduce blood pressure in both normotensive and hypertensive subjects, even though this effect was more pronounced in the latest group [et al. have shown that intakes of EPA + DHA, achievable through the consumption of 2–3 portions of oily fish/week or 2 fish oil capsules/day, reduced systolic blood pressure by 5 mm Hg in isolated systolic hypertensive adult subjects [n-3 PUFAs ability to reduce thromboxane A2 synthesis, increase NO production, and affect the autonomic nerve function [Several studies have indicated that relatively high doses of st group ,89,90,91subjects . This effunction ,93,94.In the Diet and Reinfarction Trial (DART) study, 2033 men after myocardial infarction were randomly assigned to a group instructed to increase fish intake (corresponding to about 900 mg/day of EPA + DHA) or to a control group that received no specific information and followed for 2 years. The subjects advised to eat fatty fish had a 29% reduction in 2-year all-cause mortality compared with those not advised .A double-blind, randomized, controlled trial was conducted, in which 205 patients undergoing a first percutaneous trans-luminal coronary angioplasty (PTCA) received 15 capsules per day containing 1 g of either fish oil (2.7 g/day of EPA and 1.8 g/day of DHA) or olive oil. The treatment was started 3 weeks before PTCA and continued for 6 months thereafter. Restenosis occurred significantly less often in the fish oil group (22.0%–35.6%) than in the control group (40.0%–53.3%) .n-3 PUFAs experienced a 15% reduction in the primary end point of death, nonfatal myocardial infarction, and nonfatal stroke (p < 0.02). Moreover, there was a 20% and a 45% reduction in all-cause of mortality and in sudden death, respectively [The Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico (GISSI)-Prevenzione trial enrolled 11,324 patients with recent myocardial infarction and randomized them to the following treatment groups: vitamin E (300 mg/day), or EPA and DHA ethyl esters  or no supplements . After 3.5 years of follow-up, the group given ectively .p = 0.011) and by 53% in the sub-group with TG ≥ 150 mg/dL (1.70 mmol/L) and HDL-C < 40 mg/dL (1.04 mmol/L)  [The Japanese EPA Lipid Intervention Study (JELIS) investigated the effects of purified EPA for prevention of major coronary events . A total= 0.043) .N = 7046) demonstrated a small (1.8%), but still significant, reduction for all-cause mortality in patients with clinical evidence of heart failure  who had been treated with 1 g/day of n-3 PUFAs in addition to 10 mg/day of rosuvastatin [The later GISSI-HF study  [In the randomized, double-blind, placebo-controlled Alpha Omega trial ( = 4837) , patient = 4837) .N = 3851) received 1-g capsules containing either 460 mg EPA and 380 mg DHA or placebo daily in addition to guideline-adjusted therapy and were followed for 1 year [p = 0.84) Differences in secondary end points such as major CV or cerebrovascular events were also statistically similar. Interpretation of the results from the OMEGA trial was limited because the study lacked sufficient statistical power. The sample size and event rates used in the OMEGA study were based on prior studies, but the patient population was receiving considerably improved guideline-adjusted treatment of acute myocardial infarction, and thus the number of sudden death events was lower than expected [In the OMEGA trial, patients with acute myocardial infarction (r 1 year . Rates fexpected .n-3 PUFAs (600 mg of EPA and DHA at a ratio of 2:1) for prevention of cardiovascular disease in people with a history of ischaemic heart disease or ischaemic stroke [Similar results were obtained in the Supplémentation en Folates et Omega-3 (SU.FOL.OM3) clinical study. This study does not support the use of dietary supplements containing c stroke .N = 12,536). The study found that 1 g/day of EPA/DHA did not prevent death or any CV outcomes in this patient population [The effects of long-term treatment with Lovaza (1 capsule/day) on CV events was examined in the Outcome Reduction with an Initial Glargine Intervention (ORIGIN) trial in patients with type 2 diabetes mellitus, impaired fasting glucose, or impaired glucose tolerance (pulation .n-3 PUFA supplementation (Lovaza) on the occurrence of postoperative atrial fibrillation was assessed in patients who underwent cardiac surgery (N = 1516). No significant difference was observed between patients who received the perioperative n-3 PUFA supplementation and those who received placebo [In the Omega-3 Fatty Acids for Prevention of Post-operative Atrial Fibrillation (OPERA) trial, the effects of perioperative  placebo .n-3 PUFAs supplementation on CVD, several meta-analyses have been published, but the conclusions are not straightforward. Two recent meta-analyses published in the same year for example ended with different conclusions: one stated that n-3 PUFAs are not universally associated with major CV outcomes across patient populations at increased cardiovascular risk [n-3 PUFAs, when administrated as food or as supplements for at least six months, reduce CV events by 10%, cardiac death by 9% and coronary events by 18%, while showing a trend toward a lower total mortality [With the aim of assessing the role of lar risk ; the secortality .n-3 PUFAs therapies in the context of current standards of clinical care, in sufficiently large patient populations and at higher doses. The ongoing, prospective, randomized, double-blind Reduction in Cardiovascular Events with EPA-Intervention Trial  will assess the ability of Vascepa to reduce CV outcomes in high-risk statin-treated patients with hypertriglyceridemia. Moreover, the VITAL study, an ongoing (NCT01169259) placebo-controlled trial powered to examine major cardiovascular events, as well as CVD and stroke individually, may clarify the utility of n-3 PUFAs (Lovaza) in primary prevention of CVD [In conclusion, further studies are needed to assess the efficacy of n of CVD .Numerous studies, briefly reviewed here below, have demonstrated beneficial effects of fish proteins and fish-derived peptides on CV risk factors, such as lipid disorders and hypertension .In animal studies, proteins from different fish species have been shown to display hypocholesterolemic activity when compared with casein as protein source . The exaAmong the few clinical studies evaluating the hypocholesterolemic effect of fish proteins, consumption of a cod-fish protein supplement by overweight adults had no effect on triglycerides or HDL-cholesterol levels, but significantly decreased serum LDL-cholesterol levels from baseline . Furtheret al. [The antihypertensive activity of peptides from different food sources has been demonstrated both in experimental models and in human volunteers . The diset al.  developeet al. . A signiet al. .2+ channels [ACE inhibitory peptides have been found in various other fish species, . Among tchannels . Potent channels . Among 8channels .in vitro, inhibited by 40% the endothelin-converting enzyme activity, thus potentially reducing endothelin-1 production [Subsequently, there has been growing interest in exploring the possible uses of the fish by-products or the remaining raw materials, so that they can potentially be utilized rather than posing a waste and sustainability problem . As examoduction .TM by Metagenics, Aliso Viejo, CA, USA; PeptACETM Peptides 90 by Natural Factors, Monroe, WA, USA) [Fish-derived bioactive peptides have enormous potential and have been utilized in the production of pharmaceutical products with an active functional role and effect on health; for example, blood pressure lowering capsules have been manufactured that contain Katsuobushi Oligopeptide LKPNM which is converted into its active form (LKP) by digestive enzymes  .Fish protein peptides and hydrolysates have been found to exert antioxidant effects, extensively reviewed in two recent papers ,134.Dietary cod proteins were shown to improve insulin sensitivity in insulin-resistant patients . Recentlw/w casein as protein source (control), or the same diet except for 5% w/w salmon protein hydrolysate replacing the same amount of casein (SPH) [In a recent study, apolipoprotein E deficient mice were fed for 12 weeks a high fat diet containing 21% in (SPH) . A signin-3 PUFAs and proteins/peptides, which display several beneficial effects on the cardiovascular system. The benefit of the isolated nutrients on primary and secondary prevention of CVD has still to be confirmed/proven in further clinical studies.Based on several studies reporting a lower CVD mortality in regular fish consumers, guidelines for healthy individuals encourage the consumption of fish, preferably oily types, at least twice a week. The beneficial effects of fish could be attributed to a wide array of nutrients, particularly


To identify and characterize cancer stem cells (CSC) in moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC).in situ hybridization (CISH) for NANOG, OCT4, SOX2, SALL4, and STAT3.Four micrometer-thick, formalin-fixed, paraffin-embedded MDBMSCC samples from six patients underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers, NANOG, OCT4, SALL4, SOX2, and pSTAT3; cancer stem cell marker, CD44; squamous cell carcinoma (SCC) marker, EMA; and endothelial marker, CD34. The transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, STAT3, and CD44 were studied using NanoString gene expression analysis and colorimetric +/CD44+/SOX2+/SALL4+/OCT4+/pSTAT3+/NANOG+ CSC subpopulation within the tumor nests; (2) an EMA−/CD44−/CD34−/SOX2+/OCT4+/pSTAT3+/NANOG+ subpopulation within the stroma between the tumor nests; and (3) an EMA−/CD44−/CD34+/SOX2+/SALL4+/OCT4+/pSTAT3+/NANOG+ subpopulation on the endothelium of the microvessels within the stroma. The expression of CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG was confirmed by the presence of mRNA transcripts, using NanoString analysis and NANOG, OCT4, SOX2, SALL4, and STAT3 by CISH staining.Diaminobenzidine and immunofluorescent (IF) IHC staining demonstrated the presence of (1) an EMAThis study demonstrated a novel finding of three separate CSC subpopulations within MDBMSCC: (1) within the tumor nests expressing EMA, CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG; (2) within the stroma expressing SOX2, SALL4, OCT4, pSTAT3, and NANOG; and (3) on the endothelium of the microvessels within the stroma expressing CD34, SOX2, SALL4, OCT4, pSTAT3, and NANOG. Oral cavity cancer is the sixth most common cancer worldwide , with ovOral cavity squamous cell carcinoma affects the oral tongue, floor of the mouth, buccal mucosa, hard palate, mandibular and maxillary alveolus, vestibule of mouth, and retromolar trigone , 5. WhilIt has been proposed that the development and spread of many cancers –14, inclDespite recent literature showing the presence of CSC within many cancers, including breast , brain , and panThis study investigated the expression, within moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC), of the ESC markers NANOG, SOX2, SALL4, pSTAT3, OCT4, and CD44, at both the transcriptional and translational level, to identify and characterize the putative CSC population.Previously untreated MDBMSCCs from five male and one female patients, aged 38–80  years, were used in this study, which was approved by the Central Health and Disabilities Ethics Committee (ref. no. 12/CEN/74).Hematoxylin and eosin staining was performed on 4 μm-thick, formalin-fixed, paraffin-embedded blocks of six MDBMSCC samples, to confirm the appropriate histological grading and the presence of BMSCC in the sections. 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining of the sections was then performed using the Leica Bond Rx auto-stainer , as previously described . Staininn = 2) underwent immunofluorescent (IF) IHC staining using a combination of Vectafluor Excel anti-rabbit 594  and Alexa Fluor anti-mouse 488  so as to detect combinations that included NANOG, SOX2, and pSTAT3, and Vectafluor Excel anti-mouse  and Alexa Fluor anti-rabbit 594  to detect combinations that included CD44, EMA, OCT4, or SALL4.To confirm co-expression of two proteins, representative slides of MDBMSCC (Positive control tissues used for the primary antibodies were human infantile hemangioma (IH) for NANOG , and SAL® 2.0 fluorometer  and were subject to RNA integrity analysis via the 2100 Bioanalyzer Instrument (Agilent Technologies). The samples then underwent NanoString nCounter gene expression assay performed by New Zealand Genomics  according to the manufacturer’s protocol. Probes for the genes encoding NANOG (NM_024865.2), SALL4 (NM_020436.3) SOX2 (NM_003106.2), OCT4 (NM_002701.4), CD44 (NM_001001392.1), and STAT3 (NM_139276.2) and the housekeeping gene GUSB (NM_000181.1) were designed and manufactured by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.RNA was extracted from five snap-frozen MDBMSCC samples from the same cohort of patients used for DAB IHC staining and was used for NanoString nCounter™ Gene Expression Assay . Total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue and run on a KingFisher Duo machine (Thermo Fisher Scientific). RNA samples were then quantitated on a Qubitin situ hybridization (CISH). Staining was done on the Leica Bond Rx auto-stainer and detected using the ViewRNA red stain kit , as previously described . IF IHC-stained images were captured using an Olympus FV1200 biological confocal laser-scanning microscope and processed with the cellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).Hematoxylin and eosin staining confirmed the appropriate histological grading and the presence of MDBMSCC on the slides used for DAB IHC staining (data not shown).arrowheads), OCT4 , and pSTAT3 , while cytoplasmic and nuclear expression of NANOG  was observed in these cells. The endothelium of the microvessels within the stroma also stained positively for SOX2 , SALL4 , OCT4 , pSTAT3 , and NANOG .Diaminobenzidine IHC staining showed that cells within the tumor nests stained positively with the SCC marker EMA Figure A, brown.Expected staining patterns for the NANOG , SALL4 , OCT4 , STAT3 , and NANOG  in the cells within all three MDBMSCC samples.Colorimetric +/CD44+/SOX2+/OCT4+/SALL4+/pSTAT3+/NANOG+ subpopulation within the tumor nests aligns with recent literature reporting localization of CSC in OCSCC  and vascular mimicry, which have been previously demonstrated within breast  and headgeal SCC . Alterna+ subpopulation within the stroma, consistent with the findings of Huang et al.  This study demonstrates the novel finding of three CSC subpopulations within MDBMSCC, with; (2) one CSC subpopulation within the tumor nests, expressing EMA, CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG; (3) a second CSC subpopulation is within the stroma between the tumor nests, expressing SOX2, SALL4, OCT4, pSTAT3, and NANOG; and (4) a third CSC subpopulation expressing CD34, SOX2, SALL4, OCT4, pSTAT3, and NANOG localized to the endothelium of the microvessels within the stroma.This study was approved by Central Regional Health and Disability Ethics Committee (ref. no: 12/CEN/74).TI and STT formulated the study hypothesis. TI and STT designed the study. HHY, TF, HDB, and TI interpreted the IHC data. HHY, TF, HDB, and TI interpreted the CISH data. AMC processed the tissues for NanoString analysis and interpreted the data. HHY, TI, PFD, and STT drafted the manuscript. All authors commented on and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TI, PFD, and STT are inventors of the PCT patent application (No. PCT/NZ2015/050108) Cancer Diagnosis and Therapy.