Mesenchymal stem cells (MSCs) are promising cells for cardiovascular regenerative medicine. However, their potential may be limited, because of their restricted cardiovascular differentiation potential and decline in their number and functional characteristics with increasing donor age. We have previously shown that rat fetus heart harbors primitive MSCs and administration of these cells improved left ventricular (LV) function after ischemia/reperfusion injury in rats. To evaluate their potential as a new cell type for clinical cardiovascular cell therapy, we have undertaken this study on the isolation and characterization of human fetal cardiac MSCs (hfC-MSCs).MSCs were isolated from the heart of five 14-16-week-old aborted human fetuses and studied for their growth characteristics, karyotypic stability and senescence over successive passages, expression of mesenchymal and embryonal markers by flow cytometry and immunocytochemistry, constitutive expression of cardiovascular genes by RT-PCR, differentiation into cells of the cardiovascular lineage and their immunomodulatory properties.The hfC-MSCs grew as adherent monolayer with spindle shaped morphology and exhibited rapid proliferation with an average population doubling time of 34 hours and expansion to up to more than 80 population doublings with maintenance of a normal karyotype and without senescence. Immunophenotyping showed that they had similar phenotype as human bone marrow mesenchymal stem cells (hBM-MSCs) expressing CD73, CD90, CD105 and lacking expression of CD31, CD34, CD45, HLA-DR. However, hfC-MSCs expressed significantly higher levels of CD117 and SSEA-4 compared to hBM-MSCs. In addition, hfC-MSCs expressed the embryonal markers Oct-4, Nanog and Sox-2 as compared to hBM-MSCs. Further, hfC-MSCs had significantly higher expression of the cardiovascular genes viz. ISL-1, flk-1, GATA-4, NKX2.5 and MDR-1 as compared to hBM-MSCs, and could be differentiated into major cardiovascular cells . Interestingly, hfC-MSCs markedly reduced T-lymphocyte proliferation with an increased secretion of TGF-β and IL-10.Our results show that human fetus heart is a novel source of primitive MSCs with cardiovascular commitment which may have a potential therapeutic application in cardiovascular regenerative medicine. Stem cell therapy has shown immense potential for cardiac repair and regeneration. In this context, adult bone marrow derived mesenchymal stem cells (BM-MSCs) represent the most studied cell type for cardiac repair due to their unique features including the ease in their isolation/expansion, their paracrine activity to induce neovascularization post ischemic injury and most importantly due to their immunomodulatory properties –5. Howevin vitro and in vivo, suggesting the superiority of cardiac specific cells for cardiac repair and regeneration  thymidine diluted in RPMI 1640 medium, incubated for another 18 hours at 37°C in a humidified atmosphere of 5% CO2, and then harvested onto glass fiber filters. Thymidine incorporation was measured using a liquid scintillation counter . Results were expressed as the mean counts per minute for each triplicate culture.hfC-MSCs were seeded at a density of 5×10To determine the levels of TGF-β and IL-10 in the cell-free culture supernatant, commercial ELISA kits (R&D systems) were used per the manufacturer’s instructions.p < 0.05 using Student’s t-test or analysis of variance (ANOVA) Software.Results were calculated for our data and presented as mean ± SE where indicated. Statistical significance was defined as . We next evaluated the growth kinetics of hfC-MSCs and compared it to hBM-MSCs. We empirically chose 1000 cells/cm2 seeding density and counted the cell number of both the cell types, each day for 7 days. Interestingly, hfC-MSCs exhibited greater expansion potential as compared to BM-MSCs. Specifically, at day 7, the hfC-MSC cultures had more than 2-fold cell number compared to BM-MSCs, suggesting the superior proliferative ability of hfC-MSCs . Further, hfC-MSCs could be expanded to up to more than 80 population doublings with a population doubling time of approximately 34 hours. Human fC-MSCs displayed a normal karyotype over successive passages .We compared side by side the morphology of hfC-MSCs and hBM-MSCs under phase contrast microscopy and observed that similar to hBM-MSCs, hfC-MSCs grew as spindle shaped adherent cells . However, fC-MSCs had significantly higher expression of SSEA-4 and c-kit compared to BM-MSCs, suggesting the primitive nature of the cells. Further, hfC-MSCs after treatment with adipogenic and osteogenic induction media could be induced to differentiate into adipocytes and osteoblasts as demonstrated by Oil red-O and Alizarin red staining, respectively .To evaluate the phenotype of the hfC-MSCs, we performed flow cytometric analysis. Our head to head comparison between hBM-MSCs and hfC-MSCs revealed that both the cell types expressed typical mesenchymal stem cell markers viz., CD73, CD90, CD105 whereas both cell types did not express CD31, CD34, CD45 and HLA-DR . However, the hBM-MSCs did not express these markers , highlighting the primitive nature of fC-MSCs. Furthermore hfC-MSCs consistently exhibited significantly elevated levels of GATA-4, flk-1, Isl-1 genes as compared to hBM-MSCs. Interestingly, NKX2.5 and MDR-1 expression levels were undetectable in the hBM-MSCs compared to hfC-MSCs , suggesting the tissue specific gene expression in to hfC-MSCs.We have previously shown that rat fC-MSCs express embryonal markers . SimilarFig 5E and 5F).Higher expression of cardiovascular markers by hfC-MSCs compared to hBM-MSCs prompted us to look at their cardiovascular differentiation ability. Upon induction, hfC-MSC could differentiate into major cardiovascular cell types’ viz. cardiomyocyte like cells, endothelial cells and smooth muscle cells , as revealed by multinucleated differentiated cardiomyocytes with expression of cTnT , CD31 onp < 0.05) . However, when hfC-MSCs were co-cultured with PBMCs at a higher ratio of 1:5 and 1:10, there was no effect on PBMCs proliferation. Cytokines analysis revealed that hfC-MSCs secrete regulatory cytokines TGF-β and IL-10 to inhibit PBMCs proliferation (p<0.001) . These results strongly suggest that fC-MSCs exhibit immunomodulatory properties.It has been recently shown that adult cardiac MSCs exhibit immunomodulatory properties . Therefoin vitro differentiation into cells of the three major cardiac lineages including cardiomyocyte like cells, endothelial cells and smooth muscle cells. The cells maintained a stable karyotype, undifferentiated state and multipotent differentiation potential over the successive passages. The hfC-MSCs markedly reduced T-lymphocyte proliferation with an increased secretion of TGF-β and IL-10. To the best of our knowledge this is the first report on the presence of multipotent mesenchymal stem cells exhibiting immunomodulation in human fetus heart.We have observed a novel population of mesenchymal stem cells in the heart of 14–16 weeks’ human fetus, that exhibited an extended self-renewal of up to more than 80 population doublings without any sign of senescence and karyotypic abnormality, expressed pluripotency markers namely Oct-4, Nanog, SOX-2 and SSEA-4 and exhibited an We found that hfC-MSCs exhibited typical spindle shaped morphology and were adherent to plastic, similar to our published report on rfC-MSCs , 13 and in vitro into cardiomyocyte like cells, endothelial cells and smooth muscle cells. A recent study has shown that, fetal cardiovascular precursor cells could only differentiate into cardiomyocytes and smooth muscle cells in vitro but not endothelial cells. This discrepancy between the two cells populations may be due to differences in the developmental stage of fetus hearts at which they had been obtained (12 vs 14–16 weeks) or variability within the pool of fetal cardiac stem cells. Further, future studies are warranted to investigate an optimal differentiation of hfC-MSCs into bona fide cardiomyocytes, which may also be utilized for drugs screening or cardio-toxicity studies.To further understand the differences between both hfC-MSCs and hBM-MSCs, we examined differences in the expression of cardiovascular genes including GATA-4, flk-1, Isl-1, NKX2.5 and MDR-1 by real time PCR. In corroboration with previous reports that different cardiac resident stem cells express specific cardiac markers , we founWe and others have previously shown that fetal mesenchymal stem cells from rodents and humans express embryonal markers , 20, 21.Mesenchymal stem cells are extensively studied cell types in regenerative medicine due to their immunomodulatory properties . In lineIn conclusion, this study has demonstrated that human fetus heart has a population of primitive MSCs that express embryonal markers and are committed towards cardiovascular lineage, making them an attractive cell type for cardiovascular regenerative therapy. Further, these cells exhibited intense immunomodulatory properties, suggesting that they could be a potential off the shelf allogenic source of cells for therapy. We do acknowledge that aborting of fetuses may not be legally permitted in many countries and therefore the accessibility to these tissue sources may be questioned. However, our finding could be useful in countries where abortion or medical termination of pregnancy is legal. Further studies on the therapeutic efficacy in pre-clinical animal models of cardiac diseases, will be important to bring out the translational value of these stem cells.S1 FigRepresentative photomicrographs showing normal karyotype of human fetal cardiac mesenchymal stem cells at 15th passage.(TIF)Click here for additional data file.S2 FigRepresentative photomicrographs  showing differentiation of human fetal cardiac mesenchymal stem cells into adipocytes and osteocytes: (A) control cells showing no stain; (B) cells differentiated into adipocytes positive for Oil red O; (C) control cells showing no stain; D) cells differentiated into osteocytes positive for Alizarin red stain.(TIF)Click here for additional data file.


In addition, the DA receptor-mediated calcium influx dependent presynaptic inhibition mentioned above decreases with postnatal development, with selective coupling between DA receptors and each subtype of calcium channels being unchanged. Furthermore, the precise origin of these GABAergic or glutamatergic inputs to postsynaptic neurons can be identified by recent optogenetic approaches. Thus, modulatory mechanisms in specific synaptic connections between certain types of neurons in the striatum and BF are being identified.Catecholamine receptor-mediated modulation of glutamatergic or GABAergic transmission in the striatum as well as basal forebrain (BF) has been intensively studied during these two decades. In the striatum, activation of dopamine (DA) D2 receptors in GABAergic terminals inhibits GABA release onto cholinergic interneurons by selective blockade of N-type calcium channels. In the BF, glutamatergic transmission onto cholinergic projection neurons is inhibited via DA D1-like receptors by selective blockade of P/Q-type calcium channels. On the other hand, presynaptic inhibition of the GABA release onto cholinergic neurons mediated by D1-like receptors or 5-HT Nigro-striatal dopaminergic pathway plays important roles in motor control through the interaction between dopamine (DA) and acetylcholine , whereas the inhibition remained unaffected after blocking P/Q-type Ca2+ channels by ω-Aga-IV-A -containing interneurons in the cortex  to TM.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.


Interferons (IFNs) induce anti-viral programs, regulate immune responses, and exert anti-proliferative effects. To escape anti-tumorigenic effects of IFNs, malignant cells attenuate JAK/STAT signaling and expression of IFN stimulated genes (ISGs). Such attenuation may enhance the susceptibility of tumor cells to oncolytic virotherapy. Here we studied genetic and epigenetic mechanisms of interference with JAK/STAT signaling and their contribution to susceptibility of prostate cancer cells to viral infection. Bioinformatics analysis of gene-expression in cohorts of prostate cancer patients revealed genetic and epigenetic interference with the IFN program. To correlate lack of IFN signaling and susceptibility to viral infection and oncolysis; we employed LNCaP prostate cancer cells as cellular model, and the human metapneumovirus and the epizootic hemorrhagic disease virus as infectious agents. In LNCaP cells, JAK1 is silenced by bi-allelic inactivating mutations and epigenetic silencing, which also silences ISGs. Chemical inhibition of epigenetic silencing partially restored IFN-sensitivity, induced low levels of expression of selected ISGs and attenuated, but failed to block, viral infection and oncolysis. Since viral infection was not blocked by epigenetic modifiers, and these compounds may independently-induce anti-tumor effects, we propose that epigenetic modifiers and virotherapy are compatible in treatment of prostate tumors defective in JAK1 expression and IFN signaling. Prostate cancers are the most diagnosed type of cancer and the second-highest cause of cancer-related cell deaths among men in the USA . MultiplSignals of type I IFNs (IFNα and IFNβ) are transduced via a sequence of steps, including: (i) binding of IFNs to their designated receptor , (ii) activation of receptor-associated Janus kinases (JAKs) by phosphorylation, (iii) phosphorylation of IFNAR1 and IFNAR2 intracellular tails, and creation of docking sites for latent signal transducers and activators of transcription (STATs), (iv) phosphorylation and oligomerization of STATs, in a complex which proceeds to directly bind DNA and regulate gene expression .Type I IFNs induce tumor suppressor genes , 10 and LNCaP cells were isolated from human metastatic prostate adenocarcinoma found in a lymph node  and are Paramyxoviridae family. In a recent study we have inserted a GFP expression cassette into the full-length hMPV genome, generating hMPV-GFP )hMPV-GFP . This mohMPV-GFP . As a cyhMPV-GFP . When inhMPV-GFP . NotablyhMPV-GFP , 36, posThe variability in the genetic and epigenetic etiology of prostate cancers raises the enticing prospect of personalized combination of different forms of therapy, including EpMs and virotherapy. To study the contribution of epigenetic regulation to the expression of IFN-stimulated genes (ISGs) in cells defective in IFN signaling we first explored the molecular basis of the refractoriness of LNCaP prostate cancer cells to IFN. We show that in these cells, JAK1 is silenced by both bi-allelic inactivating mutations and by epigenetic silencing. In addition, we demonstrated that the latter mechanism also plays a role in the silencing of ISGs. Furthermore, abrogation of epigenetic silencing, partially restored IFN-sensitivity, induced low levels of expression of some ISGs and attenuated, but failed to block viral infection and virally-induced cell death. Since viral infection was not blocked and EpMs may independently-induce anti-tumor effects, we propose that treatments of IFN, EpMs, and viral infection are compatible with each other in the context of JAK1 minus prostate tumor cells.−3). These data show that genetic alterations to JAK1 are present in subtypes of prostate cancer cells. To study JAK1-defective prostate tumor cells, we opted for LNCaP cells as a model system; as Rossi et al., identified two heterozygous inactivating mutations in JAK1 gene , and stained with polyclonal anti-NS3 (1:300 dilution in PBS/BSA/T). Alexa-488 or 555-conjugated goat-anti-rabbit antibodies (1:200 dilution in PBS/BSA/T) were used as secondary antibodies. To detect nuclear phosphorylated STAT1, cells were fixed  and permeabilized with ice-cold methanol (10min −20°). Cells were stained with Rabbit-anti phospho-Tyr701-STAT1 (1:100 dilution in PBS/BSA/T) for overnight at 4°C. Mounting was with Fluorescence Mounting Medium .DU145 or LNCaP were seeded  was from Beit Haemek Biological Industries, Israel (cat. #03-102-1B).2×1019001 protein coding genes were downloaded from HGNC site  to an EIn Vitro DNA Tranfection Reagent . Luciferase activity was detected with the Dual-Luciferase Reporter Assay System .DU145 and LNCaP cells were co-transfected with pISRE-Luc  and renilla luciferase plasmid  as a control. pISRE-Luc contains the firefly luciferase gene under the control of five copies of the ISRE-binding sequence, located upstream of the TATA-like promoter of the herpes simplex virus thymidine kinase. Transfections were carried out with PolyJet Data are expressed as means ± standard deviation (SD). Significant differences in mean values were assessed by 1-tailed Student's t-test. A value of p ≤ 0.05 was considered significant. All experiments were repeated at least three times.


Alternative splicing is an essential post-transcriptional process to generate multiple functional RNAs or proteins from a single transcript. Progress in RNA biology has led to a better understanding of muscle-specific RNA splicing in heart disease. The recent discovery of the muscle-specific splicing factor RNA-binding motif 20 (RBM20) not only provided great insights into the general alternative splicing mechanism but also demonstrated molecular mechanism of how this splicing factor is associated with dilated cardiomyopathy. Here, we review our current knowledge of muscle-specific splicing factors and heart disease, with an emphasis on RBM20 and its targets, RBM20-dependent alternative splicing mechanism, RBM20 disease origin in induced Pluripotent Stem Cells (iPSCs), and RBM20 mutations in dilated cardiomyopathy. In the end, we will discuss the multifunctional role of RBM20 and manipulation of RBM20 as a potential therapeutic target for heart disease. Enhancer elements mainly participate in constitutive splicing, while silencers have a dominant role in alternative splicing. Cis-regulatory elements function as enhancers or silencers according to their position and context to recruit trans-regulatory factors, which consist of RNA binding proteins (RBPs) and other splicing factors . In. In18]. and rats ,48.As discussed above, RBM20 is responsible for alternative splicing of various genes in the heart, and primarily regulates titin splicing ,19,20. TIn the middle Ig and PEVK region, the splicing patterns regulated by RBM20 have been recently studied. RBM20 acts as a splicing repressor and regulation is accomplished in a dose-dependent manner ,20. It wInterestingly, recent studies demonstrated titin splicing could be regulated by hormones such as thyroid hormone and insulin in a RBM20-dependent manner ,58. ThyrMost of the studies concerning RBM20 have been conducted with the rat model and human heart tissue. The largest obstacle to study the molecular and cellular physiology of the heart is the difficulty maintaining functional human cardiomyocytes in culture . HoweverMYLK, TEK, UNC13D), negative regulation of protein depolymerization , drug metabolism , intercalated disc , glycosaminoglycan metabolic processes  and response to amino acids . This study identified 34 genes with changes in exon usage, but the overlap between spliced and expressed genes is little. These data revealed that differential gene expression and alternative splicing could be associated with distinct signaling pathways. Significant changes have been found in exon usage of novel genes including genes associated with cardiac muscle tissue development (COL14A1), myosin complex (MYL7), ribosomal small subunit biogenesis , virion assembly , and regulation of tumor necrosis factor-mediated signaling pathway  [One recent study generated iPSC cardiomyocytes (iPSC-CMs) from a DCM patient harboring a missense mutation S635A in RBM20. The RBM20 mutant-iPSC-CMs showed the abnormal distribution of α-actinin and calcium mishandling compared to the healthy group. In addition, engineered heart muscle derived from RBM20 mutant-iPSC-CMs exhibited a decrease in both active force production and passive stress of the muscle. RBM20 mutant-iPSC-CMs showed a reduction in the expression level of the N2B titin isoform and exon skipping events in the PEVK region. Transcriptome profile revealed that RBM20 mutant-iPSC-CMs differentially expressed 161 genes (DEGs) compared to the control. The most regulated DEGs are related to cellular component as stress fiber  . Another1A, UBC) .In addition, one study showed pharmacological modulation of calcium homeostasis in RBM20 patient-derived iPSC model. They found β-adrenergic treatment in RBM20 mutant-iPSC-CMs would exacerbate calcium mishandling, apoptotic changes and sarcomere disarray. However, pretreatment with β-blockers, carvedilol, verapamil or calcium channel blocker ameliorated the pathogenic phenotype after beta-adrenergic stimulation . AltogetThe function of different muscle splicing factors is relatively well understood compared to RNA-binding proteins, whose role in normal and diseased heart have been recently demonstrated. Few cases concerning the mutations in muscle-specific factors have been related to cardiomyopathies. Until recently, only mutations in RBM20 has been linked to heart disease ,22. MutaDevelopment of RNA biology has led to the discovery of novel muscle-specific splicing factors and their function. The knowledge of the involvement of disrupted splicing factors and splice isoforms in heart disease is continuously evolving. The RBM20 associated heart disease is mainly based on the mis-splicing of pivotal cardiac genes, among which titin is recognized as a major human disease gene ,20,68. T


Helicobacter hepaticus is a member of the mouse microbiota that colonizes the lower intestine without inducing immune pathology. However, when there is a host maladaptation such as the absence of the anti-inflammatory cytokine interleukin 10 (IL-10) or its receptor IL-10R, H. hepaticus triggers aberrant IL-23-driven intestinal inflammation. This response results in major changes in the intestinal innate cell compartment, with the accumulation of inflammatory macrophages. Relying both on a bacterial trigger and on an immune defect, H. hepaticus-induced colitis in the context of IL-10/IL-10R axis deficiency shares many features of human inflammatory bowel diseases (IBD). In our study , we questioned the interactions between H. hepaticus and intestinal macrophages that promote mutualism. Our results show that H. hepaticus produces a large polysaccharide that triggers IL-10 production without a corresponding inflammatory response in macrophages. Moreover,H. hepaticus polysaccharide specifically induces an anti-inflammatory gene signature in vitro and in vivo, including transcriptional factors known as repressors of immune activation. This anti-inflammatory program depends on the TLR2/MSK/CREB pathway, which might be crucial to maintain mutualistic relationships at the intestinal interface.A high density of microbes inhabits the intestine, helping with food digestion, vitamin synthesis, xenobiotic detoxification, pathogen resistance and immune system maturation. Crucial for human health, communities of commensal bacteria (collectively termed microbiota) benefit in return from a nutrient-rich environment. Host-microbiota mutualism results from a long-term co-adaptation. At barrier surfaces, immune cells distinguish harmful from commensal bacteria and tolerate non-self organisms at close proximity to the mucosa; gut inhabitants have developed strategies to ensure beneficial conditions in their preferred niche. So far, the complex dialogue of host-microbial mutualism is poorly understood.  Strategically located at the mucosal barrier, intestinal macrophages are immune sentinels. Essential to homeostasis, they are sensors and interpreters of the intestinal microenvironment. They recognize microbial products from both commensals and pathogens through pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs). The activation of PRRs triggers the production of pro-inflammatory mediators, such as IL-6 and TNFα; but also negative feedback mechanisms and anti-inflammatory signals, such as IL-10, to prevent chronic inflammation and tissue damage. However, the signaling mechanisms employed by particular gut inhabitants to actively promote mutualism remain elusive, as is their interaction with macrophages.H. hepaticus-colonized mice, we first assessed whether colonization of Specific Pathogen Free (SPF) mice withH. hepaticus triggers IL-10 production by intestinal macrophages. Three days post-inoculation, the frequency of IL10high resident macrophages (MHCII+CD11b+CD11cint/highCD64+) was increased in the caecal lamina propria. To examine the signaling mechanisms underlying IL-10 induction, we stimulated M-CSF-differentiated bone-marrow-derived macrophages (BMDMs) and showed that H. hepaticus culture supernatant (SNHh) induces high IL-10 but low IL-6 and TNFα production compared to the whole bacteria, leading to high ratios of anti-inflammatory to inflammatory cytokines. IL-10 induction was not affected by treatment with DNase, RNase, and proteinase K followed by heat, suggesting that the active molecule in the resultant preparation (SNHht) was not a nucleic acid or a protein, but possibly a polysaccharide. Size fractionation showed that the ability to induce IL-10 in BMDMs was restricted to high molecular weight components, pointing towards a large polysaccharide. This conclusion was supported by the findings that treatment of SNHht with sodium metaperiodate, which cleaves polysaccharide chains, resulted in the loss of IL-10-inducing activity; and that the crude polysaccharide fraction isolated by cold ethanol precipitation recapitulated SNHht activity. Moreover, pre-incubation of SNHht with beads coated with the carbohydrate-binding lectin Concanavalin A depleted the IL-10-inducing activity, suggesting that the active molecule contains α-mannose and α-glucose sugars.As blockade of IL-10 signaling induces colitis inIn vivo, oral administration of SNHht increased the frequency of IL-10-producing macrophages in the caecum of SPF mice. Intraperitoneal injection of SNHht induced Il10 expression in peritoneal cells, with a lower inflammatory response compared to canonical TLR ligands such as LPS from Escherichia coli, Pam2CSK4 or Pam3CSK4.Hht triggers IL-10 production through a mechanism involving TLR2, MyD88 and ERK1/2. Unlike other TLR2 agonists that elicit a pro-inflammatory response with high IL-6 and TNFα, H. hepaticus polysaccharide preferentially induces IL-10. To assess whole genome differences in the macrophage transcriptional response to SNHht or to the canonical TLR2/1 agonist Pam3CSK4, we performed a microarray analysis of BMDMs after 3h stimulation. It revealed that Pam3CSK4 induces a large repertoire of pro-inflammatory genes associated with M1 pro-inflammatory macrophages  or involved in the recruitment, activation, and proliferation of T cells ; whereas SNHht specifically induces the transcription of genes highly expressed in tissue-resident or M2 macrophages , transcription factors repressing NF-κB  and T cell activation (Egr3), and genes involved in tissue repair . Specific targets induced by SNHht were enriched for genes  with predicted binding sites for the anti-inflammatory transcription factor CREB. Using mice with targeted disruption of the key CREB phosphorylation site , or mice with complete knock-out of MSK1/2 kinases (Msk1/2-/-) -activated by ERK1/2 and responsible for CREB phosphorylation-, we confirmed that both MSK1/2 and CREB are required for SNHht anti-inflammatory properties. SNHht also induces a CREB/MSK-dependent immunomodulatory program in intestinal tissue in vivo, raising the possibility that it contributes to mutualistic relationships in the H. hepaticus-infected gut.Experiments using BMDMs from a panel of PRR-deficient mice and pharmacological blockade of downstream kinases subsequently revealed that SNH. hepaticus polysaccharide, the genes and conditions required for its production and the strain and species specificity of its activity . Considering the high molecular weight and presence in the culture supernatant, H. hepaticus polysaccharide could be carried by Outer Membrane Vesicles (OMVs), as described for the anti-inflammatory Polysaccharide A (PSA) from Bacteroides fragilis. Electron microscopy revealed OMVs in SNHh that were not present in SNHht, probably due to the high temperature treatment. Analysis of purified OMVs is necessary to determine if the polysaccharide is a component of these structures.This work raises questions about the structure of H. hepaticus +αIL-10R model, TLR2-/- mice did not develop stronger intestinal inflammation compared to wild-type mice, possibly because the severity is already high in wild-type conditions with neutralization of IL-10. However, it is difficult to draw conclusions from this experiment as TLR2 mediates both pro- and anti-inflammatory responses. Moreover, SNHht strongly activates CREB compared to the TLR2 agonist Pam3CSK4, which suggests that SNHht signaling may result from the interaction of TLR2 with another receptor, even though we have ruled out several candidates such as C-type lectins (CLRs) and the downstream CLR-signaling protein CARD9. Further studies are required to determine the contribution of the H. hepaticus polysaccharide-driven TLR2/CREB-dependent response in macrophages to H. hepaticus infection in vivo; and address whether the CREB/MSK pathway controls intestinal inflammation in colitis models.In the . Another predominant species of the indigenous microbiota, Faecalibacterium prausnitzii from the Clostridium cluster IV, is directly associated with intestinal health and reduced in several diseases including IBD. F. prausnitzii is a strong producer of the SCFA butyrate and releases in its supernatant the protein MAM , which prevents intestinal inflammation by inhibiting NF-κb and the production of Th1/Th17 cytokines. Although Lactobacillus is a minor member of the human colonic microbiota, intestinal depletion of certain species is associated with chronic diseases. Recently, the capacity of Lactobacillus strains to metabolize tryptophan into AhR ligands was shown to attenuate intestinal inflammation in mice. Sometimes, two strains from the same species have distinct effects on host physiology depending on the expression of one virulence or tolerogenic factor. For example, B. fragilis can be highly pathogenic in mice and humans due to the production of a toxin, whereas the PSA-expressing strain induces an immunomodulatory response in murine models.Drawing the line between commensals and pathogens is difficult, but health-promoting properties of certain gut inhabitants are well-documented. By degrading indigestible fibers, anaerobic species produce Short Chain Fatty Acids (SCFAs), which exert multiple beneficial effects on cellular immunity and metabolism. Bifidobacteria are primary degraders of diet- and host-derived sugars, providing SCFAs for the host and nutrients for other community members. They also interact directly with immune cells and modulate both innate and adaptive immune processes, such as IL-10 production and T cell induction. Several immunomodulatory molecules have been identified in Bifidobacteria, including the extracellular serpin that inactivates human pro-inflammatory proteases and anti-inflammatory exopolysaccharidesH. hepaticus releases a polysaccharide inducing a specific CREB-dependent anti-inflammatory program in the intestine, which might be essential to tolerize the niche and maintain intestinal homeostasis. Helicobacter pylori, a frequent colonizer of the human gastric mucosa, can cause severe inflammation and gastric tumorigenesis, but also protects against esophageal cancer and chronic inflammatory disorders. It has evolved to skew the adaptive immune response toward immune tolerance rather than immunity, which promotes persistent infection but also protects against allergic and auto-immune diseases. For instance, due to structural modifications, H. pylori flagellin and LPS are poor activators of TLR5 and TLR4 receptors respectively; and its TLR2 ligands exhibit predominantly anti-inflammatory properties. The intimate contact with H. pylori generates a limited but constant inflammation that can become deleterious to the host, depending on strain-specific virulence determinants, host genetics, environment and surrounding microbial communities .Between the extremes of "friend" and "foe" categories, a variety of opportunistic pathogens and pathobionts thrive in the intestinal niche by escaping immune defenses. The microbes residing in the upper mucus layer stay at a safe distance from the epithelium, but the persistent colonizers living in close contact with the mucosa have to encourage immune tolerance to avoid chronic activation and deleterious tissue damage. As described above,The intestinal microbiota contains an inherent capacity to trigger immunomodulatory responses that are essential to maintain health. Characterising the diversity of factors that promote mutualism and their modes of action at the cellular and molecular level is crucial to elucidate host-microbiota crosstalk. Modulation of immunological processes in a microbe-dependent way offers the prospect of innovative strategies to treat and prevent chronic intestinal inflammation.


Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, play an important role in cellular communication during skeletal growth and homeostasis. Bioactive molecules carried by EVs are transported to neighboring and distant cells to trigger a series of signaling cascades influencing bone homeostasis. The bioactive activities of osteoclast-derived EVs include regulation of osteoclastogenesis and osteoclast–osteoblast communication. As osteoclast-derived EVs have the potential to regulate osteoclasts and osteoblasts, their application in osteoporosis and other bone metabolic disorders is currently under investigation. However, very few reviews of osteoclast-derived EVs in bone remodeling regulation have yet been published. This article aims to review recent advances in this field, summarizing a new regulator of osteoclastogenesis and osteoclast–osteoblast communication mediated by osteoclast-derived EVs. We will analyze the major challenges in the field and potential for the therapeutic application of EVs. Bone is a dynamic, living tissue continually undergoing modeling and remodeling. Many factors, including hormones, growth factors, physical activity, and drugs, can influence bone homeostasis . Bone reIntercellular communication between osteoblasts and osteoclasts plays an important role in the regulation of bone homeostasis , as mainSome factors, such as M-CSF, RANKL, FAS ligand, complement component 3a, and semaphorin produced by osteoblasts have been demonstrated to influence osteoclastic bone resorption. Besides the well-documented regulatory mechanism of osteoblast–osteoclast communication involved in bone resorption, it is now being challenged by several studies supporting the fact that osteoclasts in turn regulate osteoblastic bone formation either by direct cell–cell contact or indirectly via cytokines, however, precisely how osteoclasts regulate osteoblasts is unclear. Accumulating evidence indicates that through either direct cell–cell communication or indirect cytokine-mediated communication, osteoclasts repress osteoblastic bone formation . So far,Interestingly, communication between osteoclasts and osteoblasts may occur via small membrane-enclosed vesicular particles termed EVs which can fuse with the nearby cell membranes within circulatory pathways . The varFigure 1), but not the markers  which are common contaminants of EV. Apoptotic bodies are usually larger than exosomes (50–5000 nm in diameter) and are generated by blebbing of the surface of apoptotic cells, resulting in vesicles composed of plasma membranes and organelle with nuclear and cytoplasmic contents . The apoptosis of osteocyte has been regarded a chemotactic signal to osteoclastic bone resorption. A recent study identified that apoptotic osteocytes are engulfed by osteoclasts during bone resorption, where osteoclasts exhibited TUNEL positive apoptotic bodies share surface markers of the bone matrix, and contain osteoclast vacuoles and osteocytic lacunae.So far, two types of osteoclast-derived EVs, exosomes and apoptotic bodies, have been described . Exosomein vitro. In this study, exosome-like EVs from osteoclasts or their precursors were isolated using ExoQuickTM exosome isolation method. The diameter of exosome-like EVs was visualized by transmission electron microscopy. The marker of exosome-like EVs is detectable by Western blot  . EVs canern blot . Immunoeern blot . In addiern blot .In addition to characteristic proteins, another feature of osteoclast-derived EVs is presence of nucleic acids (such as miRNAs) . miRNAs Figure 2). Furthermore, the depletion of RANK-enriched osteoclast-derived EVs inhibits formation of osteoclast-like cells in 1,25(OH)2D3-treated bone marrow cultures. These data imply that the inhibitory effect of RANK-rich EVs could be applied in bone disease or injury treatment. However, currently we do not know the regulatory activity of RANK-rich EVs from resorbing osteoclasts, which will be of interest for future investigation.The activity of osteoblast-derived EVs on bone remodeling has been well documented , howeverThe maintenance of bone homeostasis relies heavily on cellular communication between osteoblasts and osteoclasts through the RANKL–RANK interaction . In addiin vitro osteoblast/osteoclast co-culture, expression of all the osteoblastic activity-related genes was significantly decreased in OC-miR-214-3p mice than wild type mice. Overexpression of miR-214-3p expression in osteoclasts inhibited osteoblast activity in this co-culture system. These findings suggest that exosomal miR-214-3p could be transferred from osteoclasts to osteoblasts to inhibit osteoblast activity in vitro. In a series of tracking experiments, CMV-GFP-CD63 stained osteoclast-derived exosomes were found in co-cultured osteoblasts, indicating that the extracellular osteoclast-derived exosomes effectively reached osteoblasts. In vivo injection of purified PKH67 exosomes isolated from the supernatant of OC-miR-214-3p osteoclast dramatically decreased bone formation. In contrast, inhibition of osteoclastic-specific miR-214-3P by osteoclast-targeted antagomiR-214-3p treatment significantly enhanced bone formation . Although osteoclast-derived exosomes may attach to the membrane of the objective cell to induce intracellular signaling through corresponding receptors, it is recognized as well that osteoclast-derived microvesicles and even osteoclast-derived apoptotic bodies may have largely analogous physiological and pathological roles in intercellular communication. While osteoclast-derived exosomes could be endocytosed via micropinocytosis, phagocytosis, or receptor-/raft-mediated endocytosis, to merge with the membrane of endocytic compartment to emancipate their content into the cytoplasm of the target cells, however, it need to further test in vitro or in vivo, in addition, the molecular cargo within the osteoclast-derived EVs responsible for this effect is unidentified.Moreover, compared with wild-type mice, osteoclastic-specific miR-214-3P knock-in (OC-miR-214-3p) mice has lower bone mass and poorly organized trabecular architecture at distal femur. Histomorphometric analysis of the age-related changes and Masson’s trichrome staining also showed less bone formation in OC-miR-214-3p mice than WT mice. In ormation . These rIn summary, bone remodeling is influenced by several hormonal factors. Osteoclast-derived EVs contain multiple key bone-regulatory proteins which can modulate osteoblastic bone formation. This regulative process may reveal a novel mechanism of osteoclast–osteoblast communication. Interestingly, more and more molecules have been isolated from bone-derived EVs, and have been found to influence a number of biological processes through cell–cell communication or via interacting with the extracellular matrix . The mesF-LY, Z-NM, Q-yW, and R-SX conceived the project and designed the experiments. XL and J-XY collected and analyzed the data. All authors developed analytical tools and wrote, edited, and approved the final submission of 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.


Emerging evidence suggests that dipeptidyl peptidase-4 (DPP-4) inhibitors used to treat type 2 diabetes may have nephroprotective effects beyond the reduced renal risk conferred by glycemic control. DPP-4 is a ubiquitous protein with exopeptidase activity that exists in cell membrane-bound and soluble forms. The kidneys contain the highest levels of DPP-4, which is increased in diabetic nephropathy. DPP-4 inhibitors are a chemically heterogeneous class of drugs with important pharmacological differences. Of the globally marketed DPP-4 inhibitors, linagliptin is of particular interest for diabetic nephropathy as it is the only compound that is not predominantly excreted in the urine. Linagliptin is also the most potent DPP-4 inhibitor, has the highest affinity for this protein, and has the largest volume of distribution; these properties allow linagliptin to penetrate kidney tissue and tightly bind resident DPP-4. In animal models of kidney disease, linagliptin elicited multiple renoprotective effects, including reducing albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis, independent of changes in glucagon-like peptide-1 (GLP-1) and glucose levels. At the molecular level, linagliptin prevented the pro-fibrotic endothelial-to-mesenchymal transition by disrupting the interaction between membrane-bound DPP-4 and integrin β1 that enhances signaling by transforming growth factor-β1 and vascular endothelial growth factor receptor-1. Linagliptin also increased stromal cell derived factor-1 levels, ameliorated endothelial dysfunction, and displayed unique antioxidant effects. Although the nephroprotective effects of linagliptin are yet to be translated to the clinical setting, the ongoing Cardiovascular and Renal Microvascular Outcome Study with Linagliptin in Patients with Type 2 Diabetes Mellitus (CARMELINA®) study will definitively assess the renal effects of this DPP-4 inhibitor. CARMELINA® is the only clinical trial of a DPP-4 inhibitor powered to evaluate kidney outcomes. The global burden of diabetes is escalating at an alarming rate, with an estimated 425 million people worldwide afflicted with the disease in 2017 , making Intensive glycemic control has been shown to reduce the risk of kidney disease and other microvascular complications of type 2 diabetes in large clinical outcomes studies such as the UK Prospective Diabetes Study (UKPDS), the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, and the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study . HoweverDipeptidyl peptidase-4 (DPP-4) inhibitors are also one of the newer types of oral glucose-lowering drug licensed for type 2 diabetes, with the first-in-class sitagliptin having been approved in 2006 in the United States. DPP-4 inhibitors, also known as gliptins, are orally administered medications that have moderate glycemic efficacy and carry low risk for hypoglycemia or weight gain . EmerginAmongst the globally marketed DPP-4 inhibitors, linagliptin is of particular interest with respect to pleiotropic renoprotective effects as it is the only such drug to be excreted predominantly by non-renal pathways ; hence iThis review describes the accumulating body of evidence suggesting that linagliptin may have protective effects against diabetic nephropathy, with a focus on preclinical studies and putative molecular mechanisms.DPP-4, also known as CD26 and glycoprotein gp110, is a 110-kDa glycoprotein with serine exopeptidase activity (Enzyme Commission number 3.4.14.5) . The catIn addition to the transmembrane form, DPP-4 also exists as a slightly smaller, soluble form (727 residues) that lacks the intracellular and transmembrane regions of the membrane-bound form but retains the extracellular portion, including the catalytic domain . MembranDPP-4 was discovered over 50 years ago , but itsin vitro assays, but few of these have been established as bona fide physiological substrates ..miR-29 fUsing the same STZ-induced CD-1 murine model of type 1 diabetes, the author and colleagues subsequently identified a new profibrotic molecular mechanism comprising an interaction between DPP-4 and integrin β1 in endothelial cells . This inCrucially, the same recent study that identified the interaction between integrin β1 and DPP-4 that induces EndMT also showed that this interaction is disrupted by linagliptin . In thismiR-29 binding of the 3′-UTR during normoglycemia is lost when miR-29 levels decrease in hyperglycemic conditions. Subsequently, the newly abundant membrane-bound DPP-4 forms a complex with integrin β1 , resultnditions . Importanditions . The reaLinagliptin has also been shown to reduce TGF-β signaling in human kidney proximal tubular epithelial (HK-2) cells in high glucose concentrations by a different mechanism . The catIn light of these findings, it is notable that linagliptin treatment significantly reduced serum levels of TGF-β1 in a double-blind, randomized, placebo-controlled, mechanistic study in non-diabetic, hypertensive patients .In addition to its antifibrotic properties described above, linagliptin may protect the kidneys via several other pathways, including effects on AGE-RAGE signaling, oxidative stress, inflammation, endothelial nitric oxide activity, and increased levels of the DPP-4 substrates SDF-1 and GLP-1. The studies underpinning these hypotheses are discussed as follows.Diabetes is associated with increased levels of AGEs, i.e. proteins or lipids that are glycated by the non-enzymatic Maillard reaction as a consequence of their exposure to glucose and other saccharides. AGEs contribute to development of the macro- and microvascular complications of diabetes, including chronic kidney disease − both by cross-linking to molecules in the extracellular matrix basement membrane and by binding the RAGE receptor . ActivatLinagliptin has antioxidant properties not shared by other DPP-4 inhibitors . This main vivo model of septic shock, linagliptin ameliorated vascular dysfunction as well as reducing oxidative stress and inflammation . No. No20]. in vivo study of the tissue distribution of linagliptin in wild-type and DPP-4-deficient rats using whole-body autoradiography and measurement of tissue radioactivity following administration of radiolabeled compound, the highest drug concentrations were located in the kidneys and liver . W. WIn vitnhibitor . Additio0−5 s−1) .in vivo pleiotropic effects as linagliptin. Several studies have explored this possibility. In an ex vivo study in Zucker diabetic fatty (ZDF) rats, linagliptin conferred greater vascular protection than sitagliptin despite similar effects on blood glucose levels [in vitro study, linagliptin but not sitagliptin suppressed DPP-4 enzymatic activity and protein levels in TGF-β2-treated cultured human dermal microvascular endothelial cells [miR-29a–c and VEGFR levels, suppressed TGF-β2-induced increases in integrin β1 levels, and decreased DPP-4 dimerization [ex vivo study, linagliptin elicited more sustained inhibition of in situ DPP-4 activity than sitagliptin in the proximal tubule and glomerulus of normal male Wistar rats administered single oral doses of these drugs [These pharmacological differences suggest that other DPP-4 inhibitors may not necessarily have the same e levels . In thise levels . In an ial cells . Furtherrization . In a cose drugs . Furtherse drugs .Despite the many studies described here showing renoprotective effects of linagliptin and other DPP-4 inhibitors in animal models of diabetic nephropathy and non-diabetic kidney disease, as yet there is little clinical data to support the hypothesis that these drugs have pleiotropic renal benefits. Although this may simply reflect the paucity of clinical studies designed specifically to evaluate renal outcomes with DPP-4 inhibitors, some relevant findings have been reported.post hoc analysis of the SAVOR-TIMI 53 cardiovascular safety study found clinically meaningful reductions in albuminuria with saxagliptin treatment but no concomitant improvements in either eGFR or hard renal outcomes such as initiation of dialysis or renal transplant. The albuminuria-lowering effect of saxagliptin was not associated with its effect on glycemic control [A  control . In the  control . While t control . However control . Interes control .As have other DPP-4 inhibitors, linagliptin has demonstrated glucose-lowering efficacy and tolerability in type 2 diabetes patients with kidney disease ,148. LinA mechanistic, parallel-group, randomized clinical study in 62 patients with early type 2 diabetes suggested that 4 weeks of treatment with linagliptin prevented impairment of renal endothelial function, as measured by changes in basal renal endothelial nitric oxide activity . The gloThe renoprotective effects of improving glycemic control are well established, and linagliptin and other DPP-4 inhibitors are therefore anticipated to provide such benefits via their glucose-lowering properties alone. Additionally, linagliptin has demonstrated pleiotropic renoprotective properties in diabetic and non-diabetic animal models of nephropathy – notably, antifibrotic effects mediated via interaction with miR and integrins. The antioxidant properties of linagliptin also seem likely to play a potentially unique renoprotective role. Additional effects of linagliptin in disrupting AGE-RAGE signaling, increasing levels of peptides such as GLP-1 and SDF-1, ameliorating endothelial dysfunction, and reducing inflammation are also likely to be important for renoprotection. Looking to the future, technologies such as metabolomics  and peptDespite a common mechanism of action, DPP-4 inhibitors comprise a heterogeneous class of molecules with clinically relevant differences in pharmacology. Unlike other members of this drug class, linagliptin is non-renally excreted but capable of penetrating the various compartments of the kidney. Thus, based on its pharmacology and animal studies to date, linagliptin appears to offer the greatest potential for renoprotection.As with other incretin therapies, the putative renoprotective properties of linagliptin have yet to be demonstrated convincingly in clinical trials; however, this may simply reflect the scarcity of studies designed to specifically measure renal outcomes. For this reason, there is much interest in the ongoing CARMELINA® study. This large event-driven, placebo-controlled clinical trial is the first study designed and adequately powered to robustly evaluate renal outcomes of treatment with a DPP-4 inhibitor. Almost 7000 type 2 diabetes patients with high cardiorenal risk have been enrolled in CARMELINA®  and resu


MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.Transcriptional dysregulation of the  MYC oncogene plays an important role in enhancer-promoter looping with tumor-specific super-enhancers. Perturbation of this site provides a potential therapeutic vulnerability.Schuijers et al. show that a conserved CTCF site at the promoter of the  MYC gene, which encodes c-MYC, is a hallmark of cancer initiation and maintenance. Dysregulation of MYC is often achieved through the formation of large tumor-specific super-enhancers in the region surrounding the MYC gene  bind enhancer elements and regulate transcription from the promoters of nearby or distant genes through physical contacts that involve looping of DNA between enhancers and promoters . The mecRecent studies have revealed that specific chromosome structures play important roles in gene control. Enhancer-promoter interactions generally occur within larger chromosomal loop structures formed by the interaction of CTCF proteins bound to each of the loop anchors . These lCTCF does not generally occupy enhancer and promoter elements . AnotherMYC locus in multiple cancers and identify a CTCF-occupied site at the MYC promoter that facilitates docking with essentially any enhancers that are formed within the 2.8 Mb MYC locus. The CTCF-occupied site at the MYC promoter, which we call the MYC enhancer-docking site, can be abrogated by genetic and epigenetic editing. Similar enhancer-docking sites occur at other oncogenes. This suggests a mechanism by which tumor cells can generally hijack enhancers and, with editing, a potential therapeutic vulnerability.Here, we investigate DNA looping structures in the MYC locus /Cas9  promoter /Cas9 . Attemptpromoter . This coCF motif  and S2B CF motif . RNA anall types . Furtherll types . These rMYC enhancer-docking site contributes to looping interactions with distal enhancers, then the loss of this site should cause a decrease in DNA interactions between the MYC promoter and the distal super-enhancers. We used chromosome conformation capture combined with high-throughput sequencing (4C-seq) to compare super-enhancer interactions in K562 and HCT-116 cells with normal or perturbed CTCF binding motifs. The 4C-seq data in K562 cells indicated that the MYC enhancer-docking site interacts predominantly with distal super-enhancers located ~0.3 Mb and ~2 Mb downstream of the MYC gene and that the majority of these interactions were significantly reduced when the putative CTCF motif was perturbed  tumor cells . This change was incorporated because previous work has noted that in situ ligation dramatically decreases the rate of chimeric ligations and background interactions  by PCR. Cas9 was removed from pX330-Cas9 (Addgene 42230) and replaced by DNMT3A-3L . Guide RChIP-seq datasets were generated for this study as well as collated from previous studies  and were4C-seq reads were trimmed and mapped using bowtie with options -k 1 -m 1 against the hg19 genome assembly. We only used the reads from non-blind fragments for further analysis. The normalized profile of each sample was smoothened using a 6-kb running mean at 500-bp steps across the genome. Quantification of the 4C signal counted the reads per fragment per million sequenced reads in the super-enhancers or the CTCF MACS peak calls.https://github.com/younglab/origami using version alpha20160828. The ChIA-PET datasets analyzed along with their corresponding linker sequence and called interactions in and around the MYC TAD can be found in We developed a new software pipeline and analytical method called origami to process HiChIP and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). The software and releases can be found at 12345678


Interestingly, and somewhat counter-intuitively, anti-angiogenic therapy can promote CD8+ T-cell infiltration of tumours and increase the efficacy of adoptive CD8+ T-cell therapy. Rather than inhibit tumour angiogenesis, anti-angiogenic therapy ‘normalizes’ (matures) tumour blood vessels by promoting pericyte recruitment, increasing tumour blood vessel perfusion and sensitizing tumour blood vessels to inflammatory stimuli. A number of different approaches are currently being explored to increase recruitment by manipulating the expression of homing-associated molecules on T-cells and tumour blood vessels. Future studies should address whether these approaches improve the efficacy of adoptive T-cell therapies for solid, vascularized cancers in patients.The success of adoptive T-cell therapies for the treatment of cancer patients depends on transferred T-lymphocytes finding and infiltrating cancerous tissues. For intravenously transferred T-cells, this means leaving the bloodstream (extravasation) from tumour blood vessels. In inflamed tissues, a key event in extravasation is the capture, rolling and arrest of T-cells inside blood vessels which precedes transmigration across the vessel wall and entry into tissues. This depends on co-ordinated signalling of selectins, integrins and chemokine receptors on T-cells by their respective ligands which are up-regulated on inflamed blood vessels. Clinical data and experimental studies in mice suggest that tumour blood vessels are anergic to inflammatory stimuli and the recruitment of cytotoxic CD8 Retrospective studies of resected primary tumours from colorectal cancer patients were the first to show that the presence and location of tumour-infiltrating T-lymphocytes are prognostic for patient outcome . Specifiin vitro yet the same cells fail to eradicate cancer in the patient due to tumour-induced immunosuppression. Early attempts to overcome immunosuppression included isolating tumour infiltrating lymphocytes (TILs) from resected melanoma lesions, expanding tumour-reactive T-cells and infusing large numbers back to patients with progressive metastatic melanoma [gm) to overcome dominant immunosuppression in the cancer patient [Tumour-specific T-cells isolated from cancer patients recognize and kill tumours melanoma . These g patient . The rec patient –9. Howevgm) injected into the bloodstream are recruited into cancerous tissues to bring about cancer cell killing  surface of blood vessel endothelial cells (EC) that directs entry into tissues. Homing addresses for lymph nodes and sites of inflammation in the skin and other organs have been identified . The congm T-cells. For example, transduction of CAR T-cells with chemokine receptors for human tumour cell-derived chemokines increases homing to solid cancers and controls their growth in immunodeficient mice [Some of the adhesion molecules and chemokines that regulate T-lymphocyte homing into sites of inflammation and/or lymph nodes have been implicated in homing to cancers in experimental mice –25. Knowent mice ,27.+ T-lymphocytes do not spontaneously roll or undergo activation induced arrest in tumour blood vessels even following LPS administration [+ T-cell flux through tumour blood vessels, absence of shear stresses necessary for recruitment and/or inefficient chemokine presentation could all contribute to poor CTL recruitment. Additional mechanisms include tumour-derived products which restrict expression of homing molecules, such as ICAM-1, on tumour EC [Early studies of leucocyte (neutrophil) behaviour inside blood vessels of subcutaneous tumours in immunodeficient mice demonstrated lower levels of rolling and arrest than in normal skin, even after administration of tumour necrosis factor-α (TNF-α) or LPS, which suggested that tumour blood vessels are anergic to inflammatory stimuli ,29. Subsstration . The facstration ,30. A nuumour EC  and induumour EC . EC anerumour EC .gm T-cell therapy for solid tumours is cell dose dependent [gm T-cells recognizing relevant tumour antigens will be required before applying the rules governing tumouricidal T-cell homing in transplanted syngeneic or xenogeneic tumours to human cancers.Adoptive T-cell therapy does restrict the growth of solid vascularized cancers in experimental mice and in patients so, if tumour blood vessels are poor at recruiting injected T-cells, how does it work? Intravital imaging has demonstrated that tumour specific T-cells are recruited, not from blood vessels within the tumour nest but, from blood vessels in the peritumoural area where homing-associated adhesion molecules are preferentially expressed  Figure . Recruitependent ,38 so un111indium-labelled TILs homed to melanoma lesions and TIL homing was associated with objective clinical responses in 38% (10/26) patients [+ T-cells traffic indiscriminately to lymph nodes, liver, spleen and lungs in tumour-bearing mice while still being able to control tumour growth [There have been very few clinical studies of T-cells following injection into the venous circulation of cancer patients. However, an early study showed that patients . In a phpatients . These rpatients , which, patients . In exper growth . This figm may, however, contribute to their lack of persistence which has been repeatedly reported in patients and experimental mouse models. This may be due to lack of encounter with cognate antigen following homing to non-cancerous tissues and/or lack of homing to lymph nodes where survival and/or reactivation signals are presented. Inclusion of CD28 or other co-stimulator signalling domains in so-called second and third generation CARs prolongs the persistence of CAR T-cells but whether this is related to lymph node homing remains to be determined. Checkpoint blockade inhibitors and host-conditioning using fludarabine and cyclophosphamide increase the efficacy of TILs, CAR and TCRgm T-cells but whether this is related to altered homing is also not known [Indiscriminate homing of CAR T-cells, TILs and TCRot known –45. Howeot known .+ regulatory T-cells (Tregs) [It is over 40 years since Judah Folkman first proposed that tumour growth could be restricted by blocking the growth of blood vessels that supply the tumour . Since t (Tregs) . The use (Tregs) . Vascula (Tregs) ,51. Treg (Tregs)  but a ra (Tregs) ,54, eosi (Tregs) , irradia (Tregs)  and TLR  (Tregs) , in the  (Tregs) ,32. Ther+ T- and/or NK cell secretion of LTα3 and IFN-γ which induce expression of peripheral node addressin  and the arrest chemokine CCL21 respectively. Although comprising <10% of the tumour vascular network, newly formed HEV in mice recruit adoptively transferred naïve T-cells directly from the bloodstream into the tumour where cross-presenting APC activate naïve T-cells to kill tumour tissue [gm T-cells which are predicted to have superior efficacy in adoptive cell therapies.An exquisite example of tumour blood vessel maturation is the development (neogensis) of specialized HEV blood vessels. Studies in mice suggest that, similar to tumour blood vessel normalization, HEV neogenesis is regulated by type 1 inflammation  and can r tissue . The devr tissue . Interesr tissue , as oppor tissue  which suAlthough the promise of CAR T-cell therapy for the treatment of solid cancers has yet to be realized, it is clear that adoptive immunotherapy using T-cells expressing conventional TCRs does not work for all patients. Inefficient recruitment by immature tumour blood vessels and lack of persistence of injected T-cells are important determinants of successful outcomes in experimental mice. Manipulating homing addresses on T-cells and vascular addresses on tumour blood vessels to facilitate the recruitment of tumour-specific T-cells offer a combined approach to overcome these limitations in cancer patients. However, longitudinal imaging of transferred T-cells using non-invasive imaging using PET/SPECT tracers will be required to determine whether homing and persistence of transferred T-cells correlate with therapeutic outcomes.


This deviation was slightly increased when HLA-C or HLA-DQ was omitted from the input and was substantially decreased when using two-field resolution HLA genotype data of the recipient and serological split level HLA typing of the donor. Thus, our data suggest that our computational approach is a powerful tool to estimate PIRCHE-II/eplet values when high-resolution HLA genotype data is not available.Epitope-based HLA matching has been emerged over the last few years as an improved method for HLA matching in solid organ transplantation. The epitope-based matching concept has been incorporated in both the PIRCHE-II and the HLAMatchmaker algorithm to find the most suitable donor for a recipient. For these algorithms, high-resolution HLA genotype data of both donor and recipient is required. Since high-resolution HLA genotype data is often not available, we developed a computational method which allows epitope-based HLA matching from serological split level HLA typing relying on HLA haplotype frequencies. To validate this method, we simulated a donor-recipient population for which PIRCHE-II and eplet values were calculated when using both high-resolution HLA genotype data and serological split level HLA typing. The majority of the serological split level HLA-determined Alloimmunity due to Human Leukocyte Antigens (HLA) mismatches between donor and recipient significantly impairs graft survival after solid organ transplantation –3. The rAlthough the number of HLA mismatches is a potent predictor of transplant outcome, not every HLA mismatch will have an equal effect on graft failure , 7. CumuTwo in silico methods, HLAMatchmaker and PIRCHE-II, have incorporated the epitope-based HLA matching concept in their algorithm to find the most suitable donor for a recipient. HLAMatchmaker determines differences in B-cell epitopes between donor and recipient to estimate the risk of graft failure –15. ThesTo be able to identify the unique set of donor and recipient HLA epitopes, identification of the exact polymorphisms in donor and recipient HLA is required. Low-resolution HLA typing is, however, not sufficient to identify these polymorphisms, as low-resolution HLA typing can cover numerous HLA alleles at high-resolution HLA level. Thus, low-resolution HLA typing will lead to an ambiguous epitope definition. Therefore, two-field resolution HLA genotype data of both donor and recipient is preferably required to unambiguously determine the HLA compatibility between donor and recipient at epitope level. Indeed, both HLAMatchmaker and the PIRCHE-II algorithm require high-resolution HLA genotypes of both donor and recipient as input for their algorithm. Although HLA genotyping methodologies have improved over the last few years and high throughput NGS technology became available, the quick availability of reliable high-resolution HLA genotype data remains challenging. High-resolution HLA genotyping is especially for deceased donors hardly feasible, as time is a major limiting factor for deceased organ transplantation.Instead of high-resolution HLA genotyping of donor and recipients, also alternative approaches can be used to facilitate epitope-based HLA matching. In the present study we describe a computational method to perform epitope-based HLA matching using serological split level HLA typing as input. In this computational method, the most likely high-resolution HLA genotypes that correspond to a serological split level HLA typing are identified using HLA haplotype frequency tables. For all of these high-resolution HLA genotypes, a PIRCHE-II and eplet value can be calculated, which can subsequently be weighed against the normalized frequency of the pair of HLA haplotypes in the general population. To test whether the risk estimation alters when using our computational approach, we calculated the PIRCHE-II/eplet values when using serological split level HLA typing  and compared these values with the PIRCHE-II and eplet values when using high-resolution HLA genotype data .n = 4,579) that were performed at the University Medical Center Utrecht between January 2009 until July 2016 were extracted from the lab system. HLA genotypings were performed at different resolution levels and for different loci. High-resolution HLA genotypings were performed by SBT  or NGS  whereas PCR-SSO (One Lambda) was used for lower-resolution HLA genotyping. HLA genotypes that did not have a fully unambiguous high-resolution HLA typing for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 were excluded from the population. A total of 2,373 typings had an unambiguous high-resolution HLA genotype data for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1. These typings were used to simulate a representative recipient population.To model a representative recipient population, all HLA genotypings  and two-field resolution HLA genotypes (reference group) of these donor-recipient couples were used to calculate the number of PIRCHE-II and eplets, as described below. Although donor-recipient couples were formed based on HLA-A, HLA-B, and HLA-DR only, the complete five loci-haplotypes  were used to calculate the PIRCHE-II and eplet values. PIRCHE-II and eplet values were also calculated when HLA-C or HLA-DQ was removed from the serological split level HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ typing or when using two-field resolution HLA genotype data of the recipient and serological split level HLA typing of the donor as input for the algorithms.For the serological split level HLA typings of the 2,373 donor-recipient couples, a high-resolution extrapolation method was used to identify all possible high-resolution HLA genotypes that correspond to each serological split level HLA typing. To this end, HLA haplotype frequency tables from the National Marrow Donor Program from 2007 and 2011 were both used separately to identify all potential high-resolution HLA genotypes from a serological split level HLA typing , 23.https://doi.org/10.1155/2017/9130879 shows an example of the linkage breakdown between HLA loci.For every given serologic split level HLA typing, the extrapolation algorithm started with setting up all potential two-field resolution HLA haplotype pairs  that yield the given input serological split level HLA typing. After mapping antigen/allele names for each serologic value, the haplotype frequency table was filtered for matching haplotypes. When no matching high-resolution HLA haplotype was found for a given serological split level HLA typing or subsets thereof, the selection criteria were broadened by removing the link between loci in a step-wise manner. The following order of HLA loci linkage removal was used: [i] A-B-C-DRB1-DQB1, [ii] A-B-C | DRB1-DQB1, [iii] A | B-C | DRB1-DQB1, [iv] A | B-C | DRB1 | DQB1, and [v] A | B | C | DRB1 | DQB1. It has to be noted that mode [v] is equivalent to using allele frequencies for the prediction of each individual locus. Supplementary material 1 in Supplementary Material available online at Since multiple high-resolution HLA genotypes may correspond to a single serological split level HLA typing, a list of potential high-resolution HLA genotypes was generated for both donor and recipient. For each typing, a frequency was calculated by multiplying both haplotypes' frequencies. The resulting absolute frequencies were normalized within the set of likely high-resolution HLA genotypes for the given input.This method was applied to both donors and recipients. For all these potential high-resolution HLA genotypes of donor and recipient, the PIRCHE-II and eplets values were calculated as described below. The obtained PIRCHE-II values were subsequently weighted by multiplying the normalized frequency of a certain high-resolution HLA genotype of the recipient with the normalized frequency of a certain high-resolution HLA genotype of the donor. Finally, all weighted PIRCHE-II values were summed up. The same was applied to the eplet values. Thus, the used method takes all potential high-resolution HLA genotypes that are present in the HLA haplotype frequency table into account which corresponds to a certain serological split level HLA typing. By using all these genotypes and by weighing the epitope values, multiple imputation is used to minimize bias towards common HLA genotypes.In this study, the HLA haplotype frequency tables of the Caucasian population (2007) and the European-Caucasian population (2011) were used in this study to determine all potential high-resolution HLA genotypes.50 < 1000 nM were considered as relevant HLA-DRB1 binders. These relevant HLA-DRB1 binders were only classified as a PIRCHE-II when the amino acid residues of the nonameric binding cores were not present in the amino acid sequence of recipient HLA. The PIRCHE algorithm is available via https://www.pirche.org/.For both the two-field resolution HLA genotypes (designated as reference group) and the serological split level HLA typings (designated as observation group), the number of PIRCHE-II was determined for each donor-recipient couple as described previously . Brieflyhttp://www.epitopes.net/). Mismatched eplets were defined as eplets that were present in donor HLA but absent in recipient HLA. Via interlocus HLA comparisons the number of mismatched eplets was determined.For both the two-field resolution HLA genotypes and the serological split level HLA typing, the number of mismatched eplets was determined for each donor-recipient couple using eplet definitions as provided in HLAMatchmaker algorithm version 2.1  when using the HLA haplotype frequency tables of 2007 or for 2,369 donor-recipient couples (99.8%) when using the HLA haplotype frequency tables of 2011. ln(PIRCHE-II) or ln(eplet) difference and thus a significant over- or underestimation of the hazard in graft failure. For all tested settings, we calculated the delta that corresponded to the 50% percentiles, 75% percentiles, 95% percentiles, 99% percentiles, and 99.9% percentiles to test the reliability of our estimations. For example, for the 75% percentile we calculated to which delta 25% of the values above the median stretched and to which delta 25% of the values below the median stretched. A similar approach was used for the other percentiles.The number of epitopes and alloreactivity are unlikely correlated in a linear fashion; increases from 1 epitope to 10 epitopes are likely to have a higher impact than increases from 200 epitopes to 210 epitopes and the effects are likely to plateau at some point. As such we assume an inverse logarithmic effect of epitope numbers on alloreactivity and we converted the PIRCHE-II and eplet numbers into the natural logarithm thereof. These transformed PIRCHE-II and eplet values were subsequently used to identify differences between the two-field HLA genotype-determined PIRCHE-II/eplet values, designated as “reference PIRCHE-II/eplet values,” and the serological split level HLA typing-determined PIRCHE-II/eplet values, designated as “observed PIRCHE-II/eplet values.” The delta between the observed and reference PIRCHE-II/eplet values was calculated by subtracting the log-transformed reference PIRCHE-II/eplet values from the log-transformed observed PIRCHE-II/eplet values. A delta of >+1 or <−1 was considered a high deviation, as it represents a single unit ofFor all donor-recipient couples we calculated the PIRCHE-II and eplet values when using two-field HLA genotype data and serological split HLA typing as input for the module. The PIRCHE-II/eplet values determined based on two-field resolution HLA genotypes were designated as “reference PIRCHE-II/eplet values,” whereas the PIRCHE-II/eplet values determined based on serological split HLA typing were designated as “observed PIRCHE-II/eplet values.” ln(PIRCHE-II) and ln(eplet) values and the reference ln(PIRCHE-II) and ln(eplet) values was calculated ( ln(PIRCHE-II) values did not show deviation from the reference ln(PIRCHE-II) values ( ln(PIRCHE-II) values deviated maximal 0.1 from the reference ln(PIRCHE-II) values. When using the frequency tables of 2007, 28.3% of observed ln(eplet) values did not deviate from the reference ln(eplet) values and 87.7% deviated maximal 0.1 ( ln(PIRCHE-II) and ln(eplet) values do not deviate or only slightly deviate from reference ln(PIRCHE-II) and ln(eplet) values.First we determined whether the use of serological split level HLA typing as input led to different PIRCHE-II and eplet values compared to the use of two-field resolution HLA genotype data as input. To this end, the delta between the observedlculated . When us) values . Furtherimal 0.1 . These d ln(PIRCHE-II) values did not show deviation from the reference ln(PIRCHE-II) values and 73.2% deviated maximal 0.1 ( ln(eplet) values did not deviate from the reference ln(eplet) values and 88.4% deviated maximal 0.1  values differed from the delta in ln(eplet) values. To this end, we plotted the delta for ln(PIRCHE-II) against the delta for ln(eplet). When using serological split level HLA typing of both donor and recipient, the majority of the donor-recipient couples had a comparable ln(PIRCHE-II) and ln(eplet) delta ( ln(PIRCHE-II) delta below −1, eight donor-recipient couples  had a ln(PIRCHE-II) delta above +1, and three donor-recipient couples  had a ln(eplet) delta below −1. These data suggests that the PIRCHE-II value is more often overestimated using the extrapolation approach, whereas the eplet value is more often under-estimated using the extrapolation approach. For all the donor-recipient couples who had a high deviation in the ln(PIRCHE-II) or ln(eplet) values, the corresponding ln(eplet) or ln(PIRCHE-II) values respectively were within the −1 and +1 range. This observation indicates that an increased delta in ln(PIRCHE-II) is not associated with an increased delta in ln(eplet) and vice versa.Next, we investigated whether the delta int) delta . However ln(PIRCHE-II)/ln(eplet) values. To investigate the effect of HLA-C or HLA-DQ on the PIRCHE-II and eplet estimations, we removed HLA-C or HLA-DQ from the serological split level HLA typing that was used as input for both PIRCHE-II and HLAMatchmaker.  ln(PIRCHE-II) and ln(eplet) values. When omitting HLA-C or HLA-DQ from the input, the percentage of donor-recipient couples who had a ln(PIRCHE-II) delta of zero dropped from 20.5% to 9.4% or 12.3% for omitting HLA-C or HLA-DQ, respectively ( ln(eplet) delta of zero dropped from 28.3% to 12.7% or 16.8% for omitting HLA-C or HLA-DQ, respectively  and ln(eplet) delta values for different percentiles . When omitting HLA-C or HLA-DQ from the serological split level HLA typing, the delta values for the higher percentiles slightly increased compared to delta values obtained from serological split level typing with HLA-C and HLA-DQ  delta of zero increased from 20.5% to 46.9% when using two-field resolution genotype data of the recipient ( ln(eplet), this percentage of donor-recipient couples who had a delta of zero increased from 28.3% to 50.9% ( ln(PIRCHE-II) delta and the ln(eplet) delta diminished at all percentile values when using two-field resolution genotype data of the recipient instead of serological split HLA typing  and ln(eplet) values and thus increases the reliability of the observed PIRCHE-II and eplet values. The reliability of the estimation was especially improved for PIRCHE-II and to a substantial but lesser extend for eplets. For all different settings tested, the most reliable PIRCHE-II/eplet estimation was achieved when using two-field resolution genotype data of the recipient and serological split level HLA typing of the donor.Since high-resolution HLA genotyping is a time-consuming method, high-resolution HLA genotyping of deceased donors is hardly feasible using the currently available typing methodologies. However, in most cases, high-resolution HLA genotyping of the recipient is possible. Therefore, we investigated whether a higher resolution HLA genotyping of the recipient may improve the reliability of the PIRCHE-II and eplet estimations. To this end, we used two-field resolution HLA genotype data of the recipient and serological split level HLA typing of the donor as input for the PIRCHE-II and the HLAMatchmaker algorithm. The percentage of donor-recipient couples who had aecipient . For theto 50.9% . The ln values with the delta in ln(eplet) values for the two-field resolution recipient setting, all donor-recipient couples had comparable ln(PIRCHE-II) and ln(eplet) delta values ( ln(PIRCHE-II) and ln(eplet) were observed. Thus, our data suggest that two-field resolution HLA genotyping of the recipient further improve the reliability of the PIRCHE-II and eplet estimation.When comparing the delta ina values . For allSeveral studies have shown that the number of PIRCHE-II and eplets are related to the clinical outcome after solid organ transplantation –12. To s ln(PIRCHE-II) and ln(eplet) values do not deviate or only slightly deviate from the reference ln(PIRCHE-II) and ln(eplet) values  values . These d ln(PIRCHE-II) delta and the ln(eplet) delta ( ln(PIRCHE-II)/ln(eplet) values  delta , indicat Figures  and 3. S ln(PIRCHE-II) was more often positive than negative, whereas for ln(eplet) the delta was more often negative than positive  values is not related to a high deviation in ln(eplet) values; some donor-recipient couples had a high ln(PIRCHE-II) delta and a small ln(eplet) delta and other donor-recipient couples had a high ln(eplet) delta and a small ln(PIRCHE-II) delta  delta . These ohttp://allelefrequencies.net [Our study has several limitations. First, the virtual Caucasian donor population used in our study was constructed upon the HLA haplotype frequency tables from 2007. However, these HLA haplotype frequency tables from 2007 also formed the basis for our PIRCHE-II/eplet estimation approach and therefore may cause bias in our observations or overfitting of the results. When using the updated HLA haplotype frequency tables from 2011, similar observed PIRCHE-II/eplet values were obtained , indicatcies.net , may furOur study showed that the observed PIRCHE-II and eplet values only limitedly deviate from the reference PIRCHE-II and eplet values in a quantitative manner; only differences in PIRCHE-II and eplet numbers were investigated in this study. However, although PIRCHE-II and eplet values do not differ between the observation group and the reference group, both calculations may correspond to different epitopes. Further analyses are required to identify whether the observed and reference calculations will have qualitative differences in epitopes.http://www.haplostats.org/) also estimates the most likely high-resolution HLA genotypes, but without HLA loci linkage removal when no matching high-resolution HLA haplotype is found. Thus, imputation of data can only take place when the HLA haplotypes are present in the HLA haplotype frequency tables. For the HLAMatchmaker algorithm, HLA haplotype frequency tables have been used to identify the most frequently present HLA haplotype in the population that corresponds to a given low-resolution HLA typing. This most frequently HLA haplotype is subsequently used as input for the algorithm. By using this approach, other potential less-frequent HLA haplotypes that also fit with the given low-resolution HLA typing are excluded. In our multiple imputation approach, high-resolution HLA genotypes with reduced likelihood were taken along in the PIRCHE-II and eplet calculations. Since our approach does not exclude less likely HLA haplotypes, we believe that our approach is more reliable than selecting the highest frequent high-resolution HLA haplotypes that is present in the general population.To our knowledge this is the first study that uses this computational approach for epitope-based HLA matching algorithms. Other studies have been using the HLA haplotype frequency tables in a similar way but in different settings. For example, a similar approach as our approach has been used by Madbouly et al. for the imputation of high-resolution HLA genotypes from multilocus unphased genotypes with ambiguous or missing typing data . In addiOur algorithm currently only handles serological split level HLA typing, whereas serological broad level HLA typing cannot be used as input for the algorithm. Likely, serological broad level HLA typing will further deteriorate our estimations and, consequently, is not preferred for an epitope-based HLA matching setting. Moreover, since serological split level HLA typing of both donors and recipients is currently mandatory according to the Eurotransplant guidelines , serologOur data show that although the PIRCHE-II/eplet value estimations are quite reliable, the estimations could be further improved. First, for a few donor-recipient couples, the PIRCHE-II/eplet values could not be calculated when using serological split level HLA typing, indicating that our approach cannot be used for a limited number of donor-recipient couples. One of the major improvements will be extension of the Next-Generation Sequencing-based genotype datasets that are used for establishing HLA haplotype frequency tables. As of November 2016, 11,553 HLA class-I alleles and 4,084 HLA class-II alleles are registered in the IMGT/HLA database 3.26 . These hIn conclusion, we have shown that the currently used extrapolation method is a powerful and reliable tool to estimate PIRCHE-II and eplet values. This method provides the opportunity to calculate PIRCHE-II and eplet values when using serological split level HLA typing and, thus, makes high-resolution HLA genotyping presumably redundant for the majority of the donor-recipient couples. When more next-generation sequencing-determined HLA genotype data will become available, HLA haplotype frequency tables will become more reliable and, consequently, the reliability of our epitope-based HLA matching estimations will be further improved.When no matching high-resolution HLA haplotype was found for a given serological split level HLA typing, the search criteria were broadened by removing an additional link between HLA loci. By removing an addition link, frequency calculations were adapted. A simplified example of the calculations when an additional link between HLA loci is removed is shown in Supplementary material 1.


This investigation shows that Al-polar films, as compared to N-polar ones, obtain DNPs with higher density and more homogeneously on their surfaces. The origin of these differences in density and homogeneity is discussed based on the hydrolysis behavior of AlN surfaces in aqueous suspensions.Electrostatic self-assembly of diamond nanoparticles (DNPs) onto substrate surfaces  is a notable technique, enabling chemical vapor deposition (CVD) of nanocrystalline diamond thin films on non-diamond substrates. In this study, we examine this technique onto differently polarized (either Al- or N-polar)  The vital step of this approach is the nucleation enhancement of diamond on AlN surfaces prior to the CVD process because the nucleation density on pure (untreated) AlN surfaces is extremely low (106–107 cm−2) [11 cm−2) of DNPs homogeneously on AlN surfaces as diamond nucleation sites, enabling the deposition of ultra-thin (~40 nm) and highly homogeneous (root mean square (rms) surface roughness of 12 nm) NCD films on them [c-axis oriented sputtered AlN films can be either Al- or N-polar, resulting in different surface terminations as the models of crystal structures for both Al- and N-polar cases are shown in Chemical vapor deposition (CVD) of nanocrystalline diamond (NCD) on aluminum nitride (AlN) is an epochal approach to combine NCD and AlN . This ap07 cm−2) . To dately low 10–107 cm−2 on them . One impc-axis oriented sputtered AlN film surfaces. This investigation shows that Al-polar films, as compared to N-polar ones, obtain DNPs with higher density and more homogeneously on their surfaces. The origin of these differences in density and homogeneity is discussed based on the hydrolysis behavior of AlN surfaces.In this paper, we report on electrostatic self-assembly of DNPs onto differently polarized (either Al- or N-polar) For the electrostatic self-assembly of DNPs onto AlN film surfaces, an aqueous colloid of DNPs was prepared and its properties are mentioned below. According to analyses via dynamic light scattering (DLS), the colloidal DNPs have a narrow distribution of particle sizes centering on the core size of DNPs (~3 nm), indicating that the DNPs are monodispersed in the aqueous colloid. Also, the DNPs retain negative zeta potential of −80 mV, which is required for the successful electrostatic self-assembly of DNPs onto AlN surfaces . In addi11 cm−2) are homogeneously obtained on the both Al- and N-polar AlN film surfaces. However, the seed density and its homogeneity on the Al-polar film surface seem to be slightly higher than those on the N-polar film surface although those differences are not so obvious. To make this point clear, the seed densities were statistically evaluated and the results are shown in 2 randomly chosen on each of the AlN film surface. As one can see in 11 cm−2) is more than five times higher than that on the N-polar surface (0.19 × 1011 cm−2). Therefore, it can now be stated that the polarity of c-axis oriented sputtered AlN films has an unignorably significant impact on the electrostatic self-assembly of DNPs onto AlN surfaces in terms of density and its homogeneity of the self-assembled DNPs.The electrostatic self-assembly of DNPs onto Al- and N-polar sputtered AlN film surfaces was carried out using the aforementioned aqueous colloid of DNPs. To enable clear scanning electron microscopy (SEM) observations of the DNPs assembled onto the AlN surfaces, a short-time growth of NCD grains on the AlN surfaces was additionally performed in an ellipsoidal reactor via microwave plasma assisted CVD . Figure In order to determine the influence of the observed differences in seed density and its homogeneity on CVD of NCD thin films, ultrathin NCD films were grown on both Al- and N-polar AlN film surfaces through the electrostatic self-assembly of DNPs. Since ultrathin NCD films are promising as transparent elastic electrode materials for emerging micro-opto-electro-mechanical system (MOEMS) applications such as the tunable micro-optical lenses  introducTo track the origin of the observed differences in seed density and its homogeneity between the Al- and the N-polar AlN film surfaces, the atomic concentrations of Al, N, C and O determined via X-ray photoelectron spectroscopy (XPS) are compared between the Al- and the N-polar AlN film surfaces immediately before and after the electrostatic self-assembly of DNPs as those results are shown in 2O3) polymorphs and hydrated aluminas, including polymorphs of aluminum oxide hydroxide (AlOOH) and aluminum trihydroxide (Al(OH)3) [Namely, the observed difference in atomic concentration of O indicates that the N-polar film surface is more oxidized in atmospheric moisture as compared to the Al-polar film surface. This is consistent with the previously reported result that N-polar AlN film surfaces oxidize more actively than Al-polar film surfaces do . FurtherAl(OH)3) . Such a 3) [To understand this difference in reduction rate of atomic concentration of O between the Al- and the N-polar film surfaces, a general behavior of AlN surfaces in aqueous suspensions could be considered. In principle, AlN surfaces oxidize in aqueous suspensions in the same way as they do in atmospheric moisture. However, the difference is that the amorphous AlOOH layer, which is formed by reaction 1), is dissolved in aqueous suspensions and are finally precipitated as crystalline bayerite (Al(OH)3)  while su3) . Consequ, is diss3) ,19. Mean3) . Considec-axis oriented AlN films sputtered on Si(100) substrates. The sputtered Al- and N-polar AlN films show, as an evidence of the polarity difference, opposite signs in longitudinal piezoelectric coefficients (d33) of ~+6 and ~−4 pm/V, respectively. Also, the Al- and the N-polar films show huge differences in both etching rates in an aqueous KOH solution and etched surface morphologies [In this study, commercially available detonation nanodiamond powder  and AlN nanopowder  were used to produce their aqueous colloids. The surfaces of AlN films used in this study, for both Al- and N-polar cases, were the surfaces of highly hologies . Here, ihologies  but in dhologies .7/2 peak located at 84.0 eV. The spectrometer was equipped with an hemispherical energy analyzer . The path energy was 20 eV corresponding to an energy resolution of 0.6 eV. This XPS set-up was previously used to investigate DNPs assembled onto various substrates [The detonation nanodiamond powder was initially annealed in air in order to obtain a negative zeta potential in an aqueous colloid, which is required for the successful electrostatic self-assembly of DNPs onto AlN surfaces . The annbstrates ,24,25. Ac-axis oriented sputtered AlN film surfaces. Also, ultrathin (~15 nm) NCD films were grown on both polarities of AlN film surfaces via the examined electrostatic self-assembly of DNPs. Further, the behaviors of differently polarized AlN film surfaces in atmospheric moisture and aqueous suspensions were discussed based on the atomic concentrations of Al, N, C and O on the surfaces determined via XPS before and after the electrostatic self-assembly of DNPs. These investigations and discussions provided one conclusion that Al-polar AlN film surfaces, as compared to N-polar film ones, obtain higher seed density more homogeneously due to their less active oxidation in atmospheric moisture and also more refrained hydrolysis behavior in aqueous colloids of DNPs. Therefore, it can be claimed that Al-polar films are more suitable as substrates to grow NCD thin films for the applications based on NCD/AlN unimorph structures such as surface acoustic wave resonators, mechanical micro-resonators, and tunable micro-optical lenses.In this study, electrostatic self-assembly of DNPs was examined onto differently polarized (either Al- or N-polar)


Late Gadolinium Enhancement (LGE) allows imaging of infarction by visualizing the accumulation of contrast agent within the myocardium using an inversion recovery (IR) prepared sequence. However, myocardial hyperenhancement can sometimes be poorly detected in the vicinity of epicardial fat because both fat and hyperenhanced tissue appear bright. Thus, a method for fat signal suppression is desired. One previous approach to fat-suppressed LGE was to null fat signal with appropriately timed tip-up/tip-down fat-selective RF pulses , but thi1 relaxation and longitudinal magnetization of fat signal lies below the transverse plane (Mz < 0) during acquisition  chosen to null normal myocardial tissue. Next, fat-selective tip-up and tip-down pulses are applied, with the tip-down pulse played immediately prior to data acquisition such that fat experiences minimal Tz < 0) vs. hyperenhanced tissue (Mz > 0). To achieve this the following condition for TIfat, the time between the fat tip-down pulse and the center of k-space acquisition, must be met: TIfat < -T1,fat*log(0.5 + exp(-(TI-Tt)/T1,fat) - exp + 0.5*exp), where Tt: time between IR and fat tip-up pulses, RR: R-R interval duration.After PS reconstruction fat appears black due to its opposite signal polarity  and compared to conventional fat-suppressed magnitude LGE with heart rate adaptive timing [In 7 out of 8 exams conventional fat nulling resulted in significantly reduced but not completely removed fat signal Fig. , whereasThe negative polarity of fat at readout and appropriate windowing of the image leads to complete elimination of fat signal with the new PiSCES approach. Additionally the technique is relatively insensitive to timing of the fat-selective RF pulses: fat appears black as long as fat has negative polarity, thus the method has potential to be more robust across a variety of imaging conditions.