In-training evaluation reports (ITERs) of student workplace-based learning are completed by clinical supervisors across various health disciplines. However, outside of medicine, the quality of submitted workplace-based assessments is largely uninvestigated. This study assessed the quality of ITERs in pharmacy and whether clinical supervisors could be trained to complete higher quality reports.A random sample of ITERs submitted in a pharmacy program during 2013–2014 was evaluated. These ITERs served as a historical control (control group 1) for comparison with ITERs submitted in 2015–2016 by clinical supervisors who participated in an interactive faculty development workshop (intervention group) and those who did not (control group 2). Two trained independent raters scored the ITERs using a previously validated nine-item scale assessing report quality, the Completed Clinical Evaluation Report Rating (CCERR). The scoring scale for each item is anchored at 1  and 5 (“exemplary”), with 3 categorized as “acceptable”.p = 0.84) and were worse than historical control group 1 . Mean item scores for individual CCERR items were below acceptable thresholds for 5 of the 9 domains in control group 1, including supervisor documented evidence of specific examples to clearly explain weaknesses and concrete recommendations for student improvement. Mean item scores for individual CCERR items were below acceptable thresholds for 6 and 7 of the 9 domains in control group 2 and the intervention group, respectively.Mean CCERR score for reports completed after the workshop (22.9 ± 3.39) did not significantly improve when compared to prospective control group 2  contains supplementary material, which is available to authorized users. Conducted in practice settings under the supervision of clinician mentors, students have opportunities to reinforce and shape development of existing knowledge and skills while engaged in direct patient care . The in-ciplines .Despite programs’ reliance on an ITER as an account of trainees’ clerkship performance and collectively, as a reliable summative record of a student’s demonstrated skills, knowledge, and behaviours over time, rater variability pervades WBA and is typically considered undesirable . Althougel al have demonstrated that ITER quality can be improved by both live workshop and asynchronous web-based faculty development which systematically emphasizes the written element clinical supervisors use to elaborate on physician trainee performance [The ability of rater-training to effectively mitigate such influences on WBA is mixed , 11–13. formance , 15. A nformance . ITER coformance .Unlike medicine, the literature outlining the quality of documented student workplace-based performance in pharmacy is lacking. Direct patient care clerkships represent significant proportions of pharmacy curricula and merit the same audit that campus-based assessment strategies receive ) , 18. SimA quasi-experiemental design using both retrospective and prospective controls was used to investigate the effectiveness of a faculty development workshop on the quality of narrative comments documented in pharmacy ITERs as measured by the CCERR instrument.medication therapy experts  and are akin to the CanMEDS framework for training the medical expert (physician) [“exceeds expectations”; “meets expectations”; or “below expectations” represented by 3, 2, and 1, respectively.The Doctor of Pharmacy (PharmD) program at the College of Pharmacy (CPH) at Qatar University (QU) is a Canadian-accredited post-baccalaureate degree supporting the training of graduate students to assume advanced pharmacy practice positions as integrated members of multidisciplinary teams delivering direct patient care. PharmD students complete 32 -weeks of experiential training (8 internships of 4 -weeks’ duration) for which ITERs are submitted by each internship’s clinical supervisor. The ITER used is a 25-item assessment instrument organized according to the seven domains of expected educational outcomes for graduating ysician) , 21, 22.The first cohort of ten graduate students enrolled in the QU CPH PharmD program in 2011. This small class size has fluctuated in number between 8 and 20 students over subsequent academic years. While students have the opportunity for overseas internships with various U.S. and Canadian university partners, the majority are offered locally by an expanding pool of nearly 50 clinical supervisors. To determine ITER quality in our program, we evaluated a random sample of those completed in the 2013–2014 academic year using the CCERR scoring tool  file 1. In the fall of the 2015–2016 academic year, a faculty development program was devised for all undergraduate and PharmD clinical supervisors similar in content to a workshop for medical trainee supervisors previously reported . All locThe overall and weighted mean scores for each of the nine CCERR item categories in the intervention group were independently compared to both the historic and concurrent control groups using Mann-Whitney tests for non-parametric continuous data with significant level (2-sided) set at alpha 0.05. The proportion of CCERR item categories scored below the threshold score for acceptability (less than 3) was also calculated. Inter-rater reliability was calculated mid-way and at the conclusion of ITER review for the 2013–2014 and the 2015–2016 data sample. At the conclusion of ITER review of the 2013–2014 and of the 2015–2016 sample, each research assistant was asked to re-rate the same two randomly selected ITERs. Acceptable inter- and intra-rater reliability was set at 0.75 as measured by the intraclass correlation coefficient . Ethics A random sample of 54 ITERs were reviewed as part of control group 1 , but was found to be when each was compared to control group 1 (p = 0.0001). Interrater reliability between the two independent ITER assessors for the entire data set was 0.77. Overall assessor intrarater reliability was 0.87 and 0.91, respectively.Nine 19.6%) PharmD clinical supervisors attended the faculty development workshop and 77 student internships were subsequently completed during the remainder of the academic year  and concluding with an action plan . While wDespite reinforcement through faculty development initiatives, shortcomings in the amount and nature of ITER narrative persist, especially as it pertains to constructive comments , 28. In As an objective measure of quality, the CCERR scale still relies on subjective assessment . Use of multiple independent raters for CCERR administration may mitigate this phenomenon, but inherent cognitive biases invariably persist, especially as it pertains to language use and interpretation , 32. In medication expert are formally discussed and recorded. Irrespective of undocumented verbal feedback exchanges between trainer and trainee throughout this learning experience, our program expects suitable support of the summative judgement of student performance and ultimately clerkship outcome (to pass or to fail). We have found through the low CCERR scores that this narrative element of ITER completion among our clinical supervisors can be improved. We encourage adoption of systematic ITER quality assurance processes for pharmacy programs and other health disciplines.Our orphan assessment of ITER quality in pharmacy education is concerning. The medical literature is replete with study of the validity and reliability of the ITER numeric scoring component and increasingly, scrutiny of narrative comment but thus far our work is without apparent precedent in any other health profession. , 29, 35.Although our absolute aggregate mean CCERR scores are in fact higher than those found following ITER faculty development among physicians, the small number of completed ITERs considered for evaluation following the faculty development workshop, especially in the intervention group, is a limitation to our findings . InherenThe quality of narrative comments documented in pharmacy ITERs as measured by the CCERR instrument was low and did not appreciably change following a faculty development workshop. Despite our first experience, we believe the CCERR instrument can be a useful vehicle to support improved WBA documentation in pharmacy student workplace-based training. The challenges to securing professional development attendance is widely acknowledged and our program is also exploring distance-based faculty development models that would incorporate routine personalized ITER feedback to clinical supervisors . Greater


Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock. Briefly, DNaseI-treated RNA (1 http://www.ingenuity.com) to identify significantly regulated canonical pathways in endothelial cells during LPS treatment in direct comparison to the mock-treated controls. Only pathways exhibiting significant changes (P ≤ 0.05) were considered for the analysis.To identify differentially regulated protein-coding gene pathways in HMECs during LPS treatment, we mapped our RNA-Seq reads to the human RefSeq database (GRch37/hg19) following same parameters as described above for ncRNAs. PCGs showing ≥3-fold up- or downregulation were further subjected to Ingenuity® Systems Pathway Analysis  was used for the statistical analysis. Statistical significance was accepted at the P value of ≤0.05.The D'Agostino and Pearson omnibus normality test was performed to test the normal distribution of data. Comparisons between the unmatched groups were done by the Mann–Whitney https://doi.org/10.1155/2017/3427461). Of the total RNA in eukaryotic cells, mRNAs and rRNAs represent approximately 5 and 80%, respectively, whereas the remaining 15% includes other RNA molecules comprised of spliceosomal RNAs, snoRNAs, nonpolyadenylated RNAs, tRNAs, mitochondrial RNAs, and other specialized RNAs such as LINE/SINE or B-element RNAs which are abundantly expressed and mapped to the repeat regions of the genome [To catalogue alterations in the lncRNA expression profile of HMECs subjected to LPS treatment for 3 h and 24 h, we first performed RNA sequencing. Sequencing produced approximately 28–37 million reads per library, of which nearly 31–36% and 11–15% of the reads mapped to mRNAs and ncRNAs, respectively  at 3 and 24 h following LPS treatment by determining their chromosomal location . Long noin vitro or in vivo. To accomplish this, sequences of functionally annotated lncRNAs were acquired from lncRNAdb [Since very little is known about the functions of lncRNAs in the endothelium and our analysis revealed extensive modulation of their expression, we next focused our efforts on identifying those with a defined functional role in the determination of endothelial responses lncRNAdb  and blaslncRNAdb  enabled lncRNAdb .Of the 44 lncRNA candidates identified in this study, 22 are from genomic loci of PCGs , while the remaining 22 originate from the intergenic regions. The expression of PCGs overlapping with these lncRNAs was either downregulated or remained unchanged. Specifically, the expression of ADAMTS9 , HOXA11a (homeobox A11a), and PDE7B (phosphodiesterase 7B) harboring lncRNAs ADAMTS9-AS2, Hoxa11as, and NTT on the respective antisense strand was compromised, while that of ITPR1 on the opposite strand of lncRNA EGO was unaltered at both 3 or 24 h post-LPS treatment (Supplementary Table 3).EGO, HOTAIRM1, and lnc-IL7R are three lncRNAs with known regulatory functions that were found to be differentially expressed in endothelial cells after LPS treatment. EGO and HOTAIRM1 have 3 and 5 splice variants, respectively. Among them, 2 splice variants of EGO and 4 splice variants of HOTAIRM1 were differentially expressed in response to LPS treatment for 3 and 24 h. To further validate splice variant-specific expression of EGO (transcript NONHSAT087634) and HOTAIRM1 (transcript NONHSAT119666) lncRNAs, we performed qRT-PCR analysis. In agreement with the findings from our RNA-Seq data, the expression of both NONHSAT087634 and NONHSAT119666 transcripts was significantly upregulated at 3 h post-LPS treatment. At 24 h, the expression levels of both NONHSAT087634 and NONHSAT119666 transcripts declined and were not significantly different from the basal level in mock-treated HMECs Figures . AdditioFinally, Ingenuity Pathway Analysis revealed a number of endothelial cell signaling pathways activated in response to LPS treatment for 3 and 24 h Tables . As expeLeptospira interrogans, can also signal through the TLR2 pathway [The strategic location of vascular endothelium at the interface of circulating blood and underlying tissues also renders it highly susceptible to injury or infection . In addi pathway .It is now well appreciated that despite the transcription of a major proportion (>80%) of human genome, PCGs account for only ~2%, while an overwhelming majority is transcribed into ncRNAs lacking protein-coding capacity . Of thesA “search and identify” strategy based on the comparative analysis of our datasets with lncRNAdb, a database listing functionally annotated lncRNAs , allowedHOTAIRM1 (HOX antisense intergenic RNA myeloid 1), a lincRNA located in the HOXA genomic cluster, modulates the expression of genes involved in myeloid differentiation. Recent evidence suggests increased expression of HOTAIRM1 in cardiomyocytes treated in vitro with LPS or those isolated from mice receiving LPS to induce sepsis [+ hematopoietic progenitor cells through regulation of expression of eosinophil granule protein, although its mode of action has not been defined yet [α in cardiomyocytes after LPS treatment [κB in the induced expression of inflammatory genes in mouse macrophages [A number of previous studies have highlighted the importance of HOTAIRM1 and EGO in immune cell activation. e sepsis . EGO, anined yet . Considereatment  and suggrophages . LPS trerophages , 76. Altrophages . Thus, gEndothelial activation, injury, and dysfunction represent major contributory mechanisms underlying the pathogenesis of endotoxic shock and sepsis. Hence, the expression profiles of PCGs in vascular endothelial cells activated by LPS have been extensively investigated –22. In aThe limitation of this study is that a majority of the lncRNA splice variants have not been characterized in detail as of yet, thus limiting our efforts to functionally annotate their role in vital cellular functions such as immune regulation, epigenetic modulation, cell proliferation, and growth suppression. Therefore, further investigations aimed at defining modulatory effects of specific splice variants during the pathogenesis of sepsis and septic shock syndrome should reveal important new insights into the vascular endothelium's responses to LPS in particular and pathophysiology of sepsis syndrome in general.Supplementary file 1: Sequence alignment of splice variants of EGO and HOTAIRM1 lncRNA. Supplementary Table 1: Total reads from the RNA sequencing experiments at each treatment and percentage of reads mapped to coding (mRNA) and non-coding (ncRNA) transcripts. Supplementary Table 2: Expression level of regulatory  splice variants of lncRNAs in 3 and 24 h LPS treated human microvascular endothelial cells, their genomic origins, and coding and non-coding indexes. Supplementary Table 3: Expression of Protein coding genes (PCGs) overlapping with functionally annotated lncRNAs at 3 and 24 h LPS treatment in human microvascular endothelial cells (HMECs). Supplementary Table 4: Significant canonical pathways and associated differentially regulated protein coding genes (PCGs) at 3 h in human microvascular endothelial cells (HMECs) after LPS treatment. Supplementary Table 5: Significant canonical pathways and associated differentially regulated protein coding genes (PCGs) at 24 h in human microvascular endothelial cells (HMECs) after LPS treatment.


Concomitant inhibition of BET proteins and ATR of cultured melanoma cells resulted in similar effects as recently shown in lymphoma, such as induction of apoptosis and p62, implicated in autophagy, senescence-associated secretory pathway and ER stress. In vivo, apoptosis and suppression of subcutaneous growth of patient-derived melanoma and B16F10 cells were observed. Our data suggest that ATRI/BETI combination therapies are effective in melanoma.Metastatic malignant melanoma continues to be a challenging disease despite clinical translation of the comprehensive understanding of driver mutations and how melanoma cells evade immune attack. In Myc-driven lymphoma, efficacy of epigenetic inhibitors of the bromodomain and extra-terminal domain (BET) family of bromodomain proteins can be enhanced by combination therapy with inhibitors of the DNA damage response kinase ATR. Whether this combination is active in solid malignancies like melanoma, and how it relates to immune therapy, has not previously investigated. To test efficacy and molecular consequences of combination therapies cultured melanoma cells were used. To assess tumor responses to therapies  Multiple resistance pathways have been found,7 suggesting new combination treatments that are tested in various clinical trials. Second, the discovery of immune checkpoints8 enabled the development of antibodies directed against CTLA4 and PD-1 (or its ligand PDL1), which show lower response rates but generally more durable responses.10 Taken together, these modern treatments have been successful, but to cure or make MM manageable chronic, more and safe drugs are needed.Malignant melanoma (MM) is potentially curable if diagnosed early but if the disease becomes metastatic it often is fatal. Recent advances in the molecular and immunological characterization of the disease have generated new promising avenues of therapeutic intervention.11 BET proteins regulate transcription and appear to be utilized during cancer progression to epigenetically reprogram both blood and solid cancers.12 Small-molecule inhibitors of BET proteins are in clinical trials but preclinical models already suggest that combination therapies will be needed to further the efficacy of BET inhibitors (BETIs). Indeed, we and other investigators have recently demonstrated that inhibitors targeting cell-signaling molecules, the proteasome, components of the DNA damage response and HDAC synergize with BETIs to kill B-cell malignancies.19 HDAC and BETI combination treatment is also effective in melanoma20 but whether any of the other therapies would be effective is not known.BET proteins are emerging targets for cancer therapy.21 When ATR is activated it phosphorylates the checkpoint kinase Chk1 that work together with ATR to phosphorylate components in the replication machinery to inhibit further replication. Hence, inhibition of ATR or Chk1 is detrimental to cells experiencing replication fork problems such as cancer cells expressing high levels of the MYC oncogene.25Ataxia-telangiectasia and Rad3-related (ATR) is a kinase that belongs to the PI3-kinase-like family, which also includes PI3K, mTOR, ATM and DNA-PK. ATR has a critical role in the regulation of replication and is activated by replication fork stalling. Known causes of stalling include UV-induced DNA damage and nucleotide deprivation but also excess replication fork firing exerted by oncogenic replication stress.26 We have demonstrated that Myc-induced lymphoma cells undergoing replication stress, because of ATR inhibition, are sensitive to BETIs.19 Here we wish to investigate whether or not this finding can be extended to solid cancers. By using cultured melanoma cells, patient-derived xenografts (PDXs) and syngenic transplant models we show that the therapeutic combination targeting of ATR and BET proteins is effective in melanoma.Both BETIs and Chk1 inhibitors have previously been shown to have efficacy in cultured melanoma cells and Chk1 has even been suggested to be essential for the melanocytic lineage.28). To assess the therapeutic effect of combined inhibition of ATR kinase and BET protein we cultured the melanoma cell lines A375 and MeWo in the presence of the ATR inhibitor (ATRI) VE821 and/or RVX2135.19 Both compounds were antiproliferative as assessed by microscopy, CellTiter Glo  measurements and cell counts  and ER stress.ed cells . We thered cells . Westerned cells . The apoed cells , but Bimed cells  were noted cells  it is poed cells , which c29 We recently developed a platform of highly characterized PDX models.30 To test whether ATRI/BETI would work in a PDX model end we had to use the bioavailable ATRI AZ20, as VE821 is not bioavailable in vivo. In PDX model M121218 we observed a robust reduction in subcutaneous growth and tumor size , a marker of double-stranded DNA damage that often follows ATR/Chk1 inhibition.32Cultured melanoma cells are grown in very different conditions than melanoma cells in patients or in mice.mor size , a reducmor size , and a mmor size . To inveanalysis . As seen19To investigate whether tumors from other patients would be sensitive to the ATRI/BETI combination therapy we treated three other PDX models. In two of these models models, the combination treatment blocked growth resulting in smaller tumors and induction of apoptosis . In the in vitro in the presence or absence of ATRI (VE821 or AZ20) and/or BETI (RVX2135 or iBET762). The cells were noticeably sensitive to BETI, less to ATRI but very sensitive to the combination therapy  and two DNA methyltransferase inhibitors are already approved for various malignancies. Several BETI, histone/lysine methyltransferase inhibitors and HAT inhibitors are in various stages of development. Combination therapies are an obvious approach but which therapies to combine, when to combine and how to combine to maximize efficacy and minimize side effects is still unknown. We have identified two different possible combination therapies against Myc-induced cancers, BETI/HDACI15 and BETI/ATRI . It appears as if the mechanisms are broad, resulting in cell death and large changes in transcriptional output. On the other hand, it remains to be found whether or not there are selective events that are shared between these two potent combination therapies that can be further utilized in design of new therapeutic approaches.To conclude, it is worth noticing that targeted therapies directed against the mutated driver 2. Viability following inhibitor treatment was monitored with CellTiter Glo.All cell lines were from Cell Lines Services . They were maintained in complete medium  and cultured at 37 °C with 5% CORNA preparation is carried out using the Nucleospin RNA isolation kit . Following cDNA synthesis using iScript cDNA synthesis kit  indicated genes were amplified using a SYBR green PCR mastermix . Primer sequences are available on request. The ΔΔCT method was used to calculate the relative expression.25 A unit of 50 μg of protein was resolved on 4–20% ClearPAGE gels  and transferred to nitrocellulose membrane . The membrane was blotted with specific antibodies. Antibodies against the following proteins were used: phosphorylated ATR ; cleaved PARP ; ATR ; ATF4 (Santa Crutz Biotechnology); CHOP (Santa Crutz Biotechnology); phosphorylated H2Ax , p62  and Actin .For western blot analysis of protein expression, cell pellets or tumor pieces lysed in lysis buffer as described before.5 cells mixed with equal volume of Matrigel  subcutaneously at the flank of immunocompromised, non-obese severe combined immune-deficient interleukin-2 chain receptor γ knockout mice  as described previously.30 Tumors were measured with caliper at regular time points and tumor volume calculated using the formula: tumor volume (mm3)=(length(mm)) × (width(mm))2/2. B16F10-luciferase cells were transplanted by subcutaneous injection. Seven days after transplantation, mice were injected with 100 μl of 30 mg/ml d-luciferin. Mice were sedated in an isofluran administrating chamber and then placed in an IVIS Lumina III XR machine .All animal experiments were performed in accordance with EU directive 2010/63 . The PDXs were obtained by injecting 2 × 10t-test or one-way ANOVA (with Sidak corrections) for tumor burden, and the log-rank test for survival. All mouse experiments contained 3–5 mice per group.Values are presented as mean±S.D. when data are combined. For statistical analyses, we used Graphpad Software, Inc. : multiple


Consistent with Dr. Paul Terasaki's “humoral theory of rejection” numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR) and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production .  Antibody mediated rejection (AMR) is a major contributor to rejection risk and allograft loss in solid organ transplantation , 2. AMR Pretransplant sensitization can occur from prior transplants, blood transfusions, pregnancy, and mechanical assist devices (in heart failure) resulting in autoantibody formation. Allosensitization affects approximately 6–9% of cardiac transplant candidates , 20 and After transplantation, 24% of renal allograft recipients will develop de novo HLA DSA within ten years  and appr1R) can precede de novo HLA DSA , a common histological feature across solid organ transplant [γ and cell-contact dependent manner [Leukocyte recruitment and activation are a common histological feature of AMR. Macrophage infiltration is observed in heart  and renaansplant , 6, 109.t manner . Collect1R antibodies mediate endothelial cell activation and vasoconstriction by binding to the second extracellular loop of the AT1R protein and act as an angiotensin II agonist promoting downstream activation of AP-1 and NF-κB [1R and ETAR antibodies frequently occur together in patient sera [1R antibodies suggesting that the mechanism of injury for AT1R antibodies is not the complement pathway [γ secreting T cells [There is less mechanistic data for non-HLA antibodies in the pathogenesis of AMR. However, non-HLA antibodies can also mediate EC activation and complement activation and leukocyte interaction/activation. Non-HLA antibodies that activate EC can increase the expression of HLA class I and II and have been shown to develop independently or in conjunction with HLA DSA , 111. ATnd NF-κB . AT1R anent sera –115. C4d pathway , 44, 112 pathway . Evidenc T cells . LG3 ant T cells .Experimental models and clinical experience demonstrate that anti-donor HLA and non-HLA antibodies exhibit pathogenic functions through multiple mechanisms that likely have extensive crosstalk. AMR manifests as a broad spectrum both histologically and symptomatically. Across solid organs, the microvasculature is the principal target of antibody mediated injury. A single uniform approach to prevent graft injury and loss in the setting of donor specific antibodies will probably not be effective for all patients, and personalized therapies tailored to address unique patient and donor features will be needed to protect from AMR and chronic rejection. Non-HLA antibodies have also been associated with TCMR in renal transplantation  suggestiCollectively, data on antibody pathogenicity defined by the antibody specificity, isotype, and ability to activate EC and complement can lead to different effector functions that mediate different pathological outcomes. Further studies to clarify which HLA and non-HLA antibody attributes  contribute to subclinical, acute, and chronic AMR would be useful in order to identify biomarkers of different outcomes. Employment of newer techniques, such as the “molecular microscope,” can provide additional insight into the active transcriptome in the graft tissue, allowing a measurement of the local inflammation and the transcriptome signature for AMR . Clinica


Erythroid Krüppel-like Factor (EKLF/KLF1) is an erythroid-enriched transcription factor that plays a global role in all aspects of erythropoiesis, including cell cycle control and differentiation. We queried whether its mutation might play a role in red cell malignancies by genomic sequencing of the KLF1 transcription unit in cell lines, erythroid neoplasms, dysplastic disorders, and leukemia. In addition, we queried published databases from a number of varied sources. In all cases we only found changes in commonly notated SNPs. Our results suggest that if there are mutations in KLF1 associated with erythroid malignancies, they are exceedingly rare. Its roles in ß-like globin gene regulation during terminal erythroid differentiation have been well-established using genetic, biochemical, and molecular approaches3. Specific functional properties and expression characteristics of EKLF, along with recognition of its surprisingly broad role prior to and during red cell differentiation (reviewed in8), provide the conceptual basis for the present study.Erythroid Krüppel-like Factor (EKLF/KLF1) is a red cell-enriched, zinc finger DNA binding protein that interacts with its cognate 5′CCMCRCCCN3′ element at target promoters and enhancers12, raising the possibility that mutations at these sites or within their consensus sequences could have dramatic functional effects on gene expression control.First, single amino acids and their modifications are critically important for EKLF protein-protein interactions and its function as an activator or repressor17. Induction of p21 is reminiscent of a similar up-regulation that has been observed with KLF418 and with KLF619, known tumor suppressors that function in a p53-independent manner and that are frequently inactivated or downregulated in human cancer20.Second, subtle altering of EKLF cellular levels can change the erythroid cell cycle status from proliferation to differentiation, an effect mediated, at least in part, by its direct activation of the p21, p18, p27, and E2f2 genes21. Although most abundant in the erythroid cell, EKLF is also highly expressed in the megakaryocyte/erythroid progenitor (MEP)22. Its level is downregulated as MEPs differentiate towards the megakaryocytic lineage yet remains high in the erythroid lineage22. By using both gain- and loss-of-function approaches, we22 and others25 have found that the expression levels of EKLF impacts the bipotential lineage decisions that are made by the MEP; specifically, EKLF inhibits the formation of megakaryocyte colony and cell numbers while at the same time stimulating erythroid differentiation.Third, EKLF mRNA is highly restricted in its expression pattern during development to erythropoietic organs such as the yolk sac, fetal liver, adult bone marrow, and red pulp of the spleen28. For example, the semi-dominant mouse mutation Nan , which presents with hereditary spherocytosis, was mapped to a single amino acid change (E339D) within the second zinc finger of EKLF31. The mutation alters the DNA binding specificity of EKLF such that it no longer binds promoters of a subset of its DNA targets29. In addition, recognition of a novel target DNA sequence by Nan-EKLF leads to ectopic expression of genes not normally expressed in the red cell, yielding a neomorphic phenotype with cellular and systemic consequences33.Fourth, there are now links between KLF1 mutation and altered mammalian hematology39, where the same amino acid is altered  in human EKLF/KLF1. These patients are severely anemic with highly elevated HbF and reticulocyte levels, membrane abnormalities, severe hemolytic anemia, erythroid hyperplasia with dyserythropoiesis, splenomegaly, and growth delay36. KLF1/E325K is recognized as a characteristic feature of CDA type IV40.Excitingly, this murine mutation has converged with human disease, particularly a subtype of congenital dyserythropoietic anemia (CDA)43.Additionally, the regulation of some genes are uniquely sensitive to haploinsufficient levels of KLF1 , leading to altered genetic expression patterns and hematologic parameters in humans, of which hereditary persistence of fetal hemoglobin (HPFH) is particularly relevant to clinical outcome45. On the other hand, unrestricted proliferation and impaired differentiation are characteristic of acute myeloid leukemia (AML)47. Our test hypothesis is that dysregulation of KLF1 function may contribute or lead to either of these human malignancies.Collectively these functional properties suggest that genetic mutation or altered levels of KLF1 may also be a causative factor for a specific subset of hematopoietic disease. We focused our attention on two types of disorders. Myeloproliferative neoplasms (MPN) are chronic hematological malignancies that yield an excessive proliferation of blood cells with normal differentiation48. Recent studies provide an extensive catalogue of SNP variants of consequence for red cell parameters49. Perusal of the data enable us to extract the ones most relevant to KLF1 as summarized in Table 19p13.13, the chromosomal locus of KLF1, has been associated with variation in blood cell traits in meta-analysis studies50, enabling us to interrogate its proximal promoter, 5′ UTR, introns, exons, and 3′ UTR by eight overlapping amplimers. The proximal promoter contains highly conserved transcription factor binding sequences that are critical for its expression in erythroid cells55. To begin our evaluation of human KLF1 genomic status, we focused first on sequencing human leukemia cell lines that retain erythroid and/or megakaryocytic features57. These lines are derived from CML, AML-M6 or -M7 patients , and include those with mixed erythroid/megakaryocytic features . We conclude from genomic sequence comparison of these lines to the 1000 Genomes project59 that the KLF1 genomic changes observed represent known single nucleotide polymorphisms (SNPs), but no novel mutations  and essential thrombocythemia (ET), which represent abnormalities in the proliferation of erythroid and megakaryocytic lineage respectively45. We therefore sequenced genomic DNA from a collection of individuals with PV (eighteen), ET (eleven), and MF (five) samples. No novel mutations were identified, only SNPs  as cells whose aberrant properties might result from expression of mutated KLF1. MPNs are a heterogeneous group of clonal hematological malignancies that are characterized by hypercellular bone marrow, panmyelosis, and a gradual evolution to myelofibrosis (MF) and acute leukemiaPs Table . Althoug62. We hypothesized that the mutational status of KLF1 may provide a mechanistic basis to explain the differential expression signature in these patients, and potentially predict whether they will respond to lenalidomide. We analyzed 26 samples each of responders and non-responders. All variants identified in these samples were known SNPs  patients that vary in their response to lenalidomide treatment63, including four from patients with Down syndrome (DS) and six from those without DS. AMKL is an aggressive form of leukemia where a majority of the expanding cells are abnormal megakaryoblasts64, and whose DNA methylation patterns are distinct between DS and non-DS cells65. Again, all samples contain known KLF1 SNPs (Table We next examined ten acute megakaryoblastic leukemia (AMKL) samplesPs Table .26. The ones that result in non-synonymous coding changes are predicted not to affect KLF1 function67 , some with an overall low frequency (~5%) are nonetheless highly enriched (~20%) prevalently and exclusively in selected genetic or geographic sub-populations while completely absent in all others  , and AML tabulations74, along with those derived from The Cancer Genome Atlas (TCGA) data sets76 do not reveal a role for KLF1 in any case in these blood cancers. KLF1 is not one of 142 driver genes identified from an analysis of 1699 pediatric leukemias and tumors77. An shRNA screen of AML cells lines also did not implicate a role for KLF178, and KLF1 does not appear in differential analyses related to predicting therapy resistance in AML79. Examination of COSMIC data (v83)80 indicates that, out of 3478 curated hematopoietic and lymphoid samples, only a single coding sequence variant was observed (F27V) in a CLL patient81, one predicted to be benign67.Given our hypothesis, we were surprised by the absence of mutations associated with our target samples/sources. To expand our analysis, we also considered an 26). Two, KLF1 levels vary considerably across various malignancies as judged by tabulation of RNA sequence datasets82 . This line was established decades ago91, and early on was noted to exhibit variability93. We have noted major differences in transfection efficiency upon comparing our lab K562 stock with the ATCC K562 line (unpublished observations) although we did not find any KLF1 genomic sequence differences. We suggest labs are working with dissimilar isolates, differing in levels of GATA1 and/or KLF194.K562 cells were from our original lab stockPatient samples were procured after informed consent and IRB approval within the individual institutions . All methods were performed in accordance with the relevant institutional guidelines and regulations. Mononuclear cells from MPN patients were provided by the Myeloproliferative Disorders Research Consortium Tissue Bank Core C.Genomic DNA was isolated from all samples using a Qiagen DNeasy Blood & Tissue Kit. PCR primers spanning the complete KLF1 transcription unit were used to amplify eight overlapping regions across the locus  followed alignment to the GRCh38 reference sequence using Vector NTI ContigExpress software (ThermoFisher Scientific). SNPs and any associated parameters were identified from the 1000 Genomes databasesSupplementary Table 1


Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.The online version of this article (doi:10.1186/s12915-016-0323-9) contains supplementary material, which is available to authorized users. Morphogenesis of the vertebrate head involves the close coordination of multiple cell populations that build tissue structures of the face and neck. One example of this is the intimate patterning of neural crest-derived tissues with vasculature. Neural crest cells are highly migratory, emigrate from the dorsal neural tube to peripheral locations along stereotypical migratory pathways, and contribute to multiple tissues. The neural crest cells contribute to the aortic outflow tract and arterial walls of the large blood vessels in the cardiovascular system –4. AngioDetails of the spatio-temporal pattern of cranial neural crest and endothelial cell migration remain largely undetermined due to static end-point analyses and lack of an in vivo model system to simultaneously observe the dynamics of both cell populations. Recently, observations in trunk neural crest and endothelial cell dynamics have begun to illustrate the course of events in the trunk, but cranial neural crest cells have yet to be examined . CranialIn the absence of dynamic cell behavioral data, it is not surprising that it is largely unknown whether there are distinct molecular signals in the embryonic microenvironment that direct cranial neural crest and endothelial cell movements. We have previously shown that vascular endothelial growth factor (VEGF) is expressed in the chick head ectoderm, directly overlying neural crest cell migratory pathways and in the second branchial arch (BA2) tissue . VEGF isWe discovered in a previous molecular analysis that migrating chick cranial neural crest cells express high levels of the anti-angiogenic factor angiopoietin 2 (Ang2). Ang2 is a secreted ligand that has been shown in vitro to enhance cell motility in several non-endothelial cell types –21; howeIn this study, we examined the role of Ang2 on cranial neural crest cell migration using our previously developed in vivo imaging platform . We confFertilized leghorn chicken  or quail eggs  were incubated at 38 °C in a humidified incubator until the desired stages of development . Quail wt test was used to compare samples.Electroporated embryos were incubated 24 h to HH St15 then fixed in 4% paraformaldehyde at 4 °C overnight. Embryos were imaged whole or cut in 100-μm transverse vibratome sections . Cells found inside or outside the majority neural crest pathway Fig. –c were mThe datasets generated and/or analyzed during the current study are available as unprocessed raw data in the Stowers Institute for Medical Research original data repository.Quail embryos were fixed in 4% paraformaldehyde, embedded in 7% agarose, and vibratome sectioned at 100-μm thickness. The quail monoclonal endothelial cell surface antibody  and chihttp://research.stowers.org/imagejplugins). A hand-drawn polyline was used to calculate the fluorescence intensity in a 30-pixel-wide area in each channel.Transcripts for Ang2 and FoxD3 were visualized in whole chick embryos by HCR. Embryos were fixed at HH St15 in 4% paraformaldehyde in 0.1% diethylpyrocarbonate (DEPC)-treated phosphate-buffered saline (PBS) at ambient temperature for 2 h, then washed three times in DEPC PBS. The embryos were serially dehydrated in  methanol and frozen at –20 °C until use (maximum three days). HCR was performed according to the manufacturer’s instructions . After labeling with Alexa Fluor 546 (FoxD3) and Alexa Fluor 647 (Ang2), embryos were cleared in ScaleU2 buffer  for at lEmbryos were fluorescently labeled as described above and allowed to re-incubate to HH St10. Healthy and well-labeled embryos were mounted on Early Chick (EC) culture , 36 and l-lysine  and 1 mg/mL fibronectin . At 24 h after plating, cells were fixed in 4% paraformaldehyde, and then immunohistochemistry was performed using Phospho-FAK pTyr861  and HNK1(1:500) primary antibodies with secondary goat anti-rabbit Alexa Fluor 546  or goat anti-mouse Alexa Fluor 647 . We imaged two to four fields of view of four plated neural tubes for each control, Ang2-FL, or Ang2-shRNA condition. The sizes of the phospho-FAK clusters were determined by masking the image with the HNK1 channel and variable size spot detection . Spot detection was done with 0.9-μm seed points, and the local region threshold was adjusted so that larger areas were just covered.In vitro cultures were prepared similarly to the procedure of . BrieflyChick embryos were labeled as described with control GFP expression vector at HH St 6 to 8 somites, then allowed to develop until HH St13. The r4 stream of the neural crest was isolated manually from 12 embryos and dissociated to a single cell suspension. The cell suspension underwent fluorescence-activated cell sorting (FACS) on GFP and Sytox Red (Thermo Fisher) channels and returned 497 cells which were immediately lysed. cDNA was created with the High Capacity cDNA kit (Applied Biosystems). PCR reactions for both beta-actin and Ang2 genes were performed using Ang2 primers GATTTCTGTTCAGACCAACGAG, CAGGATTATCTTAAGAACGTAGC for a 584-bp fragment and beta-actin primers CGGTTTCGCCGGGGACGATG, CGTCAGGTCACGGCCAGCCAGA for a 500-bp fragment.To characterize the spatio-temporal aspects of neural crest and endothelial cells during head development, we analyzed HNK1  and QH1  antibody-labeled cell positions in fixed quail tissue sections taken from HH St10–15  embryosPre-otic neural crest cells from the axial level of mid-rhombomere 3 (r3) to mid-r5 exit the dorsal neural tube at HH St10 Fig.  and follEndothelial cells within the neural crest cell migratory stream appeared close enough to be in physical contact with neural crest cells Fig. . We findn = 10). In a typical time-lapse imaging session, we were able to record in vivo neural crest and endothelial cell dynamics with single - cell resolution. Focusing on the pre-otic r4 neural crest cell migratory stream, we observed a multistep process to neural crest and endothelial cell migration events. First, endothelial cells from the dorsal aorta became visually distinguishable as cells moved in a ventral-to-dorsal manner to directly underneath the dorsolateral neural crest migratory pathway  with premigratory neural crest labeled with DiI from r3 to r7 (magenta). Movie begins at roughly HH St11 after some neural crest cells have already emerged from the neural tube. Both populations of cells are highly dynamic and migrate in close spatial proximity. (MOV 6586 kb)Additional file 1. Overview of r3 to r7 neural crest and endothelial cell migration. Time-lapse, confocal images of transgenic Tie1:H2B-YFP quail embryo  coalesce near the dorsal aorta and sprout dorsally towards the neural tube, brushing against the neural crest stream (magenta). Neural crest cells pass the dorsal aorta at the same time this sprouting occurs while migrating ventrally. The endothelial cells create a chain following each other in the space just ventral to the neural crest stream, then connect to the perineural vascular plexus surrounding the neural tube. (MOV 3718 kb)Additional file 2. The r4 migratory neural crest stream and endothelial cells migrate in close proximity. Time-lapse, confocal images of developing quail embryo at the r4 level of the head beginning at roughly HH St11. Visualization of post-otic neural crest and endothelial cell mixing led to surprising observations. In contrast to the r4 neural crest area, the r6 neural crest and endothelial cells move within the same migratory pathway, again in opposite directions Fig. . We saw green) and neural crest  beginning at HH St12. Endothelial cells and neural crest in the r6 stream travel in the same plane within the embryo, resulting in collisions. Endothelial cells generally move towards the neural tube, while neural crest cells move away. The marked cell in the movie highlights an endothelial cell that is attempting to move through a dense patch of neural crest, cannot, then reroutes around the neural crest ventrally to a less dense region where it continues moving towards the neural tube. (MOV 6138 kb)Additional file 3. Post-otic neural crest cells contact endothelial cells while migrating. Time-lapse confocal images of rhombomere 6 level of developing quail with endothelial cell nuclei (green) and neural crest . Movie begins at HH St11. The endothelial cell is migrating towards the neural tube encountering many neural crest cells in its path that are migrating in the opposite direction. The endothelial cell nucleus greatly deforms, squeezing through the narrow gaps in the neural crest stream as it passes. (MOV 9331 kb)Additional file 4. Endothelial cells deform nuclear shape while migrating through neural crest stream. Time-lapse confocal images of rhombomere 6 level of developing quail with endothelial cell nuclei (green) and neural crest , HH St12. An endothelial cell enters a densely packed neural crest area. The endothelial cell moves in the reverse direction until it exits the neural crest stream next to the otic vesicle. The endothelial cell then resumes migration toward the neural tube. (MOV 3622 kb)Additional file 5. Endothelial cells reverse direction of migration while in dense neural crest stream. Time-lapse confocal images of rhombomere 6 level of developing quail with endothelial cell nuclei  and inform trailing cells to follow , 40, we To analyze the in vivo mRNA expression pattern of Ang2 with higher fidelity and confirm the expression in migrating neural crest cells, we took advantage of multiplexed in situ hybridization chain reaction  and RT-n = 7) and Ang2-FL embryos (n = 10) showed a small but significant number of cells within ventromedial locations  and Ang2-FL embryos (n = 10) at the narrowest point , the neural crest migratory stream was significantly narrower than in controls , Ang2-FL (n = 8), or Ang2-shRNA (n = 4) electroporated neural crest and tracked single neural crest cells in the r4 migratory stream. We calculated the MSD curves for tracked cells and applied a statistical analysis to infer the type of cell motion in control, Ang2-FL, and Ang2-shRNA embryos. An MSD curve is determined by calculating the mean square displacement of a cell over different windows in time. The resulting curve is then fit to a model of cellular motion such as (D) random diffusion (straight line), (V) directed migration or flow (parabolic), (C) confined diffusion (asymptotic), or combinations of these . This made inherent sense, since we have previously observed run-and-tumble style neural crest cell behaviors in in vivo time-lapse analyses [To better understand the MSD curve data, we implemented the MSD-Bayes MATLAB package . This soanalyses , 43. To l-lysine, and neural crest cells migrated in all cases. The pFAK labeling appeared punctate as there were pFAK proteins scattered within each cell; some of the bright clusters of pFAK were small (<0.5 μm), and others were almost 1 μm in diameter  –20. Therter Fig. , insets.ter Fig. . We founter Fig. . In summTime-lapse imaging of cranial neural crest cells expressing control, Ang2-FL, or Ang2-shRNA revealed that the normal endothelial cell migratory pattern was disrupted. Specifically, we found that when neural crest cells are overexpressing Ang2, endothelial cells sprouted earlier from the dorsal aorta compared to wild-type embryos and prior to the neural crest cells migrating above the dorsal aorta , migrate away from the neural tube while endothelial cells (green) sprout from the dorsal aorta and migrate in the reverse direction. In general, as the neural crest passes the dorsal aorta, the endothelial cells sprout. However, with the overexpression of Ang2, the endothelial cells sprout earlier from the dorsal aorta and form a multicellular chain, but the chain collapses back to the dorsal aorta. (MOV 8304 kb)Additional file 9. Gain of Ang2 expression in neural crest cells alters timing and pattern of endothelial cell migration. Time-lapse, confocal images of developing quail embryo at the rhombomere 4 level of the head beginning HH St10. The neural crest cells, labeled with an overexpression vector for Ang2  form an unusual pattern emerging from below the otic vesicle. Normally a sprout of endothelial cells is seen extending from the dorsal aorta and moves along the caudal side of the r4 stream, but in this embryo where Ang2 is not expressed in the neural crest, a plexus of endothelial cells moves to the caudal side of the r4 neural crest stream. In addition, a multicellular structure of endothelial cells pushes into the neural crest stream, causing a few leading neural crest cells to become separated from the stream. These cells halt their forward progression until contact is made with the stream again. (MOV 9067 kb)Additional file 10. Loss of Ang2 expression in neural crest creates new vascular plexus on caudal side of neural crest stream. Time-lapse, confocal images of developing quail embryo at the rhombomere 4 level of the head beginning HH St10. The neural crest cells are marked with a vector containing an Ang2-shRNA in We used the quail model system to study the role of angiopoietin 2 (Ang2) in cranial neural crest cell migration. Gene profiling in our previous study identified Ang2 expression by microarray in migrating chick cranial neural crest cells; however, its role was unclear. Ang2 is a secreted factor and has been shown to stimulate cell migration in vitro in a variety of non-endothelial cell types –21. GiveStatic analyses of quail developmental stages showed the progression of cranial neural crest and endothelial cell positions and their close proximity, strengthening the argument for a dynamic interplay and cell sorting process during head patterning. By using the combination of the transgenic Tie1:H2B-YFP quail, neural crest cell labeling, and tissue clearing, we were able to more closely examine the spatial distribution of the endothelial cells near the neural crest cell migratory streams without relying on a perfusion technique that would have missed smaller, non-patent vessels or single cells undergoing vasculogenesis. At all stages examined (HH St9–15), at least a few endothelial cells were approximately 20 μm or closer to the r4 neural crest cell migratory stream. 3D reconstruction of images from collected z-stacks in tissue-cleared quail embryos showed how the neural crest cells surround the branchial arch arteries and the anatomical relationship between the neural crest cell migratory stream and endothelial cells that move to the perineural vascular plexus. Thus, although arising in distinct locations for the most part, neural crest and endothelial cells appeared to have a symbiotic relationship along a common migratory pathway prior to assembling structures within the peripheral branchial arches.Time-lapse analysis revealed a multistep process to neural crest and endothelial cell interactions, suggesting a stereotypical and coordinated patterning mechanism. During a typical time-lapse imaging session, individual endothelial cells were observed to initially gather near the dorsal aorta and sprout in single file chains towards the dorsal neural crest cell migratory pathway, immediately after the migratory front of the r4 neural crest cell migratory stream passed above. Endothelial cells then migrated alongside the rostro-caudal borders and underneath the neural crest cell migratory stream and in the opposite direction to connect with the perineural vascular plexus surrounding the neural tube. Given the correlation in time of the passing of the r4 neural crest cell migratory stream directly above the disassembly and the sprouting of the endothelial cells from the dorsal aorta, it is possible to speculate this event is coordinated. Furthermore, the expression of Ang2 by migrating neural crest cells and the presence of VEGF in the surface ectoderm and BA2 may provide a mechanistic basis for further study of how the neural crest and endothelial cells coordinate movements. In contrast to the pre-otic region, post-otic neural crest and endothelial cell interactions were less coordinated. The frequency of the two cell populations bumping into each other and subsequent changes in endothelial cell trajectories suggested a coarse molecular choreography dictated by the neural crest. Subsequent studies may uncover different molecular signals directing cell trafficking in this subregion of the embryo.Knowledge of the spatio-temporal expression pattern of Ang2 helped us determine whether Ang2 has a functional role on lead neural crest cells within the migratory front or on cells throughout the stream. Multiplexed fluorescence HCR confirmed the expression correlation between Ang2 and FoxD3, marking the migrating neural crest cells Fig. . The quaPerturbation of Ang2 signaling in neural crest cells led to changes in cell behavior, cell invasion into typical neural crest cell free zones, and increased FAK phosphorylation, suggesting a critical role for Ang2 in collective neural crest cell migration. In the embryo, gain of Ang2 function caused expansion of the neural crest cell migratory stream into neighboring inhibitory regions as well as individual cell invasion 100 μm or more away from the collective group and into the ventral paraxial mesoderm Fig. . MSD anaLoss of Ang2 function led to constriction of the neural crest cell migratory stream and loss of random motion or exploratory behavior. Given this trailing cell behavior evidence, it is interesting to speculate that Ang2 functions to promote cell invasion by enhancing chemokinesis or exploration. This would agree with our previous time-lapse imaging data that showed trailing neural crest cells move with a higher speed but less directionality than leaders , 43. MaiTo our knowledge, these data represent the first report of the critical function of Ang2 during collective neural crest cell migration and detailed characterization of the spatio-temporal dynamics of neural crest and endothelial cells during vertebrate head patterning. We propose a model in which Ang2 is secreted by migrating neural crest cells and stimulates cell invasiveness. Our hypothesis of the role of Ang2 to promote neural crest migration supported by these results fits well with our previously proposed cell-induced gradient model , 40 in w


Bitter (T2R) and sweet taste (T1R) receptors have emerged as regulators of upper airway immune responses. Genetic variation of these taste receptors additionally confers susceptibility to infection and has been implicated in severity of disease in chronic rhinosinusitis (CRS). Ongoing taste receptor research has identified a variety of biologically active compounds that activate T1R and T2R receptors, increasing our understanding of not only additional receptor isoforms and their function but also how receptor function may contribute to the pathophysiology of CRS. This review will discuss the function of taste receptors in mediating airway immunity with a focus on recently described modulators of receptor function and directions for future research into the potential role of genotypic and phenotypic receptor variation as a predictor of airway disease and response to therapy. The upper respiratory tract depends upon several mechanisms to detect and then mount defenses against pathogenic bacteria to prevent colonization and infection. Failure of these mechanisms is observed in many upper respiratory diseases, including chronic rhinosinusitis (CRS). While the etiology of CRS is multifactorial, defective mucociliary clearance resulting in the stasis of sinonasal secretions is a common final pathophysiologic contributor to the disease. Leading to ultimate mucociliary dysfunction are varied environmental and genetic factors   are expressed on ciliated airway epithelial cells and recognize conserved bacterial structures called pathogen-associated molecular patterns (PAMPs). Well-known PAMPs include lipopolysaccharide, a major component of the outer membrane of gram-negative bacteria, lipoteichoic acid in gram-positive bacteria, and flagellin  which is both directly bactericidal and also increases ciliary beat frequency and MCC . AHLs are quorum-sensing molecules produced by Bacillus cereus have also been found to elicit a T2R-mediated NO response  and nonfunctional (AVI) T2R38 variants  activation of T2R38 (Kim and Drayna, P. aeruginosa (Hariri et al., While T2R agonists themselves, such as PTC, could serve as possible therapeutic agents, recent studies have shown promise for a number of biologically active compounds. Flavones, a class of flavonoids produced by plants previously noted to have antibacterial effects, also activate T2Rs (Roland et al., Sweet T1R receptors on SCCs have been shown to block immune activation until infection depletes airways surface liquid glucose concentrations, decreasing T1R signaling and releasing its inhibition of T2R-mediated immune responses. The data to support this is strong. Addition of both the bitter compound denatonium and the T1R2/3 agonists glucose and sucrose to sinonasal airway cultures showed inhibition of the T2R-mediated calcium cascade while cultures derived from mice that do not express the T1R2/3 receptors (Lemon and Margolskee, Staphylococcus species are capable of activating SCC sweet taste receptors and inhibiting T2R-mediated signaling (Lee et al., Staphylococcus species are frequent sinonasal colonizers, suggesting that this may be one mechanism that allows them to evade immune eradication, T1R antagonists could be useful therapies in Staphylococcus-driven infections. Pharmacologic suppression of this T1R downregulatory pathway could thus prove to be a useful adjunct in augmenting the immune response, both in patients who would not be expected to respond to bitter agonists, but also in infection with particular bacterial species that have hijacked this pathway to evade immune detection. However, D-amino acids also inhibited P. aeruginosa biofilm formation, suggesting that immune suppression allowing commensal bacterial growth may offer protection against more pathogenic bacterial strains. Further research is needed to clarify the nature of such bacterial-host interactions prior to moving forward with T1Rs as a therapeutic target.Interestingly, bacteria may have found a way to co-opt this natural regulatory mechanism. Recent research has shown that D-amino acids produced by There is significant evidence to support the function of taste receptors as regulators of upper airway immunity. Activation of these receptors results in immediate-onset immune activation, complementing the more sustained and slower response achieved by activation of traditional innate immunity pathways such as TLRs, or at times suppression of the innate immune response. A wide range of biologically active compounds that activate T1R and T2R receptors have recently been described, increasing our understanding of not only additional receptor isoforms but also how receptor function may contribute to the pathophysiology of CRS. These findings suggest that targeting T1R and T2R receptors may allow for augmentation of the airway immune response and identify novel therapeutic targets that would allow the future treatment of CRS to be tailored to an individual patient's receptor profile. While further testing is needed, simple taste tests to determine taste perception phenotypes, which have shown a correlation with taste receptor genotype, may be used to predict not only susceptibility to disease but also potential response to a variety of therapies, allowing for truly personalized treatment.VT performed the literature search and wrote the manuscript. All authors reviewed and approved of the final 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. The reviewer RJ and handling Editor declared their shared affiliation.


T follicular helper (Tfh) cells are a subset of CD4 T cells that provide critical signals to antigen-primed B cells in germinal centers to undergo proliferation, isotype switching, and somatic hypermutation to generate long-lived plasma cells and memory B cells during an immune response. The quantity and quality of Tfh cells therefore must be tightly controlled to prevent immune dysfunction in the form of autoimmunity and, on the other hand, immune deficiency. Both Tfh and B cell perturbations appear during HIV infection resulting in impaired antibody responses to vaccines such as seasonal trivalent influenza vaccine, also seen in biologic aging. Although many of the HIV-associated defects improve with antiretroviral therapy (ART), excess immune activation and antigen-specific B and T cell responses including Tfh function are still impaired in virologically controlled HIV-infected persons on ART. Interestingly, HIV infected individuals experience increased risk of age-associated pathologies. This review will discuss Tfh and B cell dysfunction in HIV infection and highlight the impact of chronic HIV infection and aging on Tfh–B cell interactions. D-dimer, IL-6, and TNFα that correlate with occurance of age-associated diseases.Chronic infectious diseases, such as HIV infection, and the biological process of aging are known to impact humoral immune responses to vaccination and infection –5. The iD-dimer , with high surface expression of programed death receptor 1 (PD-1) and CXC chemokine receptor 5 (CXCR5) [reviewed in Refs. –37]. DurBecause of the difficulties in studying lymphoid tissue in humans, the field has increasingly relied on a circulating subset of memory CD4 T cells that partially resemble LN Tfh cells and have been designated as peripheral Tfh (pTfh) –47. The + virologically suppressed patients on ART exhibit functional impairment in their ability to provide adequate B cell help in a number of systems  B cells to an activated state with expression of activation markers such as CD71, CD80, and CD86 , 71. TheIn healthy states, antibody responses to T-dependent antigens are generated in GCs within lymphoid tissue when antigen-primed B and T cells engage in interactions to promote B cell differentiation, SHM, and class switch recombination to develop into memory B cells and plasma cells –83. Stud+ adults on ART during the 2009/H1N1 pandemic influenza outbreak  induction of IL21 gene in pTfh cells. These antigen-specific prevaccination measures strongly associated with H1N1-specific B cell responses by ELISPOT at postvaccination  on CD4 T cells, including pTfh subset, indicating an adverse influence of baseline immune activation and inflammation on vaccine induced antibody response in older age.Our group has been interested in the question of immune function of aging HIVfh cells , 97, 98.+ and HIV− adults grouped by age as young (<40 years), middle aged (40–59 years), and old (≥60 years). Following seasonal trivalent influenza vaccine (TIV), magnitude of Ab titers against each vaccine strain were found to be lower in old age compared to others, regardless of HIV status. Baseline titers in seroprotective range were higher in HIV+ but the frequency of VR was lower in HIV+ than HIV−. Interestingly the young HIV+ showed maximum variance from HIV− and more rapid decay in titer after peak at 28 days postvaccination. In statistical analysis somewhat surprisingly effect of age rather than HIV dominated the impaired immune response observed in old persons (age > 60 years), whereas HIV clearly had a strong effect on immunity at younger ages  was negatively associated with vaccine response  identified by coexpression of HLA-DR and CD38, as well as expression of PD-1, ICOS, and Ki-67 were higher in HIV as ICOS  were alrresponse .+ than in young HIV− (− individuals but underlying mechanisms appear to be distinct from that associated with biological aging (+, even with prolonged ART-induced virological suppression (Figure Prevaccination status of B cells also revealed perturbations as evidenced by alteration in markers of activation, exhaustion and immune regulation and were more prevalent in young HIVung HIV− . HIV infal aging . For exaal aging . These rn Figure . Additio+ and CD8+ T cells express several inhibitory receptor molecules, including PD-1, LAG-3, CTLA-4, and KLRG1 (Other factors that could influence the influenza vaccine response in aging also need consideration. Data from literature suggest that vaccine-induced immune responses are considerably influenced by demographic variables such as age, sex, ethnicity, and race –105. Mannd KLRG1 , 116 thand KLRG1 –120]. HoDevelopment of a protective antibody response to vaccine or infection is important for the control or eradication of many pathogenic infections. Efficient Tfh–B cell interactions are required for regulating B cell differentiation toward the development of high affinity antibodies. Immune mechanisms underlying the regulation of Tfh–B cell interactions at the inductive sites of the immune response are an active area of immunology research. Several studies have highlighted the qualitative and quantitative impairment of Tfh compartment and their subsequent impact on humoral arm of immune response in treated HIV infection , 94, 98.+ individuals.Recent advances in the field of immune checkpoint inhibitor-based immunotherapeutic approaches in cancer immunology have highlighted the importance of cell to cell interactions on immune function. Many aspects of checkpoint molecule-based regulation of humoral immune response on Tfh and B cell interactions at the GC are not known. Trials employing checkpoint inhibitors in HIV infection will need to ensure that improved Tfh–B cell interactions not associated with autoimmunity. Immune checkpoints are negative regulators of T cell activation, T cell proliferation and effector functions and inhibiting immune checkpoints could influence and disrupt the resting status of latently infected cells and reverse latency with increase in HIV replication within GC . Future All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Sputum and blood eosinophil counts have attracted attention as potential biomarkers in chronic obstructive pulmonary disease (COPD). One question regarding the use of blood eosinophils as a biomarker in COPD is whether their levels are affected by the use of inhaled corticosteroids (ICS), which are commonly prescribed for COPD.We performed a retrospective analysis of peripheral blood leucocytes from a previously completed clinical trial that examined effects of ICS in steroid-naïve patients with COPD.The data show that the ICS-containing treatment arms (containing fluticasone propionate) had a small effect on peripheral blood eosinophils in steroid-naïve patients with COPD.NCT00358358; Post-results. Inhaled corticosteroids have a small effect on peripheral blood eosinophil counts.The effect is likely small enough on average not to affect patient stratification using cut-off values.Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease for which there are limited choices with respect to therapeutic mechanisms of action. In order to direct patient-specific therapy in a disease with high prevalence and high clinical variability, the medical community needs to identify and scientifically validate COPD biomarkers.Sputum and blood eosinophil counts have attracted attention as potential biomarkers. For example, higher sputum eosinophil counts in patients with COPD predicts clinical response to inhaled corticosteroids (ICS),One question regarding the use of blood eosinophils as a biomarker in COPD is whether their levels are affected by the use of ICS, which are commonly prescribed for COPD. Our post hoc analysis of an interventional study in steroid-naïve patients with COPD evaluated the extent to which ICS affected blood eosinophil counts and compared the distribution of baseline eosinophil counts in the study with the COPD and non-COPD control cohorts from  ECLIPSE.1)/forced vital capacity (FVC) ≤70% and a post salbutamol FEV1 ≥30% and ≤70% of predicted. Individuals were excluded if they used oral corticosteroids or ICS-containing products for more than 14 consecutive days in the prior 6 months or at any time within 30 days of screening.SCO104925 (NCT00358358) was a 12-week, double-blind, parallel-group study comparing patients randomised to receive placebo, fluticasone propionate [FP]/salmeterol combination (FSC) or the individual components of FSC . Individuals were included if they had COPD as defined by a post salbutamol forced expiratory volume in 1 s  had FEV1/FVC >70% and FEV1 >85%.The design of the ECLIPSE observational study  has been published elsewhere.Both studies were sponsored by GSK and conducted according to Good Clinical Practice and in accordance with guidelines from the Declaration of Helsinki. Each study protocol was approved by relevant ethical boards and institutional review boards at participating centres.Owing to the small sample size for the study and the presence of outliers, summaries for continuous variables are provided as median and IQR; categorical summaries are presented as percentages. Hodges-Lehmann estimatesOverall, 161 persons were randomised to treatment in study SCO104925, with 80 assigned to non-ICS-containing treatments  and 81 to ICS-containing treatments (FSC or FP). One hundred and fourty-three patients completed the 12-week study; 73 from the non-ICS-treated patients and 70 from the ICS-treated patients. To remove potential confounding factors, patients who either had an exacerbation and/or were prescribed oral corticosteroids during the treatment period were excluded. Accounting for these factors, 59 patients from the non-ICS-treated arms and 53 patients from the ICS-treated arms  were used in this post hoc analysis.The primary and key secondary outcome measures for SCO104925 measured the impact of ICS on impulse oscillometry and lung imaging end points in steroid-naïve patients with COPD.In SCO104925, median (IQR) blood eosinophil count was 200 (120 to 310) cells/μL at baseline for patients who received ICS during treatment, and 230 (120 to 410) cells/μL for patients not receiving ICS during treatment. Median (IQR) changes in eosinophil count for ICS-treated patients were −30 (−90 to 10) cells/μL at week 6 and −30 (−90 to 20) cells/μL at week 12, while median (IQR) changes for non-ICS-treated patients were −20 (−80 to 70) cells/μL at week 6 and 10 (−80 to 80) cells/μL at week 12. The differences in eosinophil count changes between groups were not significant at week 6 (median (95% CI) difference −20 (–60 to 20); p=0.315) nor at week 12 (median (95% CI) difference −40 (80 to 10); p=0.090). However, this may be due to lack of power for these comparisons based on the size of the study. Study characteristics and eosinophil levels are shown in To contextualise the findings from SCO104925, the distribution of baseline blood eosinophil counts in SCO104925 patients was compared with those in the ECLIPSE study. The baseline eosinophil counts of non-COPD controls in the ECLIPSE study were distributed differently  than the eosinophil counts of patients with COPD in ECLIPSE and SCO104925 (p<0.001 for both comparisons with controls). The distribution of blood eosinophils for SCO104925 patients was not significantly different from either the ECLIPSE COPD cohort reporting ICS use (p=0.102) or those not reporting ICS use (p=0.137). Consistent with ICS having a small effect on blood eosinophil counts, there was no significant difference in the distribution of blood eosinophil counts in ECLIPSE patients reporting ICS use at baseline compared with those patients not reporting ICS use p=0.246; . SummaryAs some of the recent discussion regarding blood eosinophils as a biomarker to direct treatment revolves around threshold values, we investigated changes in peripheral blood eosinophil levels based on thresholds and found that although the majority of patients remain in their baseline category, there is some regression to the mean with respect to using thresholds .The desire to practice precision medicine has prompted a broad, intensive search for biomarkers that can be used to direct personalised therapy in COPD. Eosinophils have been part of this search for more than a decade.


Asthma gene DNA methylation may underlie the effects of air pollution on airway inflammation. However, the temporality and individual susceptibility to environmental epigenetic regulation of asthma has not been fully elucidated. Our objective was to determine the timeline of black carbon (BC) exposure, measured by personal sampling, on DNA methylation of allergic asthma genes 5 days later to capture usual weather variations and differences related to changes in behavior and activities. We also sought to determine how methylation may vary by seroatopy and cockroach sensitization and by elevated fractional exhaled nitric oxide (FeNO).Personal BC levels were measured during two 24-h periods over a 6-day sampling period in 163 New York City children (age 9–14 years), repeated 6 months later. During home visits, buccal cells were collected as noninvasive surrogates for lower airway epithelial cells and FeNO measured as an indicator of airway inflammation. CpG promoter loci of allergic asthma genes , interferon gamma (IFNγ), inducible nitric oxide synthase (NOS2A)), arginase 2 (ARG2)) were pyrosequenced at the start and end of each sampling period.−48 5 days later. The magnitude of association between BC exposure and demethylation of IL4 CpG−48 and NOS2A CpG+5099 measured 5 days later appeared to be greater among seroatopic children, especially those sensitized to cockroach allergens , compared to non-sensitized children ; however, the difference was not statistically different. In multivariable linear regression models, lower DNA methylation of IL4 CpG−48 and NOS2A CpG+5099 were associated with increased FeNO.Higher levels of BC were associated with lower methylation of IL4 promoter CpGOur results suggest that exposure to BC may exert asthma proinflammatory gene demethylation 5 days later that in turn may link to airway inflammation. Our results further suggest that seroatopic children, especially those sensitized to cockroach allergens, may be more susceptible to the effect of acute BC exposure on epigenetic changes.The online version of this article (doi:10.1186/s13148-017-0361-3) contains supplementary material, which is available to authorized users. A consistent pattern also was observed when stratified by cockroach sensitization . When we controlled for food intake and eliminated those on asthma/allergy medications, the main findings in Table 1 and BDNA2, instead of dichotomous outcome, was used in the main analysis, a similar trend was observed for IL4 CpG−48 (but not for other loci). The higher levels of BC were associated with a decrease in methylation of IL4 CpG−48 over 5 days  = −1.75 (0.048)) in multivariable linear regression model  [Our findings of an effect of short-term (24-h) BC on changes in DNA methylation and of methylation on FeNO, each 5 days later, among urban children are novel. In comparison, we did not detect the same-day effect of BC on lower methylation of IL4 CpGexhaust) , 54. Oneexhaust) . Otherwiexhaust) , 56. OurWe found seemingly delayed effects of DNA hypomethylation of two allergic asthma genes on FeNO levels measured 5 days later. Lower methylation, consistent with the potential for greater gene expression, could underlie the reported association between measures of air pollution and increases in FeNO over this time course , 40, 57.Interestingly, we observed that the associations of DNA methylations with BC and FeNO appeared stronger among seroatopic and cockroach-sensitized children, compared to non-atopic children. Several pediatric cohorts, including ours, have investigated the effects of air pollution by asthma phenotypes , 59, 60,−48, previously shown and validated as associated with urban asthma [+5099 in buccal cells, new elements in our study design included the personal exposure assessment with specific focus on BC, capture of effects of an even shorter duration of exposure (24-h average instead of 7-day cumulative average), and a delayed effect of exposure. An additional distinction was the consideration of cockroach sensitization.We focused on epigenetic changes among known allergic asthma genes and previously reported regions of altered epigenetic regulation. By doing so, we were able to replicate the importance of the IL4 CpGn asthma . We exten asthma , 56. WhiThe reductions in allergic asthma gene methylation measured here as relative risks captured the relatively small yet significant changes in levels of methylation that were predicted to occur in daily urban living and following the behaviors of older children. We acknowledge that these small differences in single CpG sites, which essentially indicate changes among a low percent of cells within the tissues, may only partially explain the underlying immunopathogenesis. But in specific contexts, they may be reasonable surrogate measures or indicate functional differences at the cellular level . Small d−326 findings are in the same direction as those of CpG−48 in the analysis with BC, especially in stratified analysis by seroatopic, although not statistically significant. One explanation may be that we had insufficient statistical power to detect a difference at CpG−326 related to personal BC exposure. The small differences in methylation by BC exposure may be difficult to differentiate small differences in methylation from noise particularly at the lowest end of the spectrum as measured for ARG2 or from those measured in duplicate data [We acknowledge several additional limitations, including the relatively small sample size necessitated by our relatively comprehensive and repeated exposure assessment. The IL4 CpGate data . We did ate data , 25–32 (ate data . We do nThese findings offer new evidence of the relatively acute and delayed temporal effects of environmental epigenetic regulation of allergic asthma genes and their differential susceptibility by allergic phenotype. Further findings suggest associations between environmental epigenetic regulation and airway inflammation. Together, these findings inform on the dynamics of environmental epigenetic regulation among a highly susceptible, mobile and difficult to capture pediatric age group.