Healthy Organ Donors Research Articles

Gene expression networks in endothelial cells from failing human hearts.

Chronic heart failure is associated with adverse remodeling of the heart that is typically characterized by cardiomyocyte hypertrophy. This requires the formation of new capillaries to maintain oxygen supply. Insufficient angiogenesis promotes the transition from compensated hypertrophy into heart failure. The aim of this study was to identify angiogenesis-related gene networks and corresponding regulatory hubs in endothelial cells from failing human hearts. We isolated left ventricular endothelial cells from patients with advanced heart failure undergoing left ventricular assist device surgery (n = 15) and healthy organ donors (n = 2) and performed RNA sequencing. Subgroup analysis revealed no impact of comorbidities on gene expression. In a weighted gene coexpression network analysis, we found 26 gene clusters, of which 9 clusters showed a significant positive or negative correlation with the presence of heart failure. We identified the transcription factors CASZ1 (castor zinc finger 1), ZNF523 (zinc finger protein 523), and NFE2L1 (nuclear factor erythroid 2-related factor 1) as hub genes of a cluster related to angiogenesis. Knockdown of CASZ1, ZNF523, or NFE2L1 in human umbilical vein endothelial cells led to a downregulation of genes from the respective cluster, including CD34 and platelet-derived growth factor-β, confirming their regulatory function. In conclusion, we assessed gene networks in endothelial cells and identified transcription factors CASZ1, ZNF532, and NFE2L1 as potential regulators of angiogenesis in failing human hearts. Our study provides insights into the transcriptional regulation of angiogenesis beyond the classical vascular endothelial growth factor signaling pathway.NEW & NOTEWORTHY Gene coexpression network analysis defined 26 gene clusters expressed in endothelial cells from failing human hearts. Transcription factors CASZ1, ZNF523, and NFE2L1 were identified as hub genes of a cluster related to angiogenesis. Knockdown of CASZ1, ZNF523, or NFE2L1 in human umbilical vein endothelial cells led to a downregulation of genes from the respective cluster, confirming their regulatory function. This provides insights into the transcriptional regulation of angiogenesis in heart failure beyond classical signaling pathways.

Germline-like TCR-α chains shared between autoreactive T cells in blood and pancreas

Human type 1 diabetes (T1D) is caused by autoimmune attack on the insulin-producing pancreatic beta cells by islet antigen-reactive T cells. How human islet antigen-reactive (IAR) CD4+ memory T cells from peripheral blood affect T1D progression in the pancreas is poorly understood. Here, we aim to determine if IAR T cells in blood could be detected in pancreas. We identify paired αβ (TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new-onset, and established T1D donors, and measured sequence overlap with TCRs in pancreata from healthy, at risk and T1D organ donors. We report extensive TRA junction sharing between IAR T cells and pancreas-infiltrating T cells (PIT), with perfect-match or single-mismatch TRA junction amino acid sequences comprising ~29% total unique IAR TRA junctions (942/3,264). PIT-matched TRA junctions were largely public and enriched for TRAV41 usage, showing significant nucleotide sequence convergence, increased use of germline-encoded versus non-templated residues in epitope engagement, and a potential for cross-reactivity. Our findings thus link T cells with distinctive germline-like TRA chains in the peripheral blood with T cells in the pancreas.

Contribution of HLA class I (A, B, C) and HLA class II (DRB1, DQA1, DQB1) alleles and haplotypes in exploring ethnic origin of central Tunisians

BackgroundEstimation of HLA (Human leukocyte Antigen) alleles’ frequencies in populations is essential to explore their ethnic origin. Anthropologic studies of central Tunisian population were rarely reported. Then, in this work, we aimed to explore the origin of central Tunisian population using HLA alleles and haplotypes frequencies.MethodsHLA class I (A, B, C) and HLA class II (DRB1, DQA1, DQB1) loci genotyping of 272 healthy unrelated organ donors was performed by Polymerase Chain Reaction-Sequence Specific Oligonucleotide (PCR-SSO). We compared central Tunisians with other populations (Arabs, Berbers, Mediterraneans, Europeans, Africans, etc.) using alleles and haplotypes frequencies, genetic distances, Neighbour-Joining dendrogram and correspondence analysis.ResultsAmong the 19 HLA A alleles, the 26 HLA B alleles, the 13 HLA C alleles, the 15 HLA DRB1 alleles, the 6 HLA DQA1 alleles and the 5 HLA DQB1 alleles identified in the studied population, HLA A*02 (22.8%), HLA B*50 (13.1%), HLA C*06 (21.8%), HLA DRB1*07 (17.8%), HLA DQA1*01 (32.1%) and HLA DQB1*03 (31.6%) were the most frequent alleles. The extended haplotypes HLA A*02-B*50-C*06-DRB1*07-DQA1*02-DQB1*02 (1.97%) was the most frequent HLA six-loci haplotype.ConclusionCentral Tunisians were very close to other Tunisian populations, to Iberians and North Africans. They were rather distant from sub-Saharan populations and eastern Mediterraneans especially Arabs although the strong cultural and religious impact of Arabs in this population.

Comparative Intra-Subject Analysis of Gene Expression and Protein Abundance of Major and Minor Drug Metabolizing Enzymes in Healthy Human Jejunum and Liver.

First pass metabolism by phase I and phase II enzymes in the intestines and liver is a major determinant of the oral bioavailability of many drugs. Several studies analyzed expressions of major drug-metabolizing enzymes (DMEs), such as CYP3A4 and UGT1A1 in the human gut and liver. However, there is still a lack of knowledge regarding other DMEs (i.e., "minor" DMEs), although several clinically relevant drugs are affected by those enzymes. Moreover, there is very limited intra-subject data on hepatic and intestinal expression levels of minor DMEs. To fill this gap of knowledge, we analyzed gene expression (quantitative real-time polymerase chain reaction) and protein abundance (targeted proteomics) of 24 clinically relevant DMEs, that is, carboxylesterases (CES), UDP-glucuronosyltransferases (UGT), and cytochrome P450 (CYP)-enzymes. We performed our analysis using jejunum and liver tissue specimens from the same 11 healthy organ donors (8 men and 3 women, aged 19-60 years). Protein amounts of all investigated DMEs, with the exception of CYP4A11, were detected in human liver samples. CES2, CYP2C18, CYP3A4, and UGT2B17 protein abundance was similar or even higher in the jejunum, and all other DMEs were found in higher amounts in the liver. Significant correlations between gene expression and protein levels were observed only for 2 of 15 jejunal, but 13 of 23 hepatic DMEs. Intestinal and hepatic protein amounts only significantly correlated for CYP3A4 and UGT1A3. Our results demonstrated a notable variability between the individuals, which was even higher in the intestines than in the liver. Our intrasubject analysis of DMEs in the jejunum and liver from healthy donors, may be useful for physiologically-based pharmacokinetic-based modeling and prediction in order to improve efficacy and safety of oral drug therapy.

Abstract 15806: The Unique Cellular Ecosystem of the Human Basilic Vein: A Single-Cell Transcriptomic Analysis

Introduction: The cells in veins that contribute to vascular disease have not been fully characterized. This study generated a cellular atlas of the human basilic vein that will help understand vein physiology and pathologies. Methods: Single-cell sequencing of four basilic veins from healthy organ donors. Results: The integrated single-cell profile of 15,747 vein cells revealed 13 populations containing canonical and non-canonical vascular markers. The SMC population represented 16% of cells, of which 8% were purely contractile and the remaining 8% had a modulated phenotype. One cluster of modulated SMCs expressed ECM genes (collagen type IV, FN1, and TNC) while retaining high expression of contractile markers (DES, MYH11, ACTG2). The other SMC cluster had unique expression of CD36, APOE, LPL, and STEAP4, characteristic of lipid-processing cells. Surprisingly, these non-diseased veins contained a high number of resident immune cells, including pro-inflammatory macrophages (9%), CD163- and HMOX1-expressing Mhem-like macrophages (18%), lymphocytes (5%), and small counts of neutrophils (0.3%) and mast cells (0.7%). The adventitial cell populations highlighted the fundamental role of this layer in vascular maintenance, remodeling, and angiogenesis. We found a significant number of vasa vasorum ECs (6.5%) which differentiated from the luminal subpopulation (3.5%) by CD34, KDR, NOTCH4, and ICAM2 expression. There were also two types of fibroblasts. Classical fibroblasts (19%) had high expression of collagen genes, while adipogenic fibroblasts (21%) were characterized by high levels of ECM-interacting fibulins, adipocyte-associated genes (ADH1B, APOD, CXCL14, IGF1), and modulators of angiogenesis (ADM and EFEMP1). CellCall analyses identified 46 overrepresented ligand-receptor communication pathways in all clusters, including CXCL, FGF, IL-6, and complement signaling. Communication between pro-inflammatory macrophages and luminal ECs was the most overrepresented signaling connection. Conclusions: This comprehensive atlas of the human vein will help identify cell subpopulations relevant to venous pathologies, without relying on extrapolations from arterial biology and focusing instead on the uniqueness of vein tissues.

Approach to optimal assessment of right ventricular remodelling in heart transplant recipients: insights from myocardial work index, T1 mapping, and endomyocardial biopsy.

Right ventricular (RV) dysfunction is an important cause of graft failure after heart transplantation (HTx). We sought to investigate relative merits of echocardiographic tools and cardiac magnetic resonance (CMR) with T1 mapping for the assessment of functional adaptation and remodelling of the RV in HTx recipients. Sixty-one complete data set of echocardiography, CMR, right heart catheterization, and biopsy were obtained. Myocardial work index (MWI) was quantified by integrating longitudinal strain (LS) with invasively measured pulmonary artery pressure. CMR derived RV volumes, T1 time, and extracellular volume (ECV) were quantified. Endomyocardial biopsy findings were used as the reference standard for myocardial microstructural changes. In HTx recipients who never had a previous allograft rejection, longitudinal function parameters were lower than healthy organ donors, while ejection fraction (EF) (52.0 ± 8.7%) and MWI (403.2 ± 77.2 mmHg%) were preserved. Rejection was characterized by significantly reduced LS, MWI, longer T1 time, and increased ECV that improved after recovery, whereas RV volumes and EF did not change MWI was the strongest determinant of rejection related myocardial damage (area under curve: 0.812, P < 0.0001, 95% CI: 0.69-0.94) with good specificity (77%), albeit modest sensitivity. In contrast, T1 time and ECV were sensitive (84%, both) but not specific to detect subclinical RV damage. Subclinical adaptive RV remodelling is characterized by preserved RV EF despite longitudinal function abnormalities, except for MWI. While ultrastructural damage is reflected by MWI, ECV, and T1 time, only MWI has the capability to discriminate functional adaptation from transition to subclinical structural damage.

A novel association of efavirenz induced severe cutaneous adverse reactions with HLA- DRB1*03:01: A case-control study from North-East India.

HIV-infected patients have a higher risk of developing cutaneous reactions to drugs than the general population. Severe cutaneous adverse reactions (SCARs) are not uncommon in patients taking antiretroviral therapy (HAART]. To evaluate HLA class I and II allele frequencies in HIV patients on HAART who develop SCARs due to nevirapine (NVP] or efavirenz (EFZ] containing regime and compare this genotype composition with HAART tolerant patients and healthy organ donors. A case-control study for 4 years was conducted with four subsets of patients hailing from north-east India:Cohort 1- HIV seropositive patients who developed SCARs due to EFZ (n = 8];Cohort 2 - HIV seropositive patients who developed SCARs due to NVP (n = 15]; Cohort 3 -HIV seropositive NVP/EFZ-tolerant patients (n = 18]; Cohort 4 - Healthy HIV seronegative organ donors (n = 169].Cohort 3 & 4 acted as control-group. These patients were genotyped for the HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1 by a sequence-based HLA typing method. HLA-DRB1*03:01 allele revealed a significant association with EFZ regimen-induced SCARs in 62.5% patients compared with only 5.56% observed in HAART-tolerant patients and 4.14% in healthy organ. HLA-B*3505was found to be significantly associated with NVP induced SCARs. We found significant novel association of HLA-DRB1*03:01 with EFZ induced SCARs in North-East Indian HIV patients. Thus, HLA-DRB*03:01 may be useful as a genetic marker to avoid EFZ induced serious cutaneous rashes. The molecular HLA characterization of these alleles may provide a novel insight into the immunological basis of the antiretroviral drug reactions.

Comprehensive analyses of B-cell compartments across the human body reveal novel subsets and a gut-resident memory phenotype

AbstractAlthough human B cells have been extensively studied, most reports have used peripheral blood as a source. Here, we used a unique tissue resource derived from healthy organ donors to deeply characterize human B-cell compartments across multiple tissues and donors. These datasets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBCs). A comprehensive antibody-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct functions defined according to surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA-sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut- and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B-cell compartments across multiple tissues.

Integrin α9 is involved in the pathopoiesis of acute aortic dissection via mediating phenotype switch of vascular smooth muscle cell

Acute aortic dissection (AAD) is a devastating disease with high mortality; however, the pathogenic mechanisms of AAD remain poorly understood. Our present study aimed to identify genes associated with AAD and explore the molecular function of candidate genes in the pathogenesis of AAD. We used a whole-genome transcriptional microarray to identify putative AAD genes using ascending aortic tissues from four patients with AAD and four healthy organ donors. The differentially expressed genes were further validated in eight patients with AAD and eight healthy organ donors. Functional assessments were conducted to analyze the effects of the identified AAD genes on the phenotype of aortic vascular smooth muscle cells (VSMCs). The whole-genome transcriptional microarray analysis found 129 dysregulated genes in the ascending aortic tissues of AAD (fold change≥2), which were mainly associated with the focal adhesion pathway and actin cytoskeleton regulation pathway. Among these genes, integrin α9 (ITGA9) was identified to be involved in both pathways and downregulated by 50% in AAD patients. The association of ITGA9 with AAD was confirmed by Western blotting analysis (P = 0.003). Functional studies showed that knocking down ITGA9 in VSMCs resulted in a decrease in contractile markers (SM22α and α-SMA) and an increase in synthetic markers (OPN and SMemb), suggesting that the VSMCs switched from a contractile to a synthetic phenotype. After overexpression of ITGA9 by a recombinant adenovirus vector in VSMCs, SM22α and α-SMA were upregulated, while SMemb was downregulated, indicating a phenotypic switch from the synthetic to contractile phenotype of VSMCs. In conclusion, our study identified ITGA9 as a novel AAD gene. This gene is downregulated in patients with AAD and is involved in the regulation of the phenotypic switch of VSMCs from a contractile to a synthetic phenotype.

2104-P: Proinflammatory Cytokines Impair Human Islet and Beta-Cell Glucose Oxidation

Pro-inflammatory cytokines contribute to beta cell dysfunction in type 1 diabetes (T1D), but the exact underlying mechanisms remain unclear. It was previously shown that cytokines inhibit mitochondrial glucose oxidation leading to impaired glucose-stimulated insulin secretion in rat INS-1E cells, however, the effect of cytokines on mitochondrial respiration in human islets has not been explored. In this study, we investigated mitochondrial dysfunction in human islets and rat beta cells following cytokine treatment. We exposed isolated human pancreatic islets and rat INS-1E cells for up to 24 h to IL-1β and IFN-γ to mimic early beta cell destruction in T1D. Mitochondrial bioenergetics were analyzed by Seahorse XFe24 technology. Human islets from three healthy organ donors exposed to cytokines showed a reduction in glucose-induced oxygen consumption and an increase in ATP-uncoupled respiration. Higher basal mitochondrial oxygen consumption was also observed, which was not associated with an increase in ATP production. In INS-1E cells, cytokines exerted a strong inhibitory effect on mitochondrial glucose oxidation in a dose-independent manner. In acute exposure experiments, no effect on oxygen consumption was observed up to eight hours after cytokine exposure. This finding proposes that cytokine-induced inhibition of mitochondrial respiration is dependent on secondary signaling mechanism and/or changes in gene transcription. To conclude, glucose-induced oxygen consumption is reduced by cytokines in both human islets and INS-1E cells indicative of impaired mitochondrial respiration. Moreover, cytokines augment mitochondrial uncoupled respiration in human islets, indicating mitochondrial dysfunction that may contribute to islet failure in T1D. Ongoing work is investigating gene expression of genes involved in mitochondrial respiration processes. Further studies are needed to clarify the possible pathogenic role of mitochondrial dysfunction in T1D and the underlying mechanisms. Disclosure J. Melo: None. V. Hirschberg Jensen: None. J. Størling: None.

Placenta-derived mesenchymal stem cells enhance human liver organoid maturation: translational implications for liver regeneration

Background & Aim The global prevalence of end-stage liver disease (ESLD) has reached very high levels, and, to date, the best treatment option is orthotopic liver transplantation (1). However, due to the complexity of this procedure and the limited numbers of healthy organ donors, new therapeutic options are being sought. Liver progenitor and multipotent stem cells offer potential cell sources that could be used clinically (2). Different studies have reported therapeutic effects of transplanted mesenchymal stem cells (MSCs) on ESLD (3). Indeed, an improvement of liver function has been achieved in patients with liver cirrhosis after autologous MSCs injection (4). Based on our recent work where we showed that a three-dimensional (3D) culture method is effective to improve MSCs paracrine activity (5), in this work, we used a human liver organoid system to study if the paracrine signal secreted by human amnion-derived MSCs (hAMSCs) affect liver stem/progenitor cells to differentiate (Fig. 1). Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ function to model and treat human disease (6). Methods, Results & Conclusion Our data show that the expanded human liver organoids had distinct traits in the ALB production, Krt19 and HNF1β expression when compared to the reference cell line HepG2 cells. On liver organoids, we tested conventional differentiation medium (DM) conditioned by both 2D (2D CM) and 3D (3D CM) hAMSCs cultures observing that ALB, NTCP (markers of mature hepatocytes), and LGR5 (stem cell marker) expression were significantly upregulated by 3D CM compared with both 2D CM and conventional DM. Furthermore, CYP3A4 expression (marker of mature hepatocytes) was upregulated in both 2D and 3D CM. These data were also confirmed by the over-production/activity of both ALB and CYP3A4 observed in organoids grown with 3D CM. Liver epithelial dysfunctional repair play a role in the development of liver disease, and, effective repair likely requires the normal functioning of liver stem/progenitor cells. Herein, we showed that hAMSCs, principally grown in 3D cultures, has the potential to increase hepatic stem/progenitor cell differentiation, demonstrating that soluble factors secreted by hAMSCs are likely responsible for the response. This work revealing a potential approach to improve cell transplantation with the ultimate goal being efficient liver regeneration.

Quantification of Target Proteins Regulated by Micrornas in Abdominal Aortic Aneurysm Tissue

Introduction: microRNAs (miRNAs) are small non-coding RNA molecules that regulate protein expression. In a previous study we identified a set of miRNAs dysregulated in tissue of Abdominal Aortic Aneurysm (AAA) patients followed by an in silico analysis of their potential targets. In the present study our aim was to confirm if some of these protein targets were also dysregulated in AAA tissue, thus revealing novel insights into the mechanisms of AAA development. Methods: We obtained abdominal aortic tissue from patients undergoing AAA open repair surgery (N=11) and from healthy organ donors (N=7). Proteins were extracted with the miRVANA PARIS® kit (Ambion). Five proteins were selected to be quantified by Western Blot using specific antibodies and their expression was normalized against that of α-tubulin. Expression values were compared between groups with a student t test. Correlation of proteins and miRNAs expression was analyzed with Pearson correlation test. Results: Increased levels of thrombospondin-2 (THBS2) were observed in AAA tissue compared with healthy tissue (10.7±2.7 vs 2.8±0.7, relative expression) (P=0.04). Moreover, the expression levels of THBS2 negatively correlated with one of their potentially regulator miRNAs, miR-195-5p (r2=0.442, ρ=-0.665, P=0.0026). Two other proteins, LIM and SH3 domain protein 1 (LASP1) and Human Integrin alpha V (ITGAV), were also slightly overexpressed in AAA patients but without significant results. No correlation with their potentially regulator miRNAs were found. Conclusion: We have confirmed the overexpression of a potential target of a dysregulated miRNA in AAA tissue. Together with other studies, our results highlight the regulatory role of miR-195-5p in AAA, however further studies need to be done to elucidate its role in the pathology. In that sense we propose THBS2, a potential target of miRNA-195-5p, as a possible mediator in AAA. THBS2 has been previously related to the regulation of angiogenesis (1) and vascular smooth muscle cells proliferation (2) thus its involvement in vascular pathologies is highly plausible. Disclosure: Nothing to disclose

Study of Dysregulated Micrornas in Abdominal Aortic Aneurysm Tissue

Introduction - microRNAs (miRs) are small non-coding RNA molecules that regulate protein expression (1). The initiation and progression of Abdominal Aortic Aneurysm (AAA) can be regulated by miRs (2), hence their expression in abdominal aortic tissue could be useful for the identification of novel therapeutic targets. In a previous study, we identified some dysregulated miRs in plasma of patients with AAA. In the current study we have measured the expression level of those miRs in AAA and in healthy tissues in order to elucidate their local dysregulation. Methods - We obtained abdominal aortic tissue from patients undergoing AAA open repair (N=21) and from healthy organ donors (N=8). Tissue samples were snap-frozen and stored in liquid N2. A cross section of the tissue was homogenized with the TissueLyser LT® (Qiagen), and total RNA was obtained with the miRVANA PARIS® kit (Ambion). cDNA was obtained from 10 ng of total RNA. Sixteen miRs were quantified by Real Time quantitative PCR (RT-qPCR)(Exiqon) and results (CTs) were normalized with the endogenous and stable miR 423-5p selected using RefFinder. Normalized results were adjusted in a linear regression model using the statistic software R (3.3.2 version). Results - Six miRs were significantly underexpressed in AAA vs healthy tissue. The expression of miR-1, miR-27-b-3p, miR-29b-3p, mir-133a-3p, miR-133b and miR-195-5p was 4.8 (p<0.001); 2 (p<0.001); 1.4 (p=0.018); 4.4 (p<0.001); 4.6 (p<0.001) and 1.6 (p=0.023) fold lower than healthy tissue, respectively. Furthermore, 3 miRs were overexpressed in AAA tissue. Thus, miR-146a-5p, miR-21-5p and miR-144-3p were overexpressed 5.8 (p<0.001); 1.9 (p=0.012) and 7.2 (p<0.001) times, respectively. Some targets of these dysregulated miRs are related to vascular wall integrity (COL), apoptosis (PTEN) or inflammation (IRAK1) pathways. Conclusion - Our preliminary study confirms the dysregulation of 9 miRs in AAA tissue. Our results support the role of these miRs in the development of AAA. Some of the target proteins of these dysregulated miRs are involved in several mechanisms related to vascular pathologies. Thus, miR-29b is involved in collagen deposition and fibrosis, miR146a-5p in inflammation and apoptosis and miR-133b or miR-21-5p in inflammation, apoptosis and cellular proliferation and differentiation (3). However, the role of other miRs such as miR-27b-3p or miR-144-3p in AAA development is yet to be elucidated. The validation of our results in a larger cohort could reveal these miRs as therapeutic targets in AAA.

Liver-Derived TGF-β Maintains the EomeshiTbetlo Phenotype of Liver Resident Natural Killer Cells

The adult human liver hosts a complex repertoire of liver resident and transient natural killer (NK) cell populations with diverse phenotypes and functions. Liver resident NK cells are CD56bright NK cells defined by a unique expression profile of transcription factors and cell surface markers (EomeshiTbetloTIGIT+CD69+CXCR6+CD49e−). Despite extensive characterization of the phenotype of liver resident NK cells, it remains unclear how factors within the liver microenvironment induce and maintain this unique phenotype. In this study, we have explored the factors regulating the phenotype of liver resident NK cells. Isolation of healthy liver resident NK cells from donor liver perfusate and in vitro culture results in the gradual loss of the characteristic Tbetlo phenotype, with the cells increasing Tbet expression significantly at day 7. This phenotypic loss could be halted through the dose-dependent addition of liver conditioned media (LCM), generated from the ex vivo culture of liver biopsies from healthy organ donors. TGF-β, but not IL-10, replicated the Tbet suppressive effects of LCM in both liver resident and peripheral blood NK cells. Furthermore, blocking TGF-β receptor signaling using the inhibitor SB431542, reversed the effect of LCM treatment on liver resident NK cells, causing the loss of tissue resident Eomeshi Tbetlo phenotype. Our findings identify liver-derived TGF-β as an important component of the liver microenvironment, which acts to regulate and maintain the phenotype of liver resident NK cells.

Spatial and Temporal Mapping of Human Innate Lymphoid Cells Reveals Elements of Tissue Specificity

Innate lymphoid cells (ILC) play critical roles in regulating immunity, inflammation, and tissue homeostasis in mice. However, limited access to non-diseased human tissues has hindered efforts to profile anatomically-distinct ILCs in humans. Through flow cytometric and transcriptional analyses of lymphoid, mucosal, and metabolic tissues from previously healthy human organ donors, here we have provided a map of human ILC heterogeneity across multiple anatomical sites. In contrast to mice, human ILCs are less strictly compartmentalized and tissue localization selectively impacts ILC distribution in a subset-dependent manner. Tissue-specific distinctions are particularly apparent for ILC1 populations, whose distribution was markedly altered in obesity or aging. Furthermore, the degree of ILC1 population heterogeneity differed substantially in lymphoid versus mucosal sites. Together, these analyses comprise a comprehensive characterization of the spatial and temporal dynamics regulating the anatomical distribution, subset heterogeneity, and functional potential of ILCs in non-diseased human tissues.

The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease.

The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40min of their removal from the patient, and in less than 5-10min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180°C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.

Abstract A011: Tissue profiling reveals lifelong specialization of dendritic cell subsets in humans

Abstract Conventional dendritic cells (cDCs) are a rare population of cells that sense the environment and whose major role is in initiating and controlling T cell immunity. The purpose of this study was to generate a map of cDC subset distribution and function over human life, which could serve as a framework for understanding and manipulating adaptive immunity. Current DC-based cancer therapies utilize DCs derived from blood monocytes, whose function may differ from tissue cDCs. We hypothesized that analysis of cDC dynamics in human tissues could uncover a hitherto uncharacterized division of labor between cDC subsets that could facilitate the development of more refined DC-based immunotherapies. To achieve this goal, we used flow cytometry and fluorescence imaging to investigate cDC frequency, maturation, migration and clustering in human peripheral and lymphoid tissues obtained from 60 healthy organ donors aged 3 months to 93 years. Utilizing a robust phenotyping scheme that combines well-established (CD1c, CD141) and tissue-optimized (CD13, CD64) markers, we were able to distinguish plasmacytoid DCs, CD14+ monocytic cells, and two cDC subsets in 14 different tissues. Frequency analysis revealed that anatomical (e.g. vascularization) and functional (e.g. antigen presentation) factors influenced tissue DC profiles, with blood-rich tissues such as lung and bone marrow dominated by CD14+ cells, and lymphoid tissues including lymph nodes (LNs) and gut-associated lymphoid tissues (GALT) enriched for CD13hi CD141+ cDC1. Parallel analysis of mucosal tissues and their draining LNs revealed that CD1c+ Sirp-α+ cDC2 displayed a propensity to undergo maturation, accumulating in T cell-rich clusters near LN B cell follicles. In contrast, cDC1 were mostly immature, and sporadically dispersed in LN T cell zones. cDC2 maturation varied between tissues, with the lung-draining LNs containing a higher proportion of mature cDC2 and larger cDC2 clusters. While cDC1 maturation was also slightly higher in the lung-draining LN, this was not associated with significant changes in cDC1 clustering. Remarkably, these maturation and migration patterns were sustained over at least 8 decades of life, shedding new light on the dynamic foundations of adaptive immunity in humans. Together, these results reveal a lifelong division of labor between human cDC subsets, with cDC1 continuously surveying T cell zones, and cDC2 patrolling peripheral tissues, dynamically maturing and aggregating in draining LN T cell-B cell border zones, where they are uniquely poised to coordinate adaptive immunity. Citation Format: Tomer Granot, Takashi Senda, Dustin Carpenter, Donna L. Farber. Tissue profiling reveals lifelong specialization of dendritic cell subsets in humans [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A011.

The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer.

Toll-like receptors (TLRs) are innate immune receptors expressed in all parts of the alimentary tract. However, analyses comparing expression in different segments and data on germ-free animals are lacking. Alimentary tract cancers show increased TLR expression. According to the field effect concept, carcinogenetic factors induce subtle cancer predisposing alterations in the whole organ. We studied TLR1 to TLR9 expression in all segments of the alimentary tract from cancer patients’ tumor-adjacent normal mucosa, healthy organ donors, and conventional and germ-free mice by using immunohistochemistry and quantitative PCR. All TLRs were expressed in all segments of the alimentary tract. Expression was most intensive in the small intestine in humans and conventional mice, but germ-free mice showed less expression in the small intestine. TLR expression levels were similar in cancer patients and organ donors. We provide systematic baseline data on the TLR expression in the alimentary tract. Normal epithelium adjacent to tumor seems to have similar TLR expression compared with healthy tissues suggesting absence of any field effect in TLR expression. Accordingly, specimens from cancer patients’ normal tumor-adjacent tissue can be used as control tissues in immunohistochemical TLR studies in gastrointestinal cancer.

Inflammatory cells, ceramides, and expression of proteases in perivascular adipose tissue adjacent to human abdominal aortic aneurysms

Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study. Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT. Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage.

Vascular Disease in Young Indians (20-40 years): Role of Dyslipidemia.

Dyslipidemia is an established risk factor for cardiovascular disease (CVD). Atherosclerosis begins early in life as suggested by "fatty streaks" observed in coronaries of healthy organ donors aged 20-29 years. Premature occurrence of coronary heart disease (CHD) in Indians, increases the risk for young individuals. Management of Dyslipidemia in the young Indian poses several challenges. In this article we provide in-depth review of prevalence, guidelines' perspective and expert comments on management of Dyslipidemia in the young (20-40 years) Indian.