RESUMO
Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of â¼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.
Assuntos
Dor Crônica , Transcriptoma , Camundongos , Animais , DNA , RNA , GéisRESUMO
The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health1. The intesting has a length of over nine metres, along which there are differences in structure and function2. The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.
Assuntos
Intestinos , Análise de Célula Única , Humanos , Diferenciação Celular/genética , Cromatina/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/citologia , Intestinos/citologia , Intestinos/imunologia , Análise da Expressão Gênica de Célula ÚnicaRESUMO
BACKGROUND AND AIMS: Comparison of donation after brain death (DBD) and donation after cardiac death (DCD) lung tissue before transplantation have demonstrated activation of pro-inflammatory cytokine pathway in DBD donors. The molecular and immunological properties of circulating exosomes from DBD and DCD donors were not previously described. METHODS: We collected plasma from 18 deceased donors (12 DBD and six DCD). Cytokines were analyzed by 30-Plex luminex Panels. Exosomes were analyzed for liver self-antigen (SAg), Transcription Factors and HLA class II (HLA-DR/DQ) using western blot. C57BL/6 animals were immunized with isolated exosomes to determine strength and magnitude of immune responses. Interferon (IFN)-γ and tumor necrosis factor-α producing cells were quantified by ELISPOT, specific antibodies to HLA class II antigens were measured by ELISA RESULTS: We demonstrate increased plasma levels of IFNγ, EGF, EOTAXIN, IP-10, MCP-1, RANTES, MIP-ß, VEGF, and interleukins - 6/8 in DBD plasma versus DCD. MiRNA isolated from exosome of DBD donors demonstrated significant increase in miR-421, which has been reported to correlate with higher level of Interleukin-6. Higher levels of liver SAg Collagen III (p = .008), pro-inflammatory transcription factors (NF-κB, p < .05; HIF1α, p = .021), CIITA (p = .011), and HLA class II (HLA-DR, p = .0003 and HLA-DQ, p = .013) were detected in exosomes from DBD versus DCD plasma. The circulating exosomes isolated from DBD donors were immunogenic in mice and led to the development of Abs to HLA-DR/DQ. CONCLUSIONS: This study provides potential new mechanisms by which DBD organs release exosomes that can activate immune pathways leading to cytokine release and allo-immune response.
Assuntos
Exossomos , MicroRNAs , Obtenção de Tecidos e Órgãos , Humanos , Camundongos , Animais , Morte Encefálica , Projetos Piloto , Camundongos Endogâmicos C57BL , Morte , Doadores de Tecidos , Citocinas , Antígenos HLA-DR , Fatores de Transcrição , Estudos Retrospectivos , Sobrevivência de EnxertoRESUMO
BACKGROUND: Robotic donor nephrectomy (RDN) has emerged as a safe alternate to laparoscopic donor nephrectomy (LDN), offering improved visualization, instrument dexterity and ergonomics. There is still concern about how to safely transition from LDN to RDN. METHODS: We performed a retrospective review of 150 consecutive living donor operations (75 LDN and 75 RDN) at our center, comparing the first 75 RDN's with the last 75 LDN's performed prior to the initiation of the robotic transplant program. Operative times and complications were used as surrogates of efficiency and safety, respectively, to estimate the learning curve with RDN. RESULTS: RDN was associated with a longer total operative time (RDN 182 vs LDN 144 min; P < 0.0001) but a significantly shorter post-operative length of stay (RDN 1.8 vs LDN 2.1 days; P = 0.0213). Donor complications and recipient outcomes were the same between both groups. Learning curve of RDN was estimated to be about 30 cases. CONCLUSIONS: RDN is a safe alternate to LDN with acceptable donor morbidity and no negative impact on recipient outcomes even during the early part of the RDN learning curve. Surgeon preferences for the robotic approach compared to traditional laparoscopy will require further scrutiny to improve ergonomics and operative efficiency.
Assuntos
Transplante de Rim , Laparoscopia , Procedimentos Cirúrgicos Robóticos , Robótica , Humanos , Nefrectomia , Estudos Retrospectivos , Doadores Vivos , Coleta de Tecidos e ÓrgãosRESUMO
Antioxidant defence mechanisms, such as the nuclear factor-erythroid 2-related-factor-2 (NRF2) axis, are integral to oxidative stress responses and ischemic injury. Hepatic antioxidant capacity is contingent on parenchymal quality, and there is a need to develop new insights into key molecular mechanisms in marginal liver allografts that might provide therapeutic targets. This study examines the clinical relevance of NRF2 in donor livers and its response to normothermic machine perfusion (NMP). Discarded donor livers (n = 40) were stratified into a high NRF2 and low NRF2 group by quantifying NRF2 expression. High NRF2 livers had significantly lower transaminase levels, hepatic vascular inflammation and peri-portal CD3+ T cell infiltration. Human liver allografts (n = 8) were then exposed to 6-h of NMP and high NRF2 livers had significantly reduced liver enzyme alterations and improved lactate clearance. To investigate these findings further, we used a rat fatty-liver model, treating livers with an NRF2 agonist during NMP. Treated livers had increased NRF2 expression and reduced transaminase derangements following NMP compared to vehicle control. These results support the association of elevated NRF2 expression with improved liver function. Targeting this axis could have a rationale in future studies and NRF2 agonists may represent a supplemental treatment strategy for rescuing marginal donor livers.
Assuntos
Transplante de Fígado , Traumatismo por Reperfusão , Aloenxertos , Animais , Fígado , Fator 2 Relacionado a NF-E2 , Preservação de Órgãos , Perfusão , RatosRESUMO
Understanding the diversity of human tissues is fundamental to disease and requires linking genetic information, which is identical in most of an individual's cells, with epigenetic mechanisms that could have tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns. Here we report high coverage methylomes that catalogue cytosine methylation in all contexts for the major human organ systems, integrated with matched transcriptomes and genomic sequence. By combining these diverse data types with each individuals' phased genome, we identified widespread tissue-specific differential CG methylation (mCG), partially methylated domains, allele-specific methylation and transcription, and the unexpected presence of non-CG methylation (mCH) in almost all human tissues. mCH correlated with tissue-specific functions, and using this mark, we made novel predictions of genes that escape X-chromosome inactivation in specific tissues. Overall, DNA methylation in several genomic contexts varies substantially among human tissues.
Assuntos
Metilação de DNA , Epigênese Genética , Fatores Etários , Alelos , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Variação Genética , Humanos , Masculino , Especificidade de ÓrgãosRESUMO
Allelic differences between the two homologous chromosomes can affect the propensity of inheritance in humans; however, the extent of such differences in the human genome has yet to be fully explored. Here we delineate allelic chromatin modifications and transcriptomes among a broad set of human tissues, enabled by a chromosome-spanning haplotype reconstruction strategy. The resulting large collection of haplotype-resolved epigenomic maps reveals extensive allelic biases in both chromatin state and transcription, which show considerable variation across tissues and between individuals, and allow us to investigate cis-regulatory relationships between genes and their control sequences. Analyses of histone modification maps also uncover intriguing characteristics of cis-regulatory elements and tissue-restricted activities of repetitive elements. The rich data sets described here will enhance our understanding of the mechanisms by which cis-regulatory elements control gene expression programs.
Assuntos
Alelos , Epigênese Genética/genética , Epigenômica , Haplótipos/genética , Acetilação , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos/genética , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos/genética , Variação Genética/genética , Histonas/metabolismo , Humanos , Motivos de Nucleotídeos , Especificidade de Órgãos/genética , Transcrição Gênica/genéticaRESUMO
To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.
Assuntos
Sequência Conservada/genética , Genoma/genética , Genômica , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
It remains controversial whether renal allografts from donation after circulatory death (DCD) have a higher risk of acute rejection (AR). In the porcine large animal kidney transplant model, we investigated the AR and function of DCD renal allografts compared to the non-DCD renal allografts and the effects of increased immunosuppression. We found that the AR was significantly increased along with elevated MHC-I expression in the DCD transplants receiving low-dose immunosuppression; however, AR and renal function were significantly improved when given high-dose immunosuppressive therapy postoperatively. Also, high-dose immunosuppression remarkably decreased the mRNA levels of ifn-g, il-6, tgf-b, il-4, and tnf-a in the allograft at day 5 and decreased serum cytokines levels of IFN-g and IL-17 at day 4 and day 5 after operation. Furthermore, Western blot analysis showed that higher immunosuppression decreased phosphorylation of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells-p65, increased phosphorylation of extracellular-signal-regulated kinase, and reduced the expression of Bcl-2-associated X protein and caspase-3 in the renal allografts. These results suggest that the DCD renal allograft seems to be more vulnerable to AR; enhanced immunosuppression reduces DCD-associated AR and improves early allograft function in a preclinical large animal model.
Assuntos
Função Retardada do Enxerto/prevenção & controle , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Terapia de Imunossupressão , Imunossupressores/uso terapêutico , Transplante de Rim/efeitos adversos , Aloenxertos , Animais , Morte , Função Retardada do Enxerto/etiologia , Função Retardada do Enxerto/patologia , Feminino , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/efeitos dos fármacos , Suínos , Doadores de Tecidos , Obtenção de Tecidos e Órgãos/métodosRESUMO
Most knowledge on NK cells is based on studies of what are now known as conventional NK cells in the mouse spleen or human peripheral blood. However, recent studies in mice indicate the presence of tissue-resident NK cells in certain organs, such as the liver, that display different markers and transcription factor dependencies as compared with conventional NK cells. In this study, we provide evidence from cytometry by time-of-flight analysis and humanized mice indicating that human CD49e- NK cells are tissue resident in the liver. Thus, these studies indicate that tissue-resident NK cells are evolutionarily conserved in humans and mice, providing a foundation to explore their role in human disease.
Assuntos
Integrina alfa5/imunologia , Células Matadoras Naturais/fisiologia , Fígado/citologia , Fígado/imunologia , Animais , Capilares/imunologia , Citometria de Fluxo , Humanos , Integrina alfa5/genética , Células Matadoras Naturais/imunologia , Fígado/irrigação sanguínea , Camundongos , Camundongos Transgênicos , Fenótipo , Fatores de TranscriçãoRESUMO
Modulation of nitric oxide activity through blockade of CD47 signaling has been shown to reduce ischemia-reperfusion injury (IRI) in various models of tissue ischemia. Here, we evaluate the potential effect of an antibody-mediated CD47 blockade in a syngeneic and an allogeneic DCD rat kidney transplant model. The donor organ was subjected to 1 hour of warm ischemia time after circulatory cessation, then flushed with a CD47 monoclonal antibody (CD47mAb) in the treatment group, or an isotype-matched immunoglobulin in the control group. We found that CD47mAb treatment improved survival rates in both models. Serum markers of renal injury were significantly decreased in the CD47mAb-treated group compared with the control group. Histologically the CD47mAb-treated group had significantly reduced scores of acute tubular injury and acute tubular necrosis. The expression of biomarkers related to mitochondrial stress and apoptosis also were significantly lower in the CD47mAb-treated groups. Overall, the protective effects of CD47 blockade were greater in the syngeneic model. Our data show that CD47mAb blockade decreased the IRI of DCD kidneys in rat transplant models. This therapy has the potential to improve DCD kidney transplant outcomes in the human setting.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/antagonistas & inibidores , Morte , Rejeição de Enxerto/prevenção & controle , Falência Renal Crônica/cirurgia , Transplante de Rim/métodos , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose , Antígeno CD47/imunologia , Taxa de Filtração Glomerular , Sobrevivência de Enxerto , Inflamação/prevenção & controle , Testes de Função Renal , Masculino , Estresse Oxidativo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Transdução de SinaisRESUMO
We investigated whether blockade of the CD47 signaling pathway could reduce ischemia-reperfusion injury (IRI) of renal allografts donated after cardiac death (DCD) in a porcine animal model of transplantation. Renal allografts were subjected to 30 minutes of warm ischemia, 3.5 hours of cold ischemia, and then perfused with a humanized anti-CD47 monoclonal antibody (CD47mAb) in the treatment group or HTK solution in the control group (n = 4/group). The animals were euthanized five days after transplantation. At the time of reperfusion, indocyanine green-based in vivo imaging showed that CD47mAb-treated organs had greater and more uniform reperfusion. On post-transplant days 3-5, the treatment group had lower values compared to the control for creatinine and blood urea nitrogen. Histological examination of allograft tissues showed a significant decrease of acute tubular injury in the CD47mAb-treated group compared to control. Compared to the control group, CD47mAb treatment significantly decreased genes expression related to oxidative stress (sod-1, gpx-1, and txn), the inflammatory response (il-2, il-6, inf-g, and tgf-b), as well as reduced protein levels of BAX, Caspase-3, MMP2, and MMP9. These data demonstrate that CD47mAb blockade decreases IRI and subsequent tissue injury in DCD renal allografts in a large animal transplant model.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/antagonistas & inibidores , Morte , Rejeição de Enxerto/prevenção & controle , Falência Renal Crônica/cirurgia , Transplante de Rim/métodos , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose , Antígeno CD47/imunologia , Modelos Animais de Doenças , Feminino , Taxa de Filtração Glomerular , Sobrevivência de Enxerto , Inflamação/prevenção & controle , Testes de Função Renal , Estresse Oxidativo , Transdução de Sinais , SuínosRESUMO
Although the similarities between humans and mice are typically highlighted, morphologically and genetically, there are many differences. To better understand these two species on a molecular level, we performed a comparison of the expression profiles of 15 tissues by deep RNA sequencing and examined the similarities and differences in the transcriptome for both protein-coding and -noncoding transcripts. Although commonalities are evident in the expression of tissue-specific genes between the two species, the expression for many sets of genes was found to be more similar in different tissues within the same species than between species. These findings were further corroborated by associated epigenetic histone mark analyses. We also find that many noncoding transcripts are expressed at a low level and are not detectable at appreciable levels across individuals. Moreover, the majority lack obvious sequence homologs between species, even when we restrict our attention to those which are most highly reproducible across biological replicates. Overall, our results indicate that there is considerable RNA expression diversity between humans and mice, well beyond what was described previously, likely reflecting the fundamental physiological differences between these two organisms.
Assuntos
DNA Intergênico/genética , Perfilação da Expressão Gênica/métodos , Especificidade de Órgãos/genética , Proteínas/genética , Animais , Epigenômica/métodos , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Especificidade da Espécie , Transcriptoma/genéticaRESUMO
Donor organ shortages have led to an increased interest in finding new approaches to recover organs from extended criteria donors (ECD). Normothermic extracorporeal liver perfusion (NELP) has been proposed as a superior preservation method to reduce ischemia/reperfusion injury (IRI), precondition suboptimal grafts, and treat ECD livers so that they can be successfully used for transplantation. The aim of this study was to investigate the beneficial effects of a modified NELP circuit on discarded human livers. Seven human livers that were rejected for transplantation were placed on a modified NELP circuit for 8 hours. Perfusate samples and needle core biopsies were obtained at hourly intervals. A defatting solution that contained exendin-4 (50 nM) and L-carnitine (10 mM) was added to the perfusate for 2 steatotic livers. NELP provided normal temperature, electrolytes, and pH and glucose levels in the perfusate along with physiological vascular flows and pressures. Functional, biochemical, and microscopic evaluation revealed no additional injuries to the grafts during NELP with an improved oxygen extraction ratio (>0.5) and stabilized markers of hepatic injury. All livers synthesized adequate amounts of bile and coagulation factors. We also demonstrated a mild reduction (10%) of macroglobular steatosis with the use of the defatting solution. Histology demonstrated normal parenchymal architecture and a minimal to complete lack of IRI at the end of NELP. In conclusion, a modified NELP circuit preserved hepatocyte architecture, recovered synthetic functions, and hepatobiliary parameters of ECD livers without additional injuries to the grafts. This approach has the potential to increase the donor pool for clinical transplantation. Liver Transplantation 22 979-993 2016 AASLD.
Assuntos
Transplante de Fígado/métodos , Fígado , Soluções para Preservação de Órgãos/uso terapêutico , Preservação de Órgãos/métodos , Perfusão/métodos , Traumatismo por Reperfusão/prevenção & controle , Sobrevivência de Tecidos , Adulto , Idoso , Aloenxertos/patologia , Biópsia com Agulha de Grande Calibre , Carnitina/uso terapêutico , Seleção do Doador/métodos , Exenatida , Fígado Gorduroso/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Preservação de Órgãos/instrumentação , Soluções para Preservação de Órgãos/química , Peptídeos/uso terapêutico , Temperatura , Peçonhas/uso terapêutico , Isquemia QuenteRESUMO
Normothermic extracorporeal liver perfusion (NELP) can decrease ischemia/reperfusion injury to the greatest degree when cold ischemia time is minimized. Warm perfusion of cold-stored livers results in hepatocellular damage, sinusoidal endothelial cell (SEC) dysfunction, and Kupffer cell activation. However, the logistics of organ procurement mandates a period of cold preservation before NELP. The aim of this study was to determine the beneficial effects of gradual rewarming of cold-stored livers by placement on NELP. Three female porcine livers were used for each group. In the immediate NELP group, procured livers were immediately placed on NELP for 8 hours. In the cold NELP group, livers were cold-stored for 4 hours followed by NELP for 4 hours. In rewarming groups, livers were cold-stored for 4 hours, then gradually rewarmed in different durations to 38°C and kept on NELP for an additional 4 hours. For comparison purposes, the last 4 hours of NELP runs were considered to be the evaluation phase. Immediate NELP livers had significantly lower concentrations of liver transaminases, hyaluronic acid, and ß-galactosidase and had higher bile production compared to the other groups. Rewarming livers had significantly lower concentrations of hyaluronic acid and ß-galactosidase compared to the cold NELP livers. In addition, there was a significant decline in international normalized ratio values, improved bile production, reduced biliary epithelial cell damage, and improved cholangiocyte function. Thus, if a NELP machine is not available at the procurement site and livers will need to undergo a period of cold preservation, a gradual rewarming protocol before NELP may greatly reduce damages that are associated with reperfusion. In conclusion, gradual rewarming of cold-preserved livers upon NELP can minimize the hepatocellular damage, Kupffer cell activation, and SEC dysfunction.
Assuntos
Isquemia Fria , Transplante de Fígado/métodos , Fígado/cirurgia , Perfusão/métodos , Traumatismo por Reperfusão/prevenção & controle , Reaquecimento/métodos , Animais , Bile/metabolismo , Coagulação Sanguínea , Isquemia Fria/efeitos adversos , Feminino , Hepatectomia , Ácido Hialurônico/metabolismo , Células de Kupffer/enzimologia , Células de Kupffer/patologia , Fígado/enzimologia , Fígado/patologia , Transplante de Fígado/efeitos adversos , Perfusão/efeitos adversos , Perfusão/instrumentação , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Reaquecimento/efeitos adversos , Reaquecimento/instrumentação , Suínos , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
Orthotopic liver transplantation (OLT) remains the standard treatment option for nonresponsive liver failure. Because ischemia/reperfusion injury (IRI) is an important impediment to the success of OLT, new therapeutic strategies are needed to reduce IRI. We investigated whether blocking the CD47/thrombospondin-1 inhibitory action on nitric oxide signaling with a monoclonal antibody specific to CD47 (CD47mAb400) would reduce IRI in liver grafts. Syngeneic OLT was performed with Lewis rats. Control immunoglobulin G or CD47mAb400 was administered to the donor organ at procurement or to both the organ and the recipient at the time of transplant. Serum transaminases, histological changes of the liver, and animal survival were assessed. Oxidative stress, inflammatory responses, and hepatocellular damage were also quantified. A significant survival benefit was not achieved when CD47mAb400 was administered to the donor alone. However, CD47mAb400 administration to both the donor and the recipient increased animal survival afterward. The CD47mAb400-treated group showed lower serum transaminases, bilirubin, oxidative stress, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, caspase-3 activity, and proinflammatory cytokine expression of tumor necrosis factor α, interleukin-1ß, and interleukin-6. Thus, CD47 blockade with CD47mAb400 administered both to the donor and the recipient reduced liver graft IRI in a rat liver transplantation model. This may translate to decreased liver dysfunction and increased survival of liver transplant recipients.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno CD47/metabolismo , Isquemia Fria/efeitos adversos , Transplante de Fígado/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/cirurgia , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Antígeno CD47/imunologia , Citoproteção , Modelos Animais de Doenças , Mediadores da Inflamação/sangue , Fígado/irrigação sanguínea , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Necrose , Estresse Oxidativo/efeitos dos fármacos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
OBJECTIVE: The effects of obesity in liver transplantation remain controversial. Earlier institutional data demonstrated no significant difference in postoperative complications or 1-year mortality. This study was conducted to test the hypothesis that obesity alone has minimal effect on longterm graft and overall survival. METHODS: A retrospective, single-institution analysis of outcomes in patients submitted to primary adult orthotopic liver transplantation was conducted using data for the period from 1 January 2002 to 31 December 2012. Recipients were divided into six groups by pre-transplant body mass index (BMI), comprising those with BMIs of <18.0 kg/m(2) , 18.0-24.9 kg/m(2) , 25.0-29.9 kg/m(2) , 30.0-35.0 kg/m(2) , 35.1-40.0 kg/m(2) and >40 kg/m(2) , respectively. Pre- and post-transplant parameters were compared. A P-value of <0.05 was considered to indicate statistical significance. Independent predictors of patient and graft survival were determined using multivariate analysis. RESULTS: A total of 785 patients met the study inclusion criteria. A BMI of >35 kg/m(2) was associated with non-alcoholic steatohepatitis (NASH) cirrhosis (P < 0.0001), higher Model for End-stage Liver Disease (MELD) score, and longer wait times for transplant (P = 0.002). There were no differences in operative time, intensive care unit or hospital length of stay, or perioperative complications. Graft and patient survival at intervals up to 3 years were similar between groups. Compared with non-obese recipients, recipients with a BMI of >40 kg/m(2) showed significantly reduced 5-year graft (49.0% versus 75.8%; P < 0.02) and patient (51.3% versus 78.8%; P < 0.01) survival. CONCLUSIONS: Obesity increasingly impacts outcomes in liver transplantation. Although the present data are limited by the fact that they were sourced from a single institution, they suggest that morbid obesity adversely affects longterm outcomes despite providing similar short-term results. Further analysis is indicated to identify risk factors for poor outcomes in morbidly obese patients.
Assuntos
Doença Hepática Terminal/cirurgia , Transplante de Fígado/efeitos adversos , Obesidade Mórbida/cirurgia , Complicações Pós-Operatórias/mortalidade , Transplantados/estatística & dados numéricos , Adulto , Índice de Massa Corporal , Estudos de Coortes , Doença Hepática Terminal/complicações , Doença Hepática Terminal/mortalidade , Feminino , Seguimentos , Rejeição de Enxerto , Sobrevivência de Enxerto , Hospitais Universitários , Humanos , Transplante de Fígado/métodos , Masculino , Pessoa de Meia-Idade , Obesidade Mórbida/complicações , Assistência Perioperatória/métodos , Complicações Pós-Operatórias/fisiopatologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Medição de Risco , Análise de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet xenotransplantation offers a promising alternative to allotransplantation, with the potential for large-scale production of on-demand, functional islets. The yield and viability of isolated islets is highly susceptible to the quality of the donor pancreas and the method of procurement, particularly the duration of warm-ischemia time. To improve organ preservation and subsequent islet yield and viability, we have developed a protocol for surgical perfusion and resection of the porcine pancreas. This protocol employs direct infrarenal aortic cannulation and organ perfusion to both minimize warm-ischemia time and simplify the procedure for operators who do not have extensive surgical expertise. Subsequent arterial perfusion of the pancreas via the aorta flushes stagnant blood from the microvasculature, thereby reducing thrombosis and oxidative damage to the tissue. This manuscript provides a detailed protocol for surgical perfusion and resection of the porcine pancreas, followed by islet isolation and purification.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Pâncreas , Perfusão , Animais , Suínos , Ilhotas Pancreáticas/citologia , Transplante das Ilhotas Pancreáticas/métodos , Perfusão/métodos , Pâncreas/cirurgia , Pâncreas/irrigação sanguínea , Pâncreas/citologia , Transplante Heterólogo/métodosRESUMO
Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex and find epigenetic signatures of variation in ATAC-Seq data in this discovery cohort. We then scale up our single-cell RNA-Seq analysis by combining our data with five recently published single nucleus RNA-Seq datasets of healthy adult hearts. We find variation such as metabolic alterations by sex and immune changes by age in differential expression tests, as well as alterations in abundance of cardiomyocytes by sex and neurons with age. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs. Our analysis represents the largest single-cell analysis of cardiac variation by age and sex to date and provides a resource for further study of healthy cardiac variation and transcriptional regulation at single-cell resolution.