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2.
Cell Mol Gastroenterol Hepatol ; 9(2): 295-312, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31606566

RESUMO

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice. METHODS: Human IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKß in IEC (Ikkß(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation. RESULTS: NF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkß(EE)IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKß facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo. CONCLUSIONS: Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD.

3.
Hepatology ; 71(2): 522-538, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31228214

RESUMO

Chronic alcohol consumption causes increased intestinal permeability and changes in the intestinal microbiota composition, which contribute to the development and progression of alcohol-related liver disease. In this setting, little is known about commensal fungi in the gut. We studied the intestinal mycobiota in a cohort of patients with alcoholic hepatitis, patients with alcohol use disorder, and nonalcoholic controls using fungal-specific internal transcribed spacer amplicon sequencing of fecal samples. We further measured serum anti-Saccharomyces cerevisiae antibodies (ASCA) as a systemic immune response to fungal products or fungi. Candida was the most abundant genus in the fecal mycobiota of the two alcohol groups, whereas genus Penicillium dominated the mycobiome of nonalcoholic controls. We observed a lower diversity in the alcohol groups compared with controls. Antibiotic or steroid treatment was not associated with a lower diversity. Patients with alcoholic hepatitis had significantly higher ASCA levels compared to patients with alcohol use disorder and to nonalcoholic controls. Within the alcoholic hepatitis cohort, patients with levels of at least 34 IU/mL had a significantly lower 90-day survival (59%) compared with those with ASCA levels less than 34 IU/mL (80%) with an adjusted hazard ratio of 3.13 (95% CI, 1.11-8.82; P = 0.031). Conclusion: Patients with alcohol-associated liver disease have a lower fungal diversity with an overgrowth of Candida compared with controls. Higher serum ASCA was associated with increased mortality in patients with alcoholic hepatitis. Intestinal fungi may serve as a therapeutic target to improve survival, and ASCA may be useful to predict the outcome in patients with alcoholic hepatitis.

4.
Nature ; 575(7783): 505-511, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723265

RESUMO

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

5.
JMIR Med Inform ; 7(3): e13627, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31271153

RESUMO

BACKGROUND: There are gaps in delivering evidence-based care for patients with chronic liver disease and cirrhosis. OBJECTIVE: Our objective was to use interactive user-centered design methods to develop the Cirrhosis Order Set and Clinical Decision Support (CirrODS) tool in order to improve clinical decision-making and workflow. METHODS: Two work groups were convened with clinicians, user experience designers, human factors and health services researchers, and information technologists to create user interface designs. CirrODS prototypes underwent several rounds of formative design. Physicians (n=20) at three hospitals were provided with clinical scenarios of patients with cirrhosis, and the admission orders made with and without the CirrODS tool were compared. The physicians rated their experience using CirrODS and provided comments, which we coded into categories and themes. We assessed the safety, usability, and quality of CirrODS using qualitative and quantitative methods. RESULTS: We created an interactive CirrODS prototype that displays an alert when existing electronic data indicate a patient is at risk for cirrhosis. The tool consists of two primary frames, presenting relevant patient data and allowing recommended evidence-based tests and treatments to be ordered and categorized. Physicians viewed the tool positively and suggested that it would be most useful at the time of admission. When using the tool, the clinicians placed fewer orders than they placed when not using the tool, but more of the orders placed were considered to be high priority when the tool was used than when it was not used. The physicians' ratings of CirrODS indicated above average usability. CONCLUSIONS: We developed a novel Web-based combined clinical decision-making and workflow support tool to alert and assist clinicians caring for patients with cirrhosis. Further studies are underway to assess the impact on quality of care for patients with cirrhosis in actual practice.

6.
Int J Clin Pract ; 73(11): e13393, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31347754

RESUMO

BACKGROUND: Hepatorenal syndrome (HRS) is a life-threatening complication of cirrhosis and early detection of evolving HRS may provide opportunities for early intervention. We developed a HRS risk model to assist early recognition of inpatient HRS. METHODS: We analysed a retrospective cohort of patients hospitalised from among 122 medical centres in the US Department of Veterans Affairs between 1 January 2005 and 31 December 2013. We included cirrhotic patients who had Kidney Disease Improving Global Outcomes criteria based acute kidney injury on admission. We developed a logistic regression risk prediction model to detect HRS on admission using 10 variables. We calculated 95% confidence intervals on the model building dataset and, subsequently, calculated performance on a 1000 sample holdout test set. We report model performance with area under the curve (AUC) for discrimination and several calibration measures. RESULTS: The cohort included 19 368 patients comprising 32 047 inpatient admissions. The event rate for hospitalised HRS was 2810/31 047 (9.1%) and 79/1000 (7.9%) in the model building and validation datasets, respectively. The variable selection procedure designed a parsimonious model involving ten predictor variables. Final model performance in the validation dataset had an AUC of 0.87, Brier score of 0.05, slope of 1.10 and intercept of 0.04. CONCLUSIONS: We developed a probabilistic risk model to diagnose HRS within 24 hours of hospital admission using routine clinical variables in the largest ever published HRS cohort. The performance was excellent and this model may help identify high-risk patients for HRS and promote early intervention.


Assuntos
Síndrome Hepatorrenal/diagnóstico , Unidades de Terapia Intensiva , Admissão do Paciente/estatística & dados numéricos , Índice de Gravidade de Doença , Lesão Renal Aguda/diagnóstico , Adulto , Área Sob a Curva , Estudos de Coortes , Feminino , Síndrome Hepatorrenal/epidemiologia , Hospitalização/estatística & dados numéricos , Humanos , Cirrose Hepática/diagnóstico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
7.
Dig Dis Sci ; 64(7): 1878-1892, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31076986

RESUMO

BACKGROUND: Alcohol-related liver disease is one of the most prevalent chronic liver diseases worldwide. Mechanisms involved in the pathogenesis of alcohol-related liver disease are not well understood. Oxylipins play a crucial role in numerous biological processes and pathological conditions. Nevertheless, oxylipins are not well studied in alcohol-related liver disease. AIMS: (1) To characterize the patterns of bioactive ω-3 and ω-6 polyunsaturated fatty acid metabolites in alcohol use disorder and alcoholic hepatitis patients and (2) to identify associations of serum oxylipins with clinical parameters in patients with alcohol-related liver disease. METHODS: We performed a comprehensive liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of serum and fecal oxylipins derived from ω-6 arachidonic acid, ω-3 eicosapentaenoic acid, and docosahexaenoic acid in a patient cohort with alcohol-related liver disease. RESULTS: Our results show profound alterations in the serum oxylipin profile of patients with alcohol use disorder and alcoholic hepatitis compared to nonalcoholic controls. Spearman correlation of the oxylipins with clinical parameters shows a link between different serum oxylipins and intestinal permeability, aspartate aminotransferase, bilirubin, albumin, international normalized ratio, platelet count, steatosis, fibrosis and model for end-stage liver disease score. Especially, higher level of serum 20-HETE was significantly associated with decreased albumin, increased hepatic steatosis, polymorphonuclear infiltration, and 90-day mortality. CONCLUSIONS: Patients with alcohol-related liver disease have different oxylipin profiles. Future studies are required to confirm oxylipins as disease biomarker or to connect oxylipins to disease pathogenesis.


Assuntos
Alcoolismo/sangue , Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-6/sangue , Fezes/química , Hepatite Alcoólica/sangue , Oxilipinas/sangue , Adulto , Idoso , Alcoolismo/diagnóstico , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Feminino , Hepatite Alcoólica/diagnóstico , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
8.
Obesity (Silver Spring) ; 27(5): 716-723, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31012292

RESUMO

OBJECTIVE: This study aimed to evaluate a possible association between the use of obesogenic medications and inadequate weight loss in a behavioral weight-management program. METHODS: This is a case-control, single-center study of 666 adult patients within a Veterans Health Administration health system who participated in the MOVE! behavioral weight-loss program. The cohort was divided into responders (n = 150), patients who achieved ≥ 5% total weight loss by the end of the MOVE! program, and nonresponders (n = 516), those who achieved < 5% total weight loss. We reviewed each patient's medical records for exposure to obesogenic medication during the time of treatment. RESULTS: Approximately 62% (n = 411) of patients entering MOVE! had a prescription for obesogenic medications. Obesogenic medication use was associated with worse weight-loss outcomes, and participants were 37% less likely to achieve a clinically meaningful (≥ 5% total weight loss) outcome at the end of the MOVE! program (odds ratio, 0.633; 95% CI: 0.427-0.937; adjusted P = 0.022). Patients who received three or more medications (n = 72) had the greatest difficulty achieving 5% weight loss compared with the control group (odds ratio, 0.265; 95% CI: 0.108-0.646; adjusted P = 0.003). CONCLUSIONS: The use of provider-prescribed obesogenic medications was associated with worse weight-loss outcomes in a behavioral weight-loss program. Closer scrutiny of patient medications is necessary to help improve outcomes of weight-loss treatments.

10.
Int J Obes (Lond) ; 43(6): 1154-1163, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30242239

RESUMO

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is one of the most commonly performed bariatric procedures and has proven effective in providing weight loss. However, considerable variance has been noted in the degree of weight loss. Physician prescription practices may be negatively affecting weight loss post-LSG and, thus, contributing to the broad range of weight loss outcomes. The aim of our study was to determine whether commonly prescribed obesogenic medications negatively affect weight loss outcomes post-LSG. SUBJECTS/METHODS: This single center retrospective cohort study performed at a University hospital included 323 patients (≥18 years) within the University California, San Diego Healthcare System who underwent LSG between 2007 and 2016. We identified a list of 32 commonly prescribed medications that have weight gain as a side effect. We compared the percent excess weight loss (%EWL) of patients divided into two groups based on post-LSG exposure to obesogenic medications. A linear regression model was used to analyze %EWL at 12 months post-LSG while controlling for age, initial body mass index (BMI), and use of leptogenic medications. RESULTS: A total of 150 patients (Meds group) were prescribed obesogenic medications within the one-year post-LSG follow up period, whereas 173 patients (Control group) were not prescribed obesogenic medications. The Meds group lost significantly less weight compared to the Control group (%EWL ± SEM at 12 months 53.8 ± 2.4 n = 78, 65.0 ± 2.6, n = 84 respectively, P = 0.002). This difference could not be attributed to differences in age, gender, initial BMI, co-morbidities, or prescription of leptogenic medications between the two groups. CONCLUSIONS: The use of provider-prescribed obesogenic medications was associated with worse weight loss outcomes post-LSG. Closer scrutiny of patient medications may be necessary to help improve outcomes of weight loss treatments.

11.
Gut ; 68(8): 1504-1515, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30448775

RESUMO

OBJECTIVE: Antimicrobial C-type lectin regenerating islet-derived 3 gamma (REG3G) is suppressed in the small intestine during chronic ethanol feeding. Our aim was to determine the mechanism that underlies REG3G suppression during experimental alcoholic liver disease. DESIGN: Interleukin 22 (IL-22) regulates expression of REG3G. Therefore, we investigated the role of IL-22 in mice subjected to chronic-binge ethanol feeding (NIAAA model). RESULTS: In a mouse model of alcoholic liver disease, we found that type 3 innate lymphoid cells produce lower levels of IL-22. Reduced IL-22 production was the result of ethanol-induced dysbiosis and lower intestinal levels of indole-3-acetic acid (IAA), a microbiota-derived ligand of the aryl hydrocarbon receptor (AHR), which regulates expression of IL-22. Importantly, faecal levels of IAA were also found to be lower in patients with alcoholic hepatitis compared with healthy controls. Supplementation to restore intestinal levels of IAA protected mice from ethanol-induced steatohepatitis by inducing intestinal expression of IL-22 and REG3G, which prevented translocation of bacteria to liver. We engineered Lactobacillus reuteri to produce IL-22 (L. reuteri/IL-22) and fed them to mice along with the ethanol diet; these mice had reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain and upregulated expression of REG3G in intestine. However, L. reuteri/IL-22 did not reduce ethanol-induced liver disease in Reg3g-/- mice. CONCLUSION: Ethanol-associated dysbiosis reduces levels of IAA and activation of the AHR to decrease expression of IL-22 in the intestine, leading to reduced expression of REG3G; this results in bacterial translocation to the liver and steatohepatitis. Bacteria engineered to produce IL-22 induce expression of REG3G to reduce ethanol-induced steatohepatitis.


Assuntos
Disbiose , Etanol , Microbioma Gastrointestinal/fisiologia , Interleucinas/imunologia , Intestino Delgado/imunologia , Lactobacillus reuteri/imunologia , Hepatopatias Alcoólicas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Modelos Animais de Doenças , Disbiose/complicações , Disbiose/etiologia , Disbiose/imunologia , Etanol/efeitos adversos , Etanol/metabolismo , Imunidade Inata , Ácidos Indolacéticos/metabolismo , Inflamação/metabolismo , Hepatopatias Alcoólicas/imunologia , Hepatopatias Alcoólicas/microbiologia , Hepatopatias Alcoólicas/terapia , Camundongos , Camundongos Knockout , Proteínas Associadas a Pancreatite/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo
12.
Proc Natl Acad Sci U S A ; 115(39): E9192-E9200, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30209212

RESUMO

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.


Assuntos
Apoptose , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , Multimerização Proteica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/genética
13.
Gastroenterology ; 155(4): 1128-1139.e6, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29981779

RESUMO

BACKGROUND & AIMS: Screening patients with cirrhosis for hepatocellular carcinoma (HCC) has been recommended. We conducted a matched case-control study within the US Veterans Affairs (VA) health care system to determine whether screening by abdominal ultrasonography (USS) and/or by measuring serum level of α-fetoprotein (AFP) was associated with decreased cancer-related mortality in patients with cirrhosis. METHODS: We defined cases (n = 238) as patients with cirrhosis who died of HCC from January 1, 2013 through August 31, 2015 and had been in VA care with a diagnosis of cirrhosis for at least 4 years before the diagnosis of HCC. We matched each case to 1 control (n = 238), defined as a patient with cirrhosis who did not die of HCC and had been in VA care for at least 4 years before the date of the matched case's HCC diagnosis. Controls were matched to cases by year of cirrhosis diagnosis, race and ethnicity, age, sex, etiology of cirrhosis, Model for End-Stage Liver Disease score, and VA medical center. We identified all USS and serum AFP tests performed within 4 years before the date of HCC diagnosis in cases or the equivalent index date in controls and determined by chart extraction (blinded to case or control status) whether these tests were performed for screening. RESULTS: There were no significant differences between cases and controls in the proportions of patients who underwent screening USS (52.9% vs 54.2%), screening measurement of serum AFP (74.8% vs 73.5%), screening USS or measurement of serum AFP (81.1% vs 79.4%), or screening USS and measurement of serum AFP (46.6% vs 48.3%) within 4 years before the index date, with or without adjusting for potential confounders. There also was no difference in receipt of these screening tests within 1, 2, or 3 years before the index date. CONCLUSIONS: In a matched case-control study of the VA health care system, we found that screening patients with cirrhosis for HCC by USS, measurement of serum AFP, either test, or both tests was not associated with decreased HCC-related mortality. We encourage additional case-control studies to evaluate the efficacy of screening for HCC in other health care systems, in which available records are sufficiently detailed to enable identification of the indication for USS and AFP tests.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidade , Detecção Precoce de Câncer/métodos , Cirrose Hepática/diagnóstico , Cirrose Hepática/mortalidade , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidade , Ultrassonografia , alfa-Fetoproteínas/análise , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/terapia , Estudos de Casos e Controles , Causas de Morte , Humanos , Cirrose Hepática/terapia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/terapia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Fatores de Tempo , Estados Unidos/epidemiologia , United States Department of Veterans Affairs
14.
Int J Med Inform ; 117: 55-65, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30032965

RESUMO

BACKGROUND & OBJECTIVES: In healthcare, the routine use of evidence-based specialty care management plans is mixed. Targeted computerized clinical decision support (CCDS) interventions can improve physician adherence, but adoption depends on CCDS' 'fit' within clinical work. We analyzed clinical work in outpatient and inpatient settings as a basis for developing guidelines for optimizing CCDS design. METHODS: The contextual design approach guided data collection, collation and analysis. Forty (40) consenting physicians were observed and interviewed in general internal medicine inpatient units and gastroenterology (GI) outpatient clinics at two academic medical centers. Data were collated using interpretive debriefing, and consolidated using thematic analysis and three work modeling approaches (communication flow, sequence and artifact models). RESULTS: Twenty-six consenting physicians were observed at Site A and 14 at Site B. Observations included attending (33%) and resident physicians. During research team debriefings, 220 of 341 unique topics were categorized into 5 CCDS-relevant themes. Resident physicians relied on patient assessment & planning processes to support their roles as communication and coordination hubs within the medical team. Artifact analysis further elucidated the evolution of assessment and planning over work shifts. CONCLUSIONS: The usefulness of CCDS tools may be enhanced in clinical care if the design: 1) accounts for clinical work that is distributed across people, space, and time; 2) targets communication and coordination hubs (specific roles) that can amplify the usefulness of CCDS interventions; 3) integrates CCDS with early clinical assessment & planning processes; and 4) provides CCDS in both electronic & hardcopy formats. These requirements provide a research agenda for future research in clinician-CCDS integration.


Assuntos
Sistemas de Apoio a Decisões Clínicas , Comunicação , Computadores , Humanos , Médicos , Software
15.
Inflamm Bowel Dis ; 24(11): 2366-2376, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29889233

RESUMO

Background: Novel therapeutics for inflammatory bowel disease (IBD) are under development, yet mechanistic readouts at the tissue level are lacking. Techniques to assess intestinal immune composition could represent a valuable tool for mechanism of action (MOA) studies of novel drugs. Mass cytometry enables analysis of intestinal inflammatory cell infiltrate and corresponding molecular fingerprints with unprecedented resolution. Here, we aimed to optimize the methodology for isolation and cryopreservation of cells from intestinal tissue to allow for the potential implementation of mass cytometry in MOA studies. Methods: We investigated key technical issues, including minimal tissue requirements, cell isolation protocols, and cell storage, using intestinal biopsies and peripheral blood from healthy individuals. High-dimensional mass cytometry was employed for the analyses of biopsy-derived intestinal cellular subsets. Results: Dithiothreitol and mechanical dissociation decreased epithelial cell contamination and allowed for isolation of adequate cell numbers from 2 to 4 colonic or ileal biopsies (6 × 104±2 × 104) after a 20-minute collagenase digestion, allowing for reliable detection of most major immune cell subsets. Biopsies and antibody-labeled mononuclear cells could be cryopreserved for later processing and acquisition (viability > 70%; P < 0.05). Conclusions: Mass cytometry represents a unique tool for deep immunophenotyping intestinal cell composition. This technique has the potential to facilitate analysis of drug actions at the target tissue by identifying specific cellular subsets and their molecular signatures. Its widespread implementation may impact not only IBD research but also other gastrointestinal conditions where inflammatory cells play a role in pathogenesis.


Assuntos
Células Epiteliais/imunologia , Citometria de Fluxo/métodos , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Leucócitos Mononucleares/imunologia , Espectrometria de Massas/métodos , Idoso , Criopreservação , Células Epiteliais/citologia , Humanos , Imunofenotipagem , Mucosa Intestinal/citologia , Pessoa de Meia-Idade
16.
Alcohol Clin Exp Res ; 2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29953169

RESUMO

BACKGROUND: Hepatitis C virus (HCV) infection and alcohol use disorder (AUD) both adversely affect the immune system resulting in alterations in immune cell signaling and inflammatory processes. The aim of this study was to investigate how comorbid AUD contributes to abnormalities in inflammatory mediators and psychiatric impairments in adults with HCV. METHODS: Alcohol use, mood, and inflammatory factors were evaluated at 3 time points (baseline, week 4, and week 12) in Veterans with HCV, with (n = 42) and without (n = 13) comorbid AUD. Peripheral indices of immune activation, blood-brain barrier (BBB) damage (S100 calcium-binding protein B [S100B]), liver function, and viral load were measured using immunoassays and polymerase chain reaction assays. RESULTS: Comorbid AUD was associated with increased symptoms of depression and anxiety, elevated levels of liver enzymes, and altered expression of inflammatory factors. Alcohol consumption was positively correlated with the severity of psychiatric symptoms. Univariate analysis identified significant group differences in interleukin (IL)-8 (p = 0.006), IL-10 (p = 0.03), and S100B (p = 0.048), with increased levels in participants with AUD, which persisted over time despite reductions in alcohol use and no significant change in HCV viral load. Statistically significant effects of study group or time were not found for the other immune factors assessed. Exploratory receiver operating characteristic curve analysis evaluated the ability of IL-8, IL-10, and S100B to differentiate between levels of alcohol consumption and generated biomarker cutoff values used to identify low risk and unhealthy alcohol use groups. CONCLUSIONS: These results demonstrate that HCV and comorbid AUD are associated with greater psychiatric impairments, potentially resulting from increased inflammation, dysregulated cytokine expression, and compromised BBB function. Alcohol-induced BBB damage may increase the risk of neuropathological consequences within the context of chronic HCV infection.

17.
J Hepatol ; 69(2): 396-405, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29654817

RESUMO

BACKGROUND & AIMS: The degree of cholestasis is an important disease driver in alcoholic hepatitis, a severe clinical condition that needs new biomarkers and targeted therapies. We aimed to identify the largely unknown mechanisms and biomarkers linked to cholestasis in alcoholic hepatitis. METHODS: Herein, we analyzed a well characterized cohort of patients with alcoholic hepatitis and correlated clinical and histological parameters and outcomes with serum bile acids and fibroblast growth factor 19 (FGF19), a major regulator of bile acid synthesis. RESULTS: We found that total and conjugated bile acids were significantly increased in patients with alcoholic hepatitis compared with controls. Serum FGF19 levels were strongly increased and gene expression of FGF19 was induced in biliary epithelial cells and ductular cells of patients with alcoholic hepatitis. De novo bile acid synthesis (CYP7A1 gene expression and C4 serum levels) was significantly decreased in patients with alcoholic hepatitis. Importantly, total and conjugated bile acids correlated positively with FGF19 and with disease severity (model for end-stage liver disease score). FGF19 correlated best with conjugated cholic acid, and model for end-stage liver disease score best with taurine-conjugated chenodeoxycholic acid. Univariate analysis demonstrated significant associations between FGF19 and bilirubin as well as gamma glutamyl transferase, and negative correlations between FGF19 and fibrosis stage as well as polymorphonuclear leukocyte infiltration, in all patients with alcoholic hepatitis. CONCLUSION: Serum FGF19 and bile acids are significantly increased in patients with alcoholic hepatitis, while de novo bile acid synthesis is suppressed. Modulation of bile acid metabolism or signaling could represent a promising target for treatment of alcoholic hepatitis in humans. LAY SUMMARY: Understanding the underlying mechanisms that drive alcoholic hepatitis is important for the development of new biomarkers and targeted therapies. Herein, we describe a molecule that is increased in patients with alcoholic hepatitis. Modulating the molecular pathway of this molecule might lead to promising targets for the treatment of alcoholic hepatitis.


Assuntos
Ácidos e Sais Biliares , Colestase , Fatores de Crescimento de Fibroblastos/sangue , Hepatite Alcoólica , Neutrófilos/patologia , Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Biomarcadores/sangue , Colestase/etiologia , Colestase/metabolismo , Correlação de Dados , Feminino , Hepatite Alcoólica/sangue , Hepatite Alcoólica/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Índice de Gravidade de Doença , Transdução de Sinais/fisiologia
18.
J Biomed Inform ; 80: 87-95, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29530803

RESUMO

OBJECTIVE: Hepatorenal Syndrome (HRS) is a devastating form of acute kidney injury (AKI) in advanced liver disease patients with high morbidity and mortality, but phenotyping algorithms have not yet been developed using large electronic health record (EHR) databases. We evaluated and compared multiple phenotyping methods to achieve an accurate algorithm for HRS identification. MATERIALS AND METHODS: A national retrospective cohort of patients with cirrhosis and AKI admitted to 124 Veterans Affairs hospitals was assembled from electronic health record data collected from 2005 to 2013. AKI was defined by the Kidney Disease: Improving Global Outcomes criteria. Five hundred and four hospitalizations were selected for manual chart review and served as the gold standard. Electronic Health Record based predictors were identified using structured and free text clinical data, subjected through NLP from the clinical Text Analysis Knowledge Extraction System. We explored several dimension reduction techniques for the NLP data, including newer high-throughput phenotyping and word embedding methods, and ascertained their effectiveness in identifying the phenotype without structured predictor variables. With the combined structured and NLP variables, we analyzed five phenotyping algorithms: penalized logistic regression, naïve Bayes, support vector machines, random forest, and gradient boosting. Calibration and discrimination metrics were calculated using 100 bootstrap iterations. In the final model, we report odds ratios and 95% confidence intervals. RESULTS: The area under the receiver operating characteristic curve (AUC) for the different models ranged from 0.73 to 0.93; with penalized logistic regression having the best discriminatory performance. Calibration for logistic regression was modest, but gradient boosting and support vector machines were superior. NLP identified 6985 variables; a priori variable selection performed similarly to dimensionality reduction using high-throughput phenotyping and semantic similarity informed clustering (AUC of 0.81 - 0.82). CONCLUSION: This study demonstrated improved phenotyping of a challenging AKI etiology, HRS, over ICD-9 coding. We also compared performance among multiple approaches to EHR-derived phenotyping, and found similar results between methods. Lastly, we showed that automated NLP dimension reduction is viable for acute illness.


Assuntos
Algoritmos , Diagnóstico por Computador/métodos , Síndrome Hepatorrenal/diagnóstico , Fenótipo , Lesão Renal Aguda , Idoso , Registros Eletrônicos de Saúde , Feminino , Síndrome Hepatorrenal/etiologia , Síndrome Hepatorrenal/fisiopatologia , Humanos , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Processamento de Linguagem Natural , Razão de Chances , Curva ROC , Estudos Retrospectivos , Máquina de Vetores de Suporte
19.
J Clin Gastroenterol ; 52(2): 91-96, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28697153

RESUMO

BACKGROUND: Mucins are heavily glycosylated glycoproteins, synthesized by mucosal surfaces and have an important role in healthy state and malignant diseases. Change in mucins synthesis or secretion may be primary event or secondary to inflammation or carcinogenesis. AIM: The aim of this study is to assess the current knowledge about mucin expression in esophageal lesions, and to establish a role for different mucin expressions as prognostic markers. METHOD: English Medical literature searches were conducted for "mucin" and "esophagus." Observational studies were included. Meta-analysis was performed using comprehensive meta-analysis software. Pooled odds ratios (OR) and 95% confidence intervals (CI) were calculated. RESULTS: In the random-effect model, mucin expression was significantly higher in esophageal lesions than in normal esophageal mucosa with OR=5.456 (95% CI, 1.883-15.807, P=0.002). Measure of heterogeneity, demonstrated in the included studies, was high: Q=287.501, df (Q)=44.00, P<0.0001, I=84.696%. There is a gradient of mucin expression and complexity in esophageal premalignant to malignant lesions, lower in Barrett's mucosa with low grade dysplasia (LGD), increased in high grade dysplasia (HGD), and highest in esophageal adenocarcinoma (EAC). MUC2, MUC3, MUC5AC, and MUC6 expression was higher in EAC than HGD, and higher in HGD than in LGD mucosa. The opposite was found for MUC1 and MUC4. CONCLUSION: Increased expression of certain mucin genes in esophageal mucosa may be further studied as a potential diagnostic tool, and this may add important information in the surveillance of Barrett's esophagus.


Assuntos
Neoplasias Esofágicas/patologia , Mucinas/genética , Lesões Pré-Cancerosas/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Lesões Pré-Cancerosas/genética , Prognóstico
20.
Nat Commun ; 8(1): 2137, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29233961

RESUMO

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 10 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.

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