A Proof-of-Concept for Epigenetic Therapy of Tissue Fibrosis: Inhibition of Liver Fibrosis Progression by 3-Deazaneplanocin A.

The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis.

Practice Patterns and Outcomes Associated With Procalcitonin Use in Critically Ill Patients With Sepsis.

BACKGROUND.: Randomized trials support use of procalcitonin (PCT)-based algorithms to decrease duration of antibiotics for critically ill patients with sepsis. However, current use of PCT and associated outcomes in real-world clinical settings is unclear. We sought to determine PCT use in critically ill patients with sepsis in the United States and to examine associations between PCT use and clinical outcomes. METHODS.: This was a retrospective cohort study of approximately 20% of patients with sepsis hospitalized in US intensive care units. Hierarchical regression models were used to determine associations of PCT use with outcomes (antibiotic-days, incidence of Clostridium difficile infection, and in-hospital mortality). Sensitivity analyses were conducted to assess robustness of findings to different methods used to address unmeasured confounding (eg, instrumental variable, difference-in-differences analyses). RESULTS.: Among 20750 critically ill patients with sepsis in 107 hospitals with PCT available, 3769 (18%) patients had PCT levels checked; 1119 (29.7%) had serial PCT measurements. PCT use was associated with increased antibiotic-days (adjusted relative risk, 1.1; 95% confidence interval [CI], 1.15-1.18) and incidence of C. difficile (adjusted odds ratio, 1.42; 95% CI, 1.09-1.85) without a change in mortality (adjusted hazard ratio, 1.05; 95% CI, 0.93-1.19). Analysis of PCT use by instrumental variable and difference-in-difference analyses showed similar lack of antibiotic or outcome improvements associated with PCT use. CONCLUSIONS.: PCT use was not associated with improved antibiotic use or other clinical outcomes in real-world settings. Programs to improve implementation of PCT-based strategies are warranted prior to widespread adoption.

Technical performance of a novel, fully automated electrochemiluminescence immunoassay for the quantitation of ß-amyloid (1-42) in human cerebrospinal fluid.

INTRODUCTION: Available assays for quantitation of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aß [1-42]) in cerebrospinal fluid demonstrate significant variability and lack of standardization to reference measurement procedures (RMPs). We report analytical performance data for the novel Elecsys ß-amyloid (1-42) assay (Roche Diagnostics). METHODS: Lot-to-lot comparability was tested using method comparison. Performance parameters were measured according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The assay was standardized to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved RMP. RESULTS: Limit of quantitation was <11.28 pg/mL, and the assay was linear throughout the measuring range (200-1700 pg/mL). Excellent lot-to-lot comparability was observed (correlation coefficients [Pearson's r] >0.995; bias in medical decision area <2%). Repeatability coefficients of variation (CVs) were 1.0%-1.6%, intermediate CVs were 1.9%-4.0%, and intermodule CVs were 1.1%-3.9%. Estimated total reproducibility was 2.0%-5.1%. Correlation with the RMP was good (Pearson's r, 0.93). DISCUSSION: The Elecsys ß-amyloid (1-42) assay has high analytical performance that may improve biomarker-based AD diagnosis.

MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis.

BACKGROUND & AIMS: Myofibroblast transdifferentiation generates hepatic myofibroblasts, which promote liver fibrogenesis. The peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of this process. We investigated epigenetic regulation of PPARgamma and myofibroblast transdifferentiation. METHODS: Chromatin immunoprecipitation (ChIP) assays assessed the binding of methyl-CpG binding protein 2 (MeCP2) to PPARgamma and chromatin modifications that silence this gene. MeCP2(-/y) mice and an inhibitor (DZNep) of the epigenetic regulatory protein EZH2 were used in the carbon tetrachloride model of liver fibrosis. Liver tissues from mice were assessed by histologic analysis; markers of fibrosis were measured by quantitative polymerase chain reaction (qPCR). Reverse transcription PCR detected changes in expression of the microRNA miR132 and its target, elongated transcripts of MeCP2. Myofibroblasts were transfected with miR132; PPARgamma and MeCP2 expressions were analyzed by qPCR or immunoblotting. RESULTS: Myofibroblast transdifferentiation of hepatic stellate cells is controlled by a combination of MeCP2, EZH2, and miR132 in a relay pathway. The pathway is activated by down-regulation of miR132, releasing the translational block on MeCP2. MeCP2 is recruited to the 5' end of PPARgamma, where it promotes methylation by H3K9 and recruits the transcription repressor HP1alpha. MeCP2 also stimulates expression of EZH2 and methylation of H3K27 to form a repressive chromatin structure in the 3' exons of PPARgamma. Genetic and pharmacologic disruptions of MeCP2 or EZH2 reduced the fibrogenic characteristics of myofibroblasts and attenuated fibrogenesis. CONCLUSIONS: Liver fibrosis is regulated by an epigenetic relay pathway that includes MeCP2, EZH2, and miR132. Reagents that interfere with this pathway might be developed to reduce fibrogenesis in chronic liver disease.

Mechanical Ventilation Training During Graduate Medical Education: Perspectives and Review of the Literature.

BACKGROUND: Management of mechanical ventilation (MV) is an important and complex aspect of caring for critically ill patients. Management strategies and technical operation of the ventilator are key skills for physicians in training, as lack of expertise can lead to substantial patient harm. OBJECTIVE: We performed a narrative review of the literature describing MV education in graduate medical education (GME) and identified best practices for training and assessment methods. METHODS: We searched MEDLINE, PubMed, and Google Scholar for English-language, peer-reviewed articles describing MV education and assessment. We included articles from 2000 through July 2018 pertaining to MV education or training in GME. RESULTS: Fifteen articles met inclusion criteria. Studies related to MV training in anesthesiology, emergency medicine, general surgery, and internal medicine residency programs, as well as subspecialty training in critical care medicine, pediatric critical care medicine, and pulmonary and critical care medicine. Nearly half of trainees assessed were dissatisfied with their MV education. Six studies evaluated educational interventions, all employing simulation as an educational strategy, although there was considerable heterogeneity in content. Most outcomes were assessed with multiple-choice knowledge testing; only 2 studies evaluated the care of actual patients after an educational intervention. CONCLUSIONS: There is a paucity of information describing MV education in GME. The available literature demonstrates that trainees are generally dissatisfied with MV training. Best practices include establishing MV-specific learning objectives and incorporating simulation. Next research steps include developing competency standards and validity evidence for assessment tools that can be utilized across MV educational curricula.

SPECT imaging of herpes simplex virus type1 thymidine kinase gene expression by [(123)I]FIAU(1).

RATIONALE AND OBJECTIVES: Introduction of suicide genes, such as herpes simplex virus type1 thymidine kinase (HSV1-tk), in tumor cells has provided a useful method for tumor gene therapy. Several L-nucleosides, such as Lamivudine (3TC) and Clevudine (L-FMAU), have been successfully tested as high-potency antiviral agents. To investigate the potential differences between D- and L-isomers of nucleosides, [(125/123)I]-2'-fluoro-2'-deoxy-1beta-D/L-arabino-furanosy-5-iodo-uracil (D/L-FIAU) have been synthesized and evaluated as potential SPECT agents for imaging HSV1-tk gene expression. MATERIALS AND METHODS: [(125/123)I]D- and L-FIAU were prepared by iododestannylation of the respective tin precursors with (125/123)I-sodium iodide. In vitro cell uptake studies were performed by incubation of [(125)I]D- and L-FIAU in RG2 cells expressing HSV1-tk (RG2TK+). In vivo studies including biodistribution and SPECT were performed in RG2TK+ and RG2TK- tumor-bearing nude mice using [(123)I]D- and L-FIAU. RESULTS: Cell uptake and biodistribution studies indicated that [(125/123)I]L-FIAU did not show any high accumulation (sensitivity) or uptake ratios (selectivity) in HSV1-TK-positive (RG2TK+) tumors as compared to control tumors. In contrast, [(125/123)I]D-FIAU displayed both sensitivity and selectivity to RG2TK+ tumors. The selective in vivo accumulation of [(123)I]D-FIAU increased with time and the tumor uptake ratios (RG2TK+/RG2TK-) for 2, 4, and 24 hours averaged 6.2, 22.7, and 58.8, respectively. High-resolution SPECT of four nude tumor-bearing mice demonstrated a very high uptake of [(123)I]D-FIAU in the RG2TK+ tumor, while no significant tracer accumulation was observed in the RG2TK- tumor and other organs. CONCLUSION: The data suggest that only the D-isomer of [(123)I]FIAU is useful for imaging HSV1-tk gene expression in mice by high-resolution SPECT imaging.

Colchicine in clinical medicine. A guide for internists.

Colchicine (COL) has been used in medicine for a long time. It is well recognized as a valid therapy in acute flares of gouty arthritis, familial Mediterranean fever (FMF), Behçet's disease, and recurring pericarditis with effusion. It has also been used to treat many inflammatory disorders prone to fibrosis, mostly with disappointing therapeutic results. The pharmacotherapeutic mechanism of action of COL in diverse diseases is not fully understood, thought it is known that the drug accumulates preferentially in neutrophils, and this effect is useful in FMF. COL shows a large interindividual bioavailability. Furthermore, interactions with drugs interfering with CYP3A4 dependent enzymes and P-glycoprotein occur and are clinically important. The dosage of COL must be reduced in patients with relevant hepatic and/or renal dysfunction. However, when appropriately used and contraindications have been excluded, oral COL is a safe treatment.

Epigenetic downregulation of human disabled homolog 2 switches TGF-beta from a tumor suppressor to a tumor promoter.

The cytokine TGF-beta acts as a tumor suppressor in normal epithelial cells and during the early stages of tumorigenesis. During malignant progression, cancer cells can switch their response to TGF-beta and use this cytokine as a potent oncogenic factor; however, the mechanistic basis for this is poorly understood. Here we demonstrate that downregulation of disabled homolog 2 (DAB2) gene expression via promoter methylation frequently occurs in human squamous cell carcinomas (SCCs) and acts as an independent predictor of metastasis and poor prognosis. Retrospective microarray analysis in an independent data set indicated that low levels of DAB2 and high levels of TGFB2 expression correlate with poor prognosis. Immunohistochemistry, reexpression, genetic knockout, and RNAi silencing studies demonstrated that downregulation of DAB2 expression modulated the TGF-beta/Smad pathway. Simultaneously, DAB2 downregulation abrogated TGF-beta tumor suppressor function, while enabling TGF-beta tumor-promoting activities. Downregulation of DAB2 blocked TGF-beta-mediated inhibition of cell proliferation and migration and enabled TGF-beta to promote cell motility, anchorage-independent growth, and tumor growth in vivo. Our data indicate that DAB2 acts as a tumor suppressor by dictating tumor cell TGF-beta responses, identify a biomarker for SCC progression, and suggest a means to stratify patients with advanced SCC who may benefit clinically from anti-TGF-beta therapies.