RNF41 orchestrates macrophage-driven fibrosis resolution and hepatic regeneration.

Hepatic inflammation is a common trigger of chronic liver disease. Macrophage activation is a predictive parameter for survival in patients with cirrhosis. Ring finger protein 41 (RNF41) negatively regulates proinflammatory cytokines and receptors; however, the precise involvement of macrophage RNF41 in liver cirrhosis remains unknown. Here, we sought to understand how RNF41 dictates macrophage fate in hepatic fibrosis and repair within the inflammatory milieu. We found that RNF41 expression is down-regulated in CD11b+ macrophages recruited to mouse fibrotic liver and to patient cirrhotic liver regardless of cirrhosis etiology. Prolonged inflammation with TNF-α progressively reduced macrophage RNF41 expression. We designed a macrophage-selective gene therapy with dendrimer-graphite nanoparticles (DGNPs) to explore the influence of macrophage RNF41 restoration and depletion in liver fibrosis and regeneration. RNF41 expression induced in CD11b+ macrophages by DGNP-conjugated plasmids ameliorated liver fibrosis, reduced liver injury, and stimulated hepatic regeneration in fibrotic mice with or without hepatectomy. This therapeutic effect was mainly mediated by the induction of insulin-like growth factor 1. Conversely, depletion of macrophage RNF41 worsened inflammation, fibrosis, hepatic damage, and survival. Our data reveal implications of macrophage RNF41 in the control of hepatic inflammation, fibrosis, and regeneration and provide a rationale for therapeutic strategies in chronic liver disease and potentially other diseases characterized by inflammation and fibrosis.

Scalable synthesis of multicomponent multifunctional inorganic core@mesoporous silica shell nanocomposites.

Integrating multiple materials with different functionalities in a single nanostructure enables advances in many scientific and technological applications. However, such highly sophisticated nanomaterials usually require complex synthesis processes that complicate their preparation in a sustainable and industrially feasible manner. Herein, we designed a simple general method to grow a mesoporous silica shell onto any combination of hydrophilic nanoparticle cores. The synthetic strategy, based on the adjustment of the key parameters of the sol-gel process for the silica shell formation, allows for the embedment of single, double, and triple inorganic nanoparticles within the same shell, as well as the size-control of the obtained nanocomposites. No additional interfacial adhesive layer is required on the nanoparticle surfaces for the embedding process. Adopting this approach, electrostatically stabilized, small-sized (from 4 to 15 nm) CeO2, Fe3O4, Gd2O3, NaYF4, Au, and Ag cores were used to test the methodology. The mean diameter of the resulting nanocomposites could be as low as 55 nm, with high monodispersity. These are very feasible sizes for biological intervention, and we further observed increased nanoparticle stability in physiological environments. As a demonstration of their increased activity as a result of this, the antioxidant activity of CeO2 cores was enhanced when in core-shell form. Remarkably, the method is conducted entirely at room temperature, atmospheric conditions, and in aqueous solvent with the use of ethanol as co-solvent. These facile and even "green" synthesis conditions favor scalability and easy preparation of multicomponent nanocomposite libraries with standard laboratory glassware and simple benchtop chemistry, through this sustainable and cost-effective fabrication process.

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and ß-Hydroxybutyrate/Acetoacetate in Biological Samples.

The metabolic ratios lactate/pyruvate and ß-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, ß-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and ß-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Mesoporous silica coated CeO2 nanozymes with combined lipid-lowering and antioxidant activity induce long-term improvement of the metabolic profile in obese Zucker rats.

Obesity is one of the most important public health problems that is associated with an array of metabolic disorders linked to cardiovascular disease, stroke, type 2 diabetes, and cancer. A sustained therapeutic approach to stop the escalating prevalence of obesity and its associated metabolic comorbidities remains elusive. Herein, we developed a novel nanocomposite based on mesoporous silica coated cerium oxide (CeO2) nanozymes that reduce the circulating levels of fatty acids and remarkably improve the metabolic phenotype in a model of obese Zucker rats five weeks after its administration. Lipidomic and gene expression analyses showed an amelioration of the hyperlipidemia and of the hepatic and adipose metabolic dysregulations, which was associated with a down-regulation of the hepatic PI3K/mTOR/AKT pathway and a reduction of the M1 proinflammatory cytokine TNF-α. In addition, the coating of the CeO2 maximized its cell antioxidant protective effects and minimized non-hepatic biodistribution. The one-pot synthesis method for the nanocomposite fabrication is implemented entirely in aqueous solution, room temperature and open atmosphere conditions, favoring scalability and offering a safe and translatable lipid-lowering and antioxidant nanomedicine to treat metabolic comorbidities associated with obesity. This approach may be further applied to address other metabolic disorders related to hyperlipidemia, low-grade inflammation and oxidative stress.

Tetracaine from urethral ointment causes false positive amphetamine results by immunoassay.

BACKGROUND: Amphetamine urine drug screening by immunoassay is prone to cross-react with other compounds leading to false positive results. Tetracaine is a local anesthetic drug used in the clinical setting as an ointment during urinary catheterization. In our laboratory, tetracaine is often detected by gas chromatography-mass spectrometry in the urine of patients admitted in the emergency department with false positive amphetamine results. The objectives of this study were to investigate if there was cross-reactivity to tetracaine in an amphetamine immunoassay and to retrospectively evaluate the potential contribution of tetracaine to false positive amphetamine results. METHODS: An interference study was conducted using negative urine samples spiked with increasing concentrations of tetracaine hydrochloride and analyzed with the CEDIA Amphetamine/Ecstasy immunoassay. Retrospectively, urine samples of patients which yielded positive amphetamine immunoassay results and were analyzed by gas chromatography-mass spectrometry were reviewed (n = 417). The presence of tetracaine and/or other drugs by gas chromatography-mass spectrometry were gathered. RESULTS: Tetracaine caused false positive amphetamine results by immunoassay (cut-off 1000 µg/L) with a concentration of above 40 mg/L. Retrospective analysis of all positive amphetamine immunoassay samples showed that in 45 out of the 417 (10.8%) urine samples no amphetamine-like derivative was identified by gas chromatography - mass spectrometry. In 37 out of 45 (82.2%) of these false positive cases tetracaine was detected, of whom 59.5% (22/37) had an estimated tetracaine concentration of ≥40 mg/L. CONCLUSIONS: This study confirmed the interference of tetracaine in the CEDIA Amphetamine/Ecstasy immunoassay and that tetracaine may have contributed to around 80% of the false positive amphetamine cases in the urine samples of patients admitted to the emergency department at our institution.

Evaluation of an immunoassay procedure to measure 6-monoacetylmorphine.

Introduction: 6-Monoacetylmorphine (6-MAM) is a specific metabolite of heroin. Thus, the presence of 6-MAM in urine is a definitive indication of heroin intake. The possibility of having an immunoassay procedure to measure 6-MAM would be a diagnosis tool to discriminate, among opiates-positive, those patients who have consumed heroin and those who have not.Methods: EMIT® II Plus 6-Acetylmorphine Assay was used to measure 6-MAM in urine. The positive opiate screening results were confirmed at the Toxicology laboratory of our hospital by GC-MS.Results: This study includes 63 urine samples from subjects admitted to emergency department with suspicion of opiate consumption. Specificity was evaluated in the two groups of samples studied. In the first group all samples which resulted negative by opiate immunoassay (n = 33) were negative for 6-MAM immunoassay test. Thus, the specificity obtained for 6-MAM immunoassay in this group was 100%. Regarding the second specificity study, performed in positive samples by opiate immunoassay which were negative to 6 MAM by GC-MS, the specificity decreased down to 75%. In the study of sensitivity all samples confirmed as positive to 6-MAM by confirmatory method (GC-MS) resulted positive by the screening method, thus sensitivity obtained was 100%.Discussion: In this study no FN for 6-MAM was observed and therefore the new Emit® II Plus 6- Acetylmorphine Assay procedure has a high NPV, thus a negative result with 6-MAM immunoassay practically excludes heroine consume. The positive results to 6-MAM by immunoassay should be confirmed by a more analytically specific method, such as GCMS.

Cerium Oxide Nanoparticles: Advances in Biodistribution, Toxicity, and Preclinical Exploration.

Antioxidant nanoparticles have recently gained tremendous attention for their enormous potential in biomedicine. However, discrepant reports of either medical benefits or toxicity, and lack of reproducibility of many studies, generate uncertainties delaying their effective implementation. Herein, the case of cerium oxide is considered, a well-known catalyst in the petrochemistry industry and one of the first antioxidant nanoparticles proposed for medicine. Like other nanoparticles, it is now described as a promising therapeutic alternative, now as threatening to health. Sources of these discrepancies and how this analysis helps to overcome contradictions found for other nanoparticles are summarized and discussed. For the context of this analysis, what has been reported in the liver is reviewed, where many diseases are related to oxidative stress. Since well-dispersed nanoparticles passively accumulate in liver, it represents a major testing field for the study of new nanomedicines and their clinical translation. Even more, many contradictory works have reported in liver either cerium-oxide-associated toxicity or protection against oxidative stress and inflammation. Based on this, finally, the intention is to propose solutions to design improved nanoparticles that will work more precisely in medicine and safely in society.

Beyond the Scavenging of Reactive Oxygen Species (ROS): Direct Effect of Cerium Oxide Nanoparticles in Reducing Fatty Acids Content in an In Vitro Model of Hepatocellular Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic accumulation of lipids. Antisteatotic effects of cerium oxide nanoparticles (CeO2NPs) have recently been shown in animal models of liver disease. However, it is unclear whether the activity of CeO2NPs is related solely to the decrease in oxidative stress or, in addition, they directly decrease liver fatty acid accumulation. To address this question, in this work, we used an in vitro model of hepatocellular steatosis, exposing HepG2 cells to oleic and palmitic acid. Cell uptake of CeO2NPs and their effect on oxidative stress and viability of hepatic cells cultured with H2O2 were also evaluated. Results show that CeO2NPs were uptaken by HepG2 cells and reduced oxidative stress and improved cell viability. Treatment with oleic and palmitic acid increased lipogenesis and the content of different fatty acids. CeO2NPs reduced palmitic and stearic acid and most fatty acids consisting of more than 18 carbon atoms. These effects were associated with significant changes in elongase and desaturase activity. In conclusion, CeO2NPs directly protected HepG2 cells from cell injury in oxidative stress conditions and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, thus showing potential in the treatment of NAFLD.

Serum CA 19.9 Levels in Patients With Benign and Malignant Disease: Correlation With the Serum Protein Electrophoretic Pattern.

AIM: To confirm that the carbohydrate antigen 19.9 (CA 19.9) protein can be evaluated by determining changes in the ß2 zone in protein electrophoresis. MATERIALS AND METHODS: A total of 75 patients (64 with cancer, 11 with benign diseases) with abnormal serum CA 19.9 level were included. RESULTS: Patients with cancer had significantly higher serum CA 19.9 concentrations than those with benign diseases (p<0.001). Similar CA 19.9 levels were observed in patients with normal (median 1129 U/ml), weakly positive (median 699 U/ml) and positive (2333 U/ml) ß2 fraction in protein electrophoresis. In contrast, changes in the protein pattern of the ß2 fraction were related to an inflammatory pattern with significantly higher C-reactive protein concentration (p<0.0001), independently of serum CA 19.9 level. CONCLUSION: The intensity of the ß2 fraction in protein electrophoresis is related to inflammation and not to CA 19.9 in patients with cancer or other diseases.

Relationship Between CA 19.9 and the Lewis Phenotype: Options to Improve Diagnostic Efficiency.

BACKGROUND/AIM: Approximately 10% of patients are unable to synthesize CA 19.9 (Lewis-negative), and these results are erroneously considered false-negatives. The aim of this study was to confirm that CA 19.9 cannot be detected by immunoassays in Lewis-negative patients. MATERIALS AND METHODS: CA 19.9 levels were measured by immunological assays and Lewis phenotype was determined by the haemagglutination reaction. RESULTS: Patients with Lewis phenotype (a+b-) or (a-b+) had significantly higher CA 19.9 levels than Lewis-negative patients with active cancer (p<0.001), no-evidence of disease (NED) patients (p<0.001) or patients with benign disease (p<0.001). Ninenty-four percent of patients (33/35) with undetectable CA 19.9 had a Lewis-negative phenotype. Additionally, 94.7% (34/36) of patients with Lewis-negative phenotypes had undetectable CA 19.9 serum levels. CONCLUSION: Patients with undetectable CA 19.9 serum levels tend to be Lewis-negative, and CA 19.9 is not useful in diagnosis or follow-up.

Graphene-Dendrimer Nanostars for Targeted Macrophage Overexpression of Metalloproteinase 9 and Hepatic Fibrosis Precision Therapy.

Fibrosis contributes to ∼45% of all deaths in industrialized nations, but no direct antifibrotic therapeutic interventions exist to date. Graphene-based nanomaterials exhibit excellent versatility in electronics, and emerging trends exploit their properties for biomedical applications, especially for drug and gene delivery. We designed constructs of graphene nanostars linked to PAMAM-G5 dendrimer for the selective targeting and delivery of a plasmid expressing the collagenase metalloproteinase 9 under the CD11b promoter into inflammatory macrophages in cirrhotic livers. Graphene nanostars preferentially accumulated in inflammatory macrophages M1 in less than 3 h in a manner unaffected by covalent linkage to dendrimers. Dendrimer-graphene nanostars efficiently delivered the plasmid encoding for metalloproteinase 9 into macrophages, allowing the synthesis and secretion of the metalloproteinase to digest adjacent collagen fibers. In turn, metalloproteinase 9 overexpression promoted the macrophage switch from inflammatory M1 to pro-regenerative M2 in 3 days. This targeted gene therapy reduced selectively and locally the presence of collagen fibers in fibrotic tracts where inflammatory macrophages accumulated in cirrhotic mice without affecting the activation state of hepatic stellate cells. Overall, this treatment significantly reduced hepatic injury and improved liver restoration in mice with liver cirrhosis treated for 10 days. Graphene-dendrimer nanostars targeted the macrophage overexpression of metalloproteinase 9, selectively reducing hepatic fibrosis, and might be a good treatment for diseases associated with fibrosis and inflammatory macrophage accumulation.

Validation of a routine gas chromatography mass spectrometry method for 2-hydroxyglutarate quantification in human serum as a screening tool for detection of idh mutations.

High circulating levels of 2-hydroxyglutarate (2HG) have been reported in patients with determinate isocitrate dehydrogenase (IDH) mutated tumors. Recent studies indicate that in malignancies such as acute myeloid leukemia (AML), measurements of 2HG in serum provide useful diagnostic and prognostic information and improve patient selection and monitoring of IDH-targeted treatments. In the current study, we validated a sensitive and specific gas chromatography mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HG in routine clinical laboratories. Extraction was liquid-liquid with ethyl acetate, and derivatization was reduced to 3 min of microwave irradiation. The analytical method was linear over a wide dynamic range, presenting acceptable intraday and day-to-day precision and accuracy. The limit of quantification was 10 ng/mL, process efficiency ranged between 38% and 49%, and recovery of added 2HG was 99-105%. 2HG was found to be stable in serum for up to 48 h at both 4 °C and at ambient temperature, and after three freeze-thaw cycles. Microwave derivatizated extracts in the autosampler were found to be stable for up to 120 h. In summary, the present method is useful for quantification of 2HG serum levels in patients with IDH mutated malignancies in clinical laboratories.

Utility of proGRP as a tumor marker in the medullary thyroid carcinoma.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor caused by a malignant transformation in the parafollicular C-cells of the thyroid, where calcitonin (CT) is released. Nowadays the main tumor markers (TM) used in the diagnosis and follow-up of MTC patients are CT and carcinoembryonic antigen (CEA). Nonetheless, progastrin releasing peptide (proGRP) has been recently proposed as a TM useful in the MTC. Our aims were to investigate the release of proGRP in thyroid tumors, its role in the assessment of advanced MTC and its utility in the differential diagnosis between MTC and non-MTC thyroid tumors. METHODS: Serum samples from 22 patients with MTC and 16 with non-MTC were collected. Patients were classified into advanced cancer or no evidence of disease (NED). ProGRP was performed by Architect (Abbot Diagnostics), CT by Liaison (Diasorin) and CEA by Cobas E601(Roche Diagnostics). RESULTS: ProGRP median concentration in advanced MTC was significantly higher (1398.4 pg/mL) when compared with non-MTC, either in advanced disease (24.9 pg/mL) or NED (14.6 pg/mL). In non-MTC patients, proGRP median concentration was below its cutoff level (50 pg/mL). Similar to CT, proGRP was able to detect 88.9% of MTC patients, but with a slightly lower specificity of 76.9%. Using proGRP together with CT the sensitivity increased to 100%. CONCLUSIONS: The low prevalence of this malignancy strongly recommends further collaborative studies, mainly focused on monitoring proGRP during tyrosine kinase inhibitors treatment for early detection of resistance and assessing its usefulness to avoid the observed false positive fluctuations that occur with CT and CEA.

Verificación e implantación en un laboratorio de urgencias de un sistema de medición de los índices séricos (hemólisis, ictericia y lipidemia) en un Dimension® EXL(TM) / Verification and implementation of a serum index measurement system (haemolysis, icterus, and lipaemia) in a Dimension® EXL(TM) in an emergency laboratory

Introducción. La hemólisis, ictericia y lipidemia son los principales interferentes que pueden producir errores analíticos en la medición de magnitudes bioquímicas. Muchos analizadores incorporan sistemas de detección de interferentes, sin embargo no suelen estar verificados. El objetivo del estudio es verificar el sistema de medición HIL del analizador Dimension® EXL(TM) y comprobar la adecuada asignación por el proveedor de los valores de alerta. Material y métodos. Se ha evaluado el efecto de la hemoglobina, bilirrubina y triglicéridos en los resultados, comparando el valor de la magnitud en la muestra sin interferente con los valores obtenidos en la misma muestra con concentraciones crecientes del mismo. Se ha seguido el procedimiento recomendado por la Comisión de Metrología y Sistemas Analíticos de la SEQC. Asimismo, se ha elaborado un algoritmo de cuándo informar la presencia de interferencias (criterios clínicos y técnicos). Resultados. Todos los resultados de los índices hemolíticos incluyeron la concentración esperada del interferente, para la ictericia hubo ligeras diferencias, mientras que para la lipidemia el analizador proporcionó resultados más bajos de los esperados. En el estudio de los índices de alerta HIL hubo diferencias entre los resultados obtenidos y la información del fabricante. Se presenta el algoritmo para informar la presencia de estas interferencias. Conclusiones. La incorporación de estos índices de alerta sin una previa verificación de los mismos puede llevar a cometer errores. Una correcta verificación de estos sistemas permitiría detectar la falta de veracidad en la medición de estos interferentes o el inadecuado establecimiento de algunos índices de alerta (AU)

Introduction. Haemolysis, icterus (bilirubin) and lipaemia (triglycerides) (HIL) are the main interferences that can lead to analytical errors in the measurement of biological substances. Many analysers incorporate interference detection systems, which nonetheless are often not verified. The main objective is to verify the HIL measurement system of the Dimension® EXL(TM) analyser, and to check the correct assignment of the alert values by the supplier. Material and methods. The effect of the haemolysis, bilirubin and triglycerides on the results has been assessed by comparing the value of the quantity in a sample without interference with the values obtained in the same sample with increasing concentrations of interfering substances. The procedure recommended by the Comisión de Metrología y Sistemas Analíticos of the SEQC has been followed. An algorithm to inform of interferences, based on clinical and technical aspects, has been developed. Results. All haemolytic index results included the expected concentration of the interfering substance. Few errors were found for icterus, while for lipaemia the analyser gave results lower than expected. In the study of the HIL alert indexes, differences were found between the results obtained and the information provided by the supplier. Finally the algorithm followed in our laboratory to inform the presence of interfering substances is presented. Conclusions. The introduction of these alert indexes without a prior verification of them can lead to potential errors. Proper verification of these systems would enable detecting the lack of trueness in the measurement of the interfering substances or the inadequate establishment of some alert indexes (AU)

Improvement in the stability of serum samples stored in an automated refrigerated module.

OBJECTIVES: In clinical laboratories it is necessary to know for how long the analytes are stable in the samples with specific storage conditions. Our laboratory has implemented the new Aptio Automation System (AAS) (Siemens Healthcare Diagnostics) where the analyzed samples are stored in a refrigerated storage module (RSM) after being sealed. The aim of the study was to evaluate the stability of serum samples with the AAS and comparing the results with a previous study using a conventional refrigerated system. DESIGN AND METHODS: Serum samples from a total of 50 patients were collected and for each of them 27 biochemical analytes were analyzed. The samples were divided in 5 sets of 10 samples. Each set was re-analyzed at one of the following times: 24, 48, 72, 96 and 120h. Stability was evaluated according to the Total Limit of Change (TLC) criteria, which combine both analytical and biologic variation. RESULTS: A total of 26 out of 27 analytes were stable at the end of the study according to TLC criteria. Lactate dehydrogenase was not stable at 48h observing a decrease in its concentration until the end of the study. In the previous study (conventional storage system) 9 biochemical analytes were not stable with an increase of their levels due to the evaporation process. CONCLUSIONS: The RSM connected to the AAS improves the stability of serum samples. This system avoids the evaporation process due to the sealing of samples and allows better control of the samples during their storage.