Ezetimibe prevents ischemia/reperfusion-induced oxidative stress and up-regulates Nrf2/ARE and UPR signaling pathways

BACKGROUND: While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress. Nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) are protective against oxidative stress and endoplasmic reticulum (ER) stress. Ezetimibe, a cholesterol absorption inhibitor, has been shown to activate the AMP-activated protein kinase (AMPK)/Nrf2 pathway. In this study we evaluated whether Ezetimibe affects oxidative stress and Nrf2 and UPR gene expression in cellular models of ischemia-reperfusion (IR). METHODS: Cultured cells were subjected to simulated IR with or without Ezetimibe. RESULTS: IR significantly increased reactive oxygen species (ROS) production and the percentage of apoptotic cells without the up-regulation of Nrf2, of the related antioxidant response element (ARE) gene expression or of the pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Ezetimibe significantly decreased the cellular ROS formation and apoptosis induced by IR. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. We also found that Nrf2 activation was dependent on AMPK, since Compound C, a pan inhibitor of p-AMPK, blunted the activation of Nrf2. CONCLUSIONS: Ezetimibe counteracts IR-induced oxidative stress and induces Nrf2 and UPR pathway activation

Plasma bile acid profile in patients with and without Type 2 Diabetes

A paucity of information currently exists on plasma bile acid (BA) profiles in patients with and without type 2 diabetes mellitus (T2DM). We assayed 14 plasma BA species in 224 patients with T2DM and in 102 nondiabetic individuals with metabolic syndrome. Plasma BA levels were measured with ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) technique. Multivariable linear regression analyses were undertaken to assess associations between measured plasma BA species and T2DM status after adjustment for confounding factors. The presence of T2DM was significantly associated with higher plasma concentrations of both primary BAs (adjusted-standardized beta coefficient: 0.279, p = 0.005) and secondary BAs (standardized beta coefficient: 0.508, p < 0.001) after adjustment for age, sex, adiposity measures, serum alanine aminotransferase and use of statins or metformin. More specifically, the presence of T2DM was significantly associated with higher levels of plasma taurochenodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, hyodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, deoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid and glycodeoxycholic acid (adjusted-standardized beta coefficients ranging from 0.315 to 0.600; p < 0.01 or less), as well as with lower plasma levels of cholic acid (adjusted-standardized beta coefficient: -0.250, p = 0.013) and taurocholic acid (adjusted-standardized beta coefficient: -0.309, p = 0.001). This study shows that there are marked differences in plasma BA profiles between patients with and without T2DM. Further research will be needed to better understand how these differences in plasma BA profiles may interplay with the pathophysiology of T2DM

Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

Dexamethasone scheduling in MNC culture. The supplement was added in standard medium until the detection of MSC colonies (n = 16 CB units) or alternatively added for the first week of MNC culture only (n = 34). (DOCX 120 kb

SARS-CoV-2 infection in health workers: analysis from Verona SIEROEPID Study during the pre-vaccination era

Background: To report the baseline phase of the SIEROEPID study on SARS-CoV-2 infection seroprevalence among health workers at the University Hospital of Verona, Italy, between spring and fall 2020; to compare performances of several laboratory tests for SARS-CoV-2 antibody detection. Methods: 5299 voluntary health workers were enrolled from 28 April 2020 to 28 July 2020 to assess immunological response to SARS-CoV-2 infection throughout IgM, IgG and IgA serum levels titration by four laboratory tests. Association of antibody titre with several demographic variables, swab tests and performance tests (sensitivity, specificity, and agreement) were statistically analyzed. Results: The overall seroprevalence was 6%, considering either IgG and IgM, and 4.8% considering IgG. Working in COVID-19 Units was not associated with a statistically significant increase in the number of infected workers. Cohen's kappa of agreement between MaglumiTM and VivaDiagTM was quite good when considering IgG only (Cohen's kappa = 78.1%, 95% CI 74.0-82.0%), but was lower considering IgM (Cohen's kappa = 13.3%, 95% CI 7.8-18.7%). Conclusion: The large sample size with high participation (84.7%), the biobank and the longitudinal design were significant achievements, offering a baseline dataset as the benchmark for risk assessment, health surveillance and management of SARS-CoV-2 infection for the hospital workforce, especially considering the ongoing vaccination campaign. Study results support the national regulator guidelines on using swabs for SARS-CoV-2 screening with health workers and using the serological tests to contribute to the epidemiological assessment of the spread of the virus

CONDIÇÕES DE TRABALHO NO CONTEXTO DE CATADORAS DE MATERIAIS RECICLÁVEIS: DESAFIOS E PERSPECTIVAS PARA O TRABALHO SEGURO

RESUMO O estudo objetivou descrever as condições de trabalho segundo a percepção de catadoras de materiais recicláveis associadas, destacando os desafios e perspectivas para o trabalho seguro a partir da ação assistencial de enfermagem. Trata-se de estudo qualitativo, fundamentado na Pesquisa Convergente-Assistencial, realizado com 11 catadoras de materiais recicláveis de uma associação do Sul do Brasil. Os dados foram produzidos por meio de observação participante, entrevistas semiestruturadas e grupo de convergência. A análise foi realizada por meio dos passos estabelecidos pelo método: apreensão, síntese, teorização e transferência. Como resultados emergiram três categorias: “Organização do trabalho como elemento agravante das condições de trabalho: desencontros no processo de comunicação e colaboração”; “As condições de trabalho agravadas pelo rodízio de atividades e sobrecarga laboral”; e “Perspectivas para o trabalho seguro: problematizando as condições de trabalho”. Os dados evidenciam que as catadoras vivenciam precárias condições de trabalho relacionadas, principalmente, à execução das tarefas, dificultada por falhas nos processo de comunicação e colaboração entre os membros da equipe e com os fornecedores de materiais recicláveis. As precárias condições de trabalho culminam em sobrecarga, dor, automedicação e acidentes. A problematização das condições trabalho mostrou-se uma exitosa prática assistencial para a promoção do trabalho seguro. Concluiu-se que a mediação de um espaço de diálogo e pactuação pode oferecer perspectivas para a otimização do trabalho seguro

EZETIMIBE PREVENTS ISCHEMIA/REPERFUSION-INDUCED OXIDATIVE STRESS THROUGH UP-REGULATION OF NRF2/ARE AND UPR SIGNALING PATHWAYS AND INDUCTION OF AUTOPHAGY

While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress and cell damages through production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Evidences indicate that nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) pathways are protective against oxidative stress (OS) and ER stress. Ezetimibe (Eze), a cholesterol absorption inhibitor, has been shown to activate Nrf2 pathway in a p62-dependent manner. In this study, we evaluated whether Eze may affect OS as well as Nrf2 and UPR gene expression in cellular models (THP-1 monocytic cells and cardiomyocytes) of I-R. After an overnight (ON) treatment with 50\u3bcM of Eze, cells were subjected to ischemia (2 hours and 24 hours respectively) followed by reperfusion (1 hour and 24 hours respectively). I-R significantly increased ROS production and % apoptotic cells without up-regulation of Nrf2, related antioxidant response element (ARE) gene expression and pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Eze significantly decreased cellular ROS formation and apoptosis induced by I-R. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. Concerning the mechanism of Nrf2 activation, we also found that Eze significantly increased the phosphorylation of p62 that was dependent on adenosine 5\u2019-monophosphate (AMP)-activated protein kinase (AMPK) since Compound C (CC), a pan inhibitor of AMPK, blunted the activation of p62 and hence Nrf2. Since p62 phosphorylation has been shown to be dependent also on mTORC1 kinase, we performed further experiments using Rapamycin (Rap), a mTORC1 inhibitor. Despite the inhibition of mTORC1-dependent p62 phosphorylation by Rap, Nrf2 was activated. On the contrary, the co-treatment with Eze plus Rap reduced Nrf2 nuclear translocation because of the interaction of p62 with autophagic LC3B protein, which is basically increased both by Eze and Rap treatment, leading to the activation of autophagy. Finally, we tested our I-R model in human cardiomyocytes finding that, as in THP-1 cells, Eze significantly reduced ROS formation as well as I-R-induced apoptosis increasing Nrf2 nuclear translocation. Taken together, data demonstrated the new pleiotropic effects of Eze in counteracting I-R-induced oxidative stress through Nrf2 and UPR pathway activation as well as autophagy induction

Carbamazepine and Carbamazepine-10,11-epoxide measurement with the reference LC-MS/MS method and a routine immunoassay

Introduction: Carbamazepine is a common antiepileptic drug used for treatment of epilepsy and other neurologic conditions. Carbamazepine-10,11-epoxide, one of its metabolites, is pharmacologically active and its values increase with concomitant use of other anticonvulsants. This study is aimed to compare the method used in our routine laboratory with a reference LC-MS/MS method for monitoring Carbamazepine, and to compare the concentrations of the drug to one of its metabolites. Methods: plasma samples were collected from patients for whom Carbamazepine measurement had been requested. For each plasma sample, Carbamazepine was assayed on Roche Cobas with Cobas CARB4 kit (immunoassay). Carbamazepine and Carbamazepine-10,11-epoxide determination were then performed with a validated and verified LC-MS/MS technique. Results: the study population consisted of 30 subjects. No significant differences were found between the routine immunoassay and the LC-MS/MS technique using Passing-Bablok regression and Bland-Altman graph, whilst the concentrations of plasma Carbamazepine and its epoxide measured with LC-MS/MS displayed a very modest correlation (r=0.639). The ratio calculated between Carbamazepine and its epoxide displayed a broad range of values (3.37-12.55). Discussion: considering the clinical significance of Carbamazepine measurement as part of TDM, we confirmed the validity of our routinely used immunoassay as easier and faster alternative to the reference method for routine quantification of plasma Carbamazepine concentration. Considering that levels of the epoxide are unpredictable and independent from the parent drug concentrations, a more comprehensive assessment of Carbamazepine metabolites should be considered, especially when patients have uncontrolled symptoms or display challenging dose adjustment

Comprehensive assessment of humoral response after Pfizer BNT162b2 mRNA Covid-19 vaccination: a three-case series

Objectives: Since universal vaccination is a pillar against coronavirus disease 2019 (COVID-19), monitoring anti-SARS-CoV-2 neutralizing antibodies is essential for deciphering post-vaccination immune response. Methods: Three healthcare workers received 30 \u3bcg BNT162b2 mRNA Covid-19 Pfizer Vaccine, followed by a second identical dose, 21 days afterwards. Venous blood was drawn at baseline and at serial intervals, up to 63 days afterwards, for assessing total immunoglobulins (Ig) anti-RBD (receptor binding domain), anti-S1/S2 and anti-RBD IgG, anti-RBD and anti-N/S1 IgM, and anti-S1 IgA. Results: All subjects were SARS-CoV-2 seronegative at baseline. Total Ig anti-RBD, anti-S1/S2 and anti-RBD IgG levels increased between 91 and 368 folds until 21 days after the first vaccine dose, then reached a plateau. The levels raised further after the second dose (by 3c30-, 3c8- and 3c8-fold, respectively), peaking at day 35, but then slightly declining and stabilizing 3c50 days after the first vaccine dose. Anti-S1 IgA levels increased between 7 and 11 days after the first dose, slightly declined before the second dose, after which levels augmented by 3c24-fold from baseline. The anti-RBD and anti-N/S1 IgM kinetics were similar to that of anti-S1 IgA, though displaying substantially weaker increases and modest peaks, only 4- to 7-fold higher than baseline. Highly significant inter-correlation was noted between total Ig anti-RBD, anti-S1/S2 and anti-RBD IgG (all r=0.99), whilst other anti-SARS-CoV-2 antibodies displayed lower, though still significant, correlations. Serum spike protein concentration was undetectable at all-time points. Conclusions: BNT162b2 mRNA vaccination generates a robust humoral immune response, especially involving anti-SARS-Cov-2 IgG and IgA, magnified by the second vaccine dose

Comparison of five commercial anti-SARS-CoV-2 total antibodies and IgG immunoassays after vaccination with BNT162b2 mRNA

BNT162b2 mRNA Covid-19 vaccine, immune response, antibodies, immunoassays compariso

Plasma Bile Acid Profiling and Modulation of Secreted Mucin 5AC in Cholangiocarcinoma

Studies investigating the potential role of circulating bile acids (BAs) as diagnostic biomarkers for cholangiocarcinoma (CCA) are sparse and existing data do not adjust for confounding variables. Furthermore, the mechanism by which BAs affect the expression of the oncogenic mucin 5AC (MUC5AC) has never been investigated. We performed a case–control study to characterise the profile of circulating BAs in patients with CCA (n = 68) and benign biliary disease (BBD, n = 48) with a validated liquid chromatography–tandem mass spectrometry technique. Odd ratios (OR) for CCA associations were calculated with multivariable logistic regression models based on a directed acyclic graph structure learning algorithm. The most promising BAs were then tested in an in vitro study to investigate their interplay in modulating MUC5AC expression. The total concentration of BAs was markedly higher in patients with CCA compared with BBD controls and accompanied by a shift in BAs profile toward a higher proportion of primary conjugated BAs (OR = 1.50, CI: 1.14 to 1.96, p = 0.003), especially taurochenodeoxycholic acid (TCDCA, OR = 42.29, CI: 3.54 to 504.63, p = 0.003) after multiple adjustments. Western blot analysis of secreted MUC5AC in human primary cholangiocytes treated with primary conjugated BAs or with TCDCA alone allowed us to identify a novel 230 kDa isoform, possibly representing a post-translationally modified MUC5AC specie