Evaluation of IRES-mediated expression and different signal peptides for the development of CHO clones producing an anti-PCSK9 monoclonal antibody

High levels of low-density lipoprotein (LDL) in the blood are associated to an increased risk of cardiovascular diseases, which are a leading cause of death worldwide. Despite this scenario, only minor progress was made since the introduction of statins as lipid-lowering synthetic drugs in the 1980s. Among patients suffering from high cholesterol levels, about 20% present strong adverse effects to statins or don’t manage to decrease their LDL levels to normality. Since 2015, a new class of monoclonal antibodies (mAbs) was approved by FDA and EMA. These mAbs target the enzyme PCSK9 that is involved in the regulation of LDL cholesterol homeostasis, allow a higher amount of LDL receptors to be available on the surface of hepatic cells and thus enable a significant reduction of the circulating cholesterol levels. In this study, we selected signal peptides and IRES elements from the literature to use for the construction of different tricistronic expression vectors. For the light chain, two different signal peptides were evaluated (SP1 and SP2), whereas for the heavy chain just one signal peptide was used (SP3). An EMCV IRES element was placed between the light chain (LC) gene and SP3. Downstream of the heavy chain (HC) gene, an attenuated IRES (att-IRES) element was inserted, followed by the antibiotics resistance gene for selection. In order to enable the evaluation of a stringent double selection (two different antibiotics) upon co-transfection with two plasmids, two sets of vectors were constructed, having either geneticin (neo) or hygromycin B as selection marker. Thus, a total of 4 vectors were constructed, having the following tricistronic cassette structure: CMV-IE promoter, SP1 or SP2, LC gene, EMCV IRES, SP3, HC gene, att-IRES, neo or hygro. CHO.K1 (ATCC, USA) previously adapted to suspension culture in TC-LECC medium (Xell AG, Germany) were transfected (or co-transfected) using Lipofectamine 3000 (Gibco, USA), using either circular or linearized plasmids. Cell culture supernatants harvested 48h post transfection showed a similar expression level for all constructs. After approximately 2 months under selection pressure with the respective antibiotics, the stable cell pool that had been transfected with circular SP1-LC-SP3-HC-NEO was chosen for further studies. Cells were cultivated in shake flasks, attaining 13.9E6 cells/mL on day 7 and showing an specific growth rate of 1.2 d-1 during the exponential growth phase. The mAb was purified from cell culture supernatant using protein A affinity chromatography, showing high purity and homogeneity. Affinity of the purified mAb to PCSK9 was tested, confirming the success of the approach adopted in this work

Multicenter survey on the use of device-assisted enteroscopy in Portugal

Background: Device-assisted enteroscopies (DAEs) are recent endoscopic techniques that enable direct endoscopic smallbowel evaluation. Objective: The objective of this article is to evaluate the implementation of DAEs in Portugal and assess the main indications, diagnoses, diagnostic yield, therapeutic yield and complication rate. Methods: We conducted a multicenter retrospective series using a national Web-based survey on behalf of the Portuguese Small-Bowel Study Group. Participants were asked to fill out two online databases regarding procedural data, indications, diagnoses, endoscopic therapy and complications using prospectively collected institutional data records. Results: A total of eight centers were enrolled in the survey, corresponding to 1411 DAEs. The most frequent indications were obscure gastrointestinal bleeding (OGIB), inflammatory bowel disease and small-bowel tumors. The pooled diagnostic yield was 63%. A relation between the diagnostic yield and the indications was clear, with a diagnostic yield for OGIB of 69% (p ¼ 0.02) with a 52% therapeutic yield. Complications occurred in 1.2%, with a major complication rate of 0.57%. Perforations occurred in four patients (0.28%). Conclusion: DAEs are safe and effective procedures, with complication rates of 1.2%, the most serious of which is perforation. Most procedures are performed in the setting of OGIB. Diagnostic and therapeutic yields are dependent on the indication, hence appropriate patient selection is crucial.info:eu-repo/semantics/publishedVersio

LINGUAGEM: MÚSICA, ARTE, CULTURA E INFLUÊNCIA NO EMI, CAMPUS CHAPECÓ

Este trabalho apresenta uma pesquisa com a finalidade de analisar de forma científica a música como forma de linguagem e expressão artística e a influência que ela exerce nos alunos do Ensino Médio Integrado,IF-SC Campus Chapecó

Population Genetics of Streptococcus dysgalactiae Subspecies equisimilis Reveals Widely Dispersed Clones and Extensive Recombination

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America.The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred approximately 4.4 times more frequently than by point mutation.A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population

Response of high-risk of recurrence/progression bladder tumours expressing sialyl-Tn and sialyl-6-T to BCG immunotherapy

High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Guérin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of this work is to determine the efficiency of BCG immunotherapy against tumours expressing sTn and sTn-related antigen sialyl-6-T (s6T). METHODS: In a retrospective design, 94 tumours from patients treated with BCG were screened for sTn and s6T expression. In vitro studies were conducted to determine the interaction of BCG with high-grade bladder cancer cell line overexpressing sTn. RESULTS: From the 94 cases evaluated, 36 had recurrence after BCG treatment (38.3%). Treatment outcome was influenced by age over 65 years (HR=2.668; (1.344-5.254); P=0.005), maintenance schedule (HR=0.480; (0.246-0.936); P=0.031) and multifocality (HR=2.065; (1.033-4.126); P=0.040). sTn or s6T expression was associated with BCG response (P=0.024; P<0.0001) and with increased recurrence-free survival (P=0.001). Multivariate analyses showed that sTn and/or s6T were independent predictive markers of recurrence after BCG immunotherapy (HR=0.296; (0.148-0.594); P=0.001). In vitro studies demonstrated higher adhesion and internalisation of the bacillus to cells expressing sTn, promoting cell death. CONCLUSION: s6T is described for the first time in bladder tumours. Our data strongly suggest that BCG immunotherapy is efficient against sTn- and s6T-positive tumours. Furthermore, sTn and s6T expression are independent predictive markers of BCG treatment response and may be useful in the identification of patients who could benefit more from this immunotherapy

Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial

<p>Abstract</p> <p>Background</p> <p>Blood pressure (BP) within pre-hypertensive levels confers higher cardiovascular risk and is an intermediate stage for full hypertension, which develops in an annual rate of 7 out of 100 individuals with 40 to 50 years of age. Non-drug interventions to prevent hypertension have had low effectiveness. In individuals with previous cardiovascular disease or diabetes, the use of BP-lowering agents reduces the incidence of major cardiovascular events. In the absence of higher baseline risk, the use of BP agents reduces the incidence of hypertension. The PREVER-prevention trial aims to investigate the efficacy, safety and feasibility of a population-based intervention to prevent the incidence of hypertension and the development of target-organ damage.</p> <p>Methods</p> <p>This is a randomized, double-blind, placebo-controlled clinical trial, with participants aged 30 to 70 years, with pre-hypertension. The trial arms will be chlorthalidone 12.5 mg plus amiloride 2.5 mg or identical placebo. The primary outcomes will be the incidence of hypertension, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new sub-clinical atherosclerosis, and sudden death. The study will last 18 months. The sample size was calculated on the basis of an incidence of hypertension of 14% in the control group, a size effect of 40%, power of 85% and P alpha of 5%, resulting in 625 participants per group. The project was approved by the Ethics committee of each participating institution.</p> <p>Discussion</p> <p>The early use of blood pressure-lowering drugs, particularly diuretics, which act on the main mechanism of blood pressure rising with age, may prevent cardiovascular events and the incidence of hypertension in individuals with hypertension. If this intervention shows to be effective and safe in a population-based perspective, it could be the basis for an innovative public health program to prevent hypertension in Brazil.</p> <p>Trial Registration</p> <p>Clinical Trials <a href="http://www.clinicaltrials.gov/ct2/show/NCT00970931">NCT00970931</a>.</p