RESUMO
Gaucher Disease (GD) is an inherited metabolic disorder caused by mutations in the GBA1 gene. It can manifest with severe neurodegeneration and visceral pathology. The most acute neuronopathic form (nGD), for which there are no curative therapeutic options, is characterised by devastating neuropathology and death during infancy. In this study, we investigated the therapeutic benefit of systemically delivered AAV9 vectors expressing the human GBA1 gene at two different doses comparing a neuronal-selective promoter with ubiquitous promoters. Our results highlight the importance of a careful evaluation of the promoter sequence used in gene delivery vectors, suggesting a neuron-targeted therapy leading to high levels of enzymatic activity in the brain but lower GCase expression in the viscera, might be the optimal therapeutic strategy for nGD.
Assuntos
Dependovirus , Doença de Gaucher , Terapia Genética , Vetores Genéticos , Glucosilceramidase , Regiões Promotoras Genéticas , Doença de Gaucher/genética , Doença de Gaucher/terapia , Doença de Gaucher/patologia , Vetores Genéticos/genética , Terapia Genética/métodos , Humanos , Regiões Promotoras Genéticas/genética , Dependovirus/genética , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Animais , Camundongos , Encéfalo/metabolismo , Encéfalo/patologia , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Transferência de GenesRESUMO
Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.
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Doença de Gaucher/terapia , Terapia Genética , Neurônios/metabolismo , Sinapsinas/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Dependovirus/genética , Modelos Animais de Doenças , Doença de Gaucher/genética , Doença de Gaucher/patologia , Humanos , Camundongos , Neurônios/patologia , Regiões Promotoras Genéticas/genética , Sinapsinas/uso terapêuticoRESUMO
Cardiac simulation has moved from early life-saving pacemakers meant only to prevent asystole to current devices capable of physiologic stimulation for the treatment of heart rhythm and heart failure, that are also intended for remote patient and disease-progression monitoring. The actual vision of contemporary pacing aims to correct the electrophysiologic roots of mechanical inefficiency regardless of underlying structural heart diseases. The awareness of the residual cardiac dyssynchrony related to customary cardiac pacing has changed the concept of what truly represents "physiologic pacing". On a different perspective, leadless stimulation to abolish CIED surgery and prevent lead-related complications is becoming a priority both for young device recipients and for frail, elderly patients. Careful clinical evaluation attempts to bridge decision-making to patient-tailored therapy.
Assuntos
Insuficiência Cardíaca , Marca-Passo Artificial , Idoso , Desenho de Equipamento , Previsões , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/terapia , HumanosRESUMO
Background: we sought to review the evolution in the diagnosis and treatment of Arrhythmogenic Cardiomyopathy (ACM), a clinically multifaceted entity beyond the observation of ventricular arrhythmias, and the outcome of therapies aiming at sudden death prevention in a single center experience. Methods: retrospective analysis of the data of consecutive patients with an implanted cardioverter-defibrillator (ICD) and a confirmed diagnosis of ACM according to the proposed Padua Criteria, who were referred to our center from January 1992 to October 2021. Results: we enrolled 72 patients (66% males, mean age at implant 46 ± 16 years), 63.9% implanted for primary prevention. At the time of ICD implant, 29 (40.3%) patients had a right ventricular involvement, 24 (33.3%) had a dominant LV involvement and 19 (26.4%) had a biventricular involvement. After a median follow-up of 6,1 years [IQR: 2.5-9.9], 34 patients (47.2%) had 919 sustained episodes of ventricular arrhythmias (VA). 27 patients (37.5%) had 314 episodes of life-threatening arrhythmias (LT-VA), defined as sustained ventricular tachycardia ≥ 200 beats/min. Considering only the patients with an ICD capable of delivering ATP, 80.4% of VA and 65% of LT-VA were successfully terminated with ATP. 16 (22.2%) patients had an inappropriate ICD activation, mostly caused by atrial fibrillation, while in 9 patients (12.5%) there was a complication needing reintervention (in 3 cases there was a loss of ventricular sensing dictating lead revision). During the follow-up 11 (15.3%) patients died, most of them due to heart failure, and 8 (11.1%) underwent heart transplantation. Conclusions: ACM is increasingly diagnosed owing to heightened suspicion at ECG examination and to improved imaging technology and availability, though the diagnostic workflow is particularly challenging in the earliest disease stages. ICD therapy is the cornerstone of sudden death prevention, albeit its efficacy is not based on controlled studies, and VT ablation/medical therapy are complementary to this strategy. The high burden of ATP-terminated VA makes shock-only devices debatable. The progressive nature of ACM leads to severe biventricular enlargement and refractory heart failure, which pose significant treatment issues when a predominant RV dysfunction occurs owing to the reduced possibility for mechanical circulatory assistance.
RESUMO
Cardiac implantable electronic device (CIED) infection represents a dramatic event with a high mortality rate (>3x) despite antibiotic therapy and device extraction; therefore, the real winning strategy in this situation could be represented by prevention. Antibiotic prophylaxis and antibiotic-releasing envelope are effective in improving patient outcome; however, healthcare costs related to CIED infections remain high over the years. In this review we would keep the attention on a pre-surgical checklist to reduce the risk of CIED infections. In fact, checklist is an effective instrument for medical care quality improvement mainly used in surgery, but not very commonly in cath-lab and electrophysiology procedures. All steps of this checklist are of proven effectiveness in reducing the risk of CIED infections but, up till now, they are not considered together in a pre-surgical approach.
Assuntos
Lista de Checagem , Desfibriladores Implantáveis , Marca-Passo Artificial , Infecções Relacionadas à Prótese/prevenção & controle , Antibioticoprofilaxia , Humanos , Período Pré-OperatórioRESUMO
Congenital complete atrioventricular block (CCAVB) is usually due to failure of atrioventricular nodal conduction with preservation of the His-Purkinje system. Most patients with CCAVB ultimately require pacemaker therapy to restore physiologic heart rates, dealing with the detrimental effects of chronic right ventricular (RV) pacing on cardiac structure and function. The ideal stimulation pattern aims to mimic the normal conduction to restore electromechanical coupling, preventing the harmful effects of lack of atrioventricular and inter-intraventricular synchrony. This can be done through conduction system pacing. Using His bundle pacing (HBP) for cardiac resynchronization therapy in two complete congenital atrioventricular block patients, we have reported better exercise tolerance and echocardiographic improvements related to reversible left ventricular dysfunction that can be corrected by restoration of the normal activation pathway via the His-Purkinje network.
Assuntos
Bloqueio Atrioventricular , Terapia de Ressincronização Cardíaca , Bloqueio Atrioventricular/terapia , Fascículo Atrioventricular , Doença do Sistema de Condução Cardíaco , Estimulação Cardíaca Artificial , Eletrocardiografia , Bloqueio Cardíaco/congênito , Humanos , Resultado do TratamentoRESUMO
Photoplethysmographic (PPG) signals are mainly employed for heart rate estimation but are also fascinating candidates in the search for cardiovascular biomarkers. However, their high susceptibility to motion artifacts can lower their morphological quality and, hence, affect the reliability of the extracted information. Low reliability is particularly relevant when signals are recorded in a real-world context, during daily life activities. We aim to develop two classifiers to identify PPG pulses suitable for heart rate estimation (Basic-quality classifier) and morphological analysis (High-quality classifier). We collected wrist PPG data from 31 participants over a 24 h period. We defined four activity ranges based on accelerometer data and randomly selected an equal number of PPG pulses from each range to train and test the classifiers. Independent raters labeled the pulses into three quality levels. Nineteen features, including nine novel features, were extracted from PPG pulses and accelerometer signals. We conducted ten-fold cross-validation on the training set (70%) to optimize hyperparameters of five machine learning algorithms and a neural network, and the remaining 30% was used to test the algorithms. Performances were evaluated using the full features and a reduced set, obtained downstream of feature selection methods. Best performances for both Basic- and High-quality classifiers were achieved using a Support Vector Machine (Acc: 0.96 and 0.97, respectively). Both classifiers outperformed comparable state-of-the-art classifiers. Implementing automatic signal quality assessment methods is essential to improve the reliability of PPG parameters and broaden their applicability in a real-world context.
Assuntos
Fotopletismografia , Punho , Algoritmos , Artefatos , Frequência Cardíaca/fisiologia , Humanos , Fotopletismografia/métodos , Reprodutibilidade dos Testes , Processamento de Sinais Assistido por Computador , Punho/fisiologiaRESUMO
The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and pre-clinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.
Assuntos
Vetores Genéticos/administração & dosagem , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/terapia , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Humanos , Injeções Intraventriculares , Proteínas de Membrana/metabolismo , Camundongos , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Resultado do TratamentoRESUMO
OBJECTIVE: We aimed to investigate the effectiveness of isopropanolic extract of Cimicifuga Racemosa (iCR) on reducing menopausal symptoms. MATERIALS AND METHODS: A single-center observational prospective case-control study was performed to assess the improvement of menopausal symptoms in menopausal women undergone iCR administration (cases) or no treatment (controls). Menopausal symptoms were assessed through a modified version of the Menopause Rating Scale questionnaire (mMRS) at T0 (baseline), T1 (1-month follow-up), and T2 (3 months follow-up). Univariate comparisons between cases and controls were performed by using the unpaired T test for two-tailed P value with α = 0.05 significance level. RESULTS: A total of 163 women (83 cases and 80 controls) were enrolled in the study. The difference in menopausal symptoms between cases and controls from T0 to T2, and from T0 to T1, was found significant for all analyses. In particular, the difference in all menopausal symptoms was 20.56 ± 0.90 points (95%CI: 18.77-22.33, p < .001) from T0 to T2, and 10.69 ± 0.6 (95%CI: 9.49-11.88, p < .001) from T0 to T1. CONCLUSION: iCR may be effective in reducing menopausal symptoms, both after 1 month and after 3 months of treatment. The improvement was higher in vasomotor symptoms, sleep problems, and irritability.
Assuntos
Cimicifuga , Menopausa , Fitoterapia , Extratos Vegetais/uso terapêutico , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.
Assuntos
Encéfalo/metabolismo , Epilepsia/terapia , Terapia Genética , Canal de Potássio Kv1.1/genética , Convulsões/terapia , Animais , Modelos Animais de Doenças , Epilepsia/genética , Vetores Genéticos , Canal de Potássio Kv1.1/metabolismo , Masculino , Ratos , Convulsões/genéticaRESUMO
Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.
Assuntos
Adenoviridae/genética , Proteínas de Transporte/administração & dosagem , Modelos Animais de Doenças , Transtornos Neurológicos da Marcha/prevenção & controle , Terapia Genética , Longevidade/genética , Glicoproteínas de Membrana/administração & dosagem , Doença de Niemann-Pick Tipo C/prevenção & controle , Animais , Proteínas de Transporte/fisiologia , Transtornos Neurológicos da Marcha/genética , Transtornos Neurológicos da Marcha/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Mutação , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologiaRESUMO
OBJECTIVE: To compare women's experience of first-trimester combined screening (FTCS), with women's experience of an approach that uses the combination of a detailed early anatomy scan and cell-free DNA (cfDNA) analysis. METHODS: This was single-center, open label, parallel group, randomized clinical trial. Pregnant women were randomized at the time of their first prenatal visit to either a policy of first-trimester risk assessment based on FTCS, or to a policy of first-trimester risk assessment based on ultrasound findings and cfDNA. FTCS included ultrasound evaluation with crown-rump length, nuchal translucency (NT) measurement, and a detailed ultrasound scan, along with biochemistry (PAPP-A and free beta hCG). In this group, invasive diagnostic testing was offered to patients with risk >1 in 100, or NT >3.5 mm, or any fetal abnormalities on ultrasound. Women randomized in the intervention group received an approach of first-trimester risk assessment based on ultrasound findings and cfDNA. cfDNA analysis included a simultaneous microarray-based assay of non-polymorphic (chromosomes 13, 18, 21, X and Y) and polymorphic loci to estimate chromosome proportion and fetal fraction. In the intervention group, invasive diagnostic testing was offered to patients with abnormal cfDNA screening results, or NT >3.5 mm, or any fetal abnormalities on ultrasound. Participants received pre-test and post-test questionnaires regarding to measure reassurance, satisfaction, and anxiety. The primary outcome was the post-test reassurance, defined as mean score of reassurance post-test questionnaire. The effect of the assigned screening test on the mean of each outcome was quantified as mean difference (MD) with 95% confidence interval (CI). RESULTS: Forty women with singleton gestations were enrolled in the trial. Mean score for reassurance was significantly higher in the cfDNA group compared to the FTCS group in the pre-test questionnaire (MD 0.80 points, 95% CI 0.27 to 1.33) and in the post-test questionnaire (MD 16.50 points, 95% CI 2.18 to 30.82). Women randomized to the cfDNA group had higher satisfaction and lower mean anxiety score as assessed in the STAI pre-test questionnaire. CONCLUSIONS: First-trimester risk assessment for fetal aneuploidy with a combination of a detailed ultrasound examination and cfDNA is associated with better maternal reassurance and better maternal satisfaction compared to the standard first-trimester combined screening with nuchal translucency, and biochemistry. TRIAL REGISTRATION: Clinicaltrials.gov NCT04077060.
Assuntos
Teste Pré-Natal não Invasivo , Primeiro Trimestre da Gravidez/psicologia , Ultrassonografia Pré-Natal/psicologia , Adulto , Ácidos Nucleicos Livres/análise , Feminino , Humanos , Gravidez , Adulto JovemRESUMO
Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid ß-glucosidase (GCase) using the irreversible inhibitor conduritol B-epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE-treated mice and a genetic GD mouse model, the Gba(flox/flox) ;nestin-Cre mouse, with 120 of the 144 genes up-regulated in CBE-treated mice also up-regulated in Gba(flox/flox) ;nestin-Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time-consuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Doença de Gaucher/patologia , Animais , Modelos Animais de Doenças , Doença de Gaucher/induzido quimicamente , Doença de Gaucher/genética , Perfilação da Expressão Gênica , Inositol/análogos & derivados , CamundongosRESUMO
BACKGROUND: The subcutaneous implantable cardioverter-defibrillator (S-ICD) is used in patients at risk of sudden death. Our aim was to assess clinical predictors of electrocardiographic ineligibility for S-ICD, and the impact of exercise on S-ICD eligibility in an unselected series of patients requiring ICD therapy. METHODS: 102 patients at risk of sudden death were evaluated at rest and during exercise. Electrocardiograph screening using limb lead electrodes (to simulate the S-ICD sensing vectors) was performed at rest and during bicycle ergometer exercise. RESULTS: R wave amplitude in lead D3 during exercise >16mV, baseline QTc and the sum of amplitudes of the R waves at supine >30mV were predictors of ineligibility for S-ICD. Eligibility increased from 90% to 100% of patients when evaluated with an "any of the three leads" criterion compared to current recommendations. A more restrictive criterion based on two of three ECG leads caused an eligibility drop at 66%, that further decreased to 56% during exercise; these figures improved to 79% and 81%, respectively, when an "any 2 of 3 leads" criterion was used. CONCLUSIONS: Huge ECG amplitude and QTc duration are associated with ineligibility in the current S-ICD release. By performing exercise testing, lead suitability changes in one patient out of 14 (7% of tested patients) and eligibility is decreased by use of a more stringent criterion for eligibility (ECG criteria satisfied in two of three leads). A dynamic selection of sensing vectors aiming at situation-specific suitability (any of three leads) would increase S-ICD eligibility to 100% of patients.
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Desfibriladores Implantáveis , Eletrocardiografia/métodos , Teste de Esforço/métodos , Insuficiência Cardíaca , Idoso , Feminino , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
PURPOSE: Oncological patients are at increasing risk of QT prolongation, a risk factor for ventricular arrhythmia. We assessed impact and risk factors for corrected QT (QTc) prolongation during multiple-cycle chemotherapy. METHODS: We enrolled 100 outpatients initiating chemotherapy in a university center specializing in female cancer. Clinical, drug, laboratory, and 12-lead ECG data collection at baseline and at each chemotherapy cycle was performed. RESULTS: Enrolled patients were followed for 992 chemotherapy cycles (median 7; interquartile range 6-13); 2438 ECGs were recorded (20; 18-31) 36.8% pre-therapy, 36.8% following chemotherapy, and 22.5% 7-10 days after chemotherapy. Maximum QTc (Max-QTc) was recorded after 4 chemotherapy administrations in >50% of the entire cohort and also within every subset of patients with prolonged QTc (57% 471-480 ms; 54% 481-500 ms; 66% >500 ms). No cumulative effect on QTc was shown. QTc prolongation was comparable among the various protocols. Prophylactic/supportive drugs were not associated with additional QTc prolongation. Variables independently associated with QTc prolongation >470 ms were age (OR 1.056 95% CI 1.006-1.108, p = 0.028) and the baseline-first chemotherapy averaged QTc (BC-QTc) (OR 1.092 95% CI 1.051-1.136), a novel parameter devised for this study. Only BC-QTc maintained significance for QTc >480 ms. BC-QTc >435 ms identified 100 % of patients with Max-QTc >500 ms, 96% with Max-QTc 481-500 ms, and 66% with Max-QTc 471-480 ms. Only 29% of patients with Max-QTc ≤470 ms presented a BC-QTc >435 ms. CONCLUSIONS: Our results confirm the high prevalence of QTc prolongation after chemotherapy. Most of the patients reached Max-QTc after several cycles. BC-QTc may help in stratifying arrhythmic risk in real-world clinical practice.
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Antineoplásicos/efeitos adversos , Síndrome do QT Longo/induzido quimicamente , Neoplasias/tratamento farmacológico , Adulto , Antineoplásicos/uso terapêutico , Eletrocardiografia , Feminino , Humanos , Síndrome do QT Longo/epidemiologia , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Pacientes Ambulatoriais , Prevalência , Estudos ProspectivosAssuntos
Potenciais de Ação , Arritmias Cardíacas/terapia , Fascículo Atrioventricular/fisiopatologia , Estimulação Cardíaca Artificial , Frequência Cardíaca , Marca-Passo Artificial , Tecnologia de Sensoriamento Remoto/instrumentação , Processamento de Sinais Assistido por Computador , Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologia , Automação , Fascículo Atrioventricular/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Humanos , Valor Preditivo dos Testes , Fatores de Tempo , Resultado do TratamentoRESUMO
We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.
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Fluoreto de Fosfato Acidulado , Vetores Genéticos , Lentivirus , Animais , Humanos , Lentivirus/genética , Vetores Genéticos/genética , Células HEK293 , Transfecção , DNA/genética , Tetraciclina , Mamíferos/genéticaRESUMO
In recent years, advances in melanoma treatment have renewed patient hope. This comprehensive review emphasizes the evolving treatment landscape, particularly highlighting first-line strategies and the interplay between immune-checkpoint inhibitors (ICIs) and targeted therapies. Ipilimumab plus nivolumab has achieved the best median overall survival, exceeding 70 months. However, the introduction of new ICIs, like relatlimab, has added complexity to first-line therapy decisions. Our aim is to guide clinicians in making personalized treatment decisions. Various features, including brain metastases, PD-L1 expression, BRAF mutation, performance status, and prior adjuvant therapy, significantly impact the direction of advanced melanoma treatment. We also provide the latest insights into the treatment of rare melanoma subtypes, such as uveal melanoma, where tebentafusp has shown promising improvements in overall survival for metastatic uveal melanoma patients. This review provides invaluable insights for clinicians, enabling informed treatment choices and deepening our understanding of the multifaceted challenges associated with advanced melanoma management.
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Melanoma , Neoplasias Uveais , Humanos , Melanoma/genética , Melanoma/terapia , Melanoma/patologia , Neoplasias Uveais/diagnóstico , Neoplasias Uveais/genética , Neoplasias Uveais/terapia , Ipilimumab , NivolumabeRESUMO
BACKGROUND: Cardiac resynchronization therapy (CRT) represents an effective heart failure treatment, associated with reduction in mortality and heart failure hospitalizations. This Italian survey aimed to address relevant CRT issues. METHODS: An online survey was administered to AIAC members. RESULTS: One hundred and five electrophysiologists participated, with a median of 40 (23-70) CRT implantations/year (33% in high-volume centres). Forty-five percent of respondents (especially working in high-volume centres) reported an increase in CRT implantations in the last 2 years, in 16% a decrease, and in 38% CRT remained stable. Seventy-five percent of respondents implanted CRT only in patients with European Heart Rhythm Association (EHRA) class I indications. All operators collected ECG and echocardiography before implantation. Eighty-five percent of respondents selected coronary sinus target vein empirically, whereas 10% used mechanical and/or electrical delay techniques. Physicians working in high-volume centres reported a lower failure rate compared with others (16 vs. 34%; Pâ=â0.03). If the coronary sinus lead could not be positioned in the target branch, 80% placed it in another vein, whereas 16% opted for a surgical approach or for conduction system pacing (CSP). Eighty percent accomplished CRT optimization in all patients, 17% only in nonresponders. Regarding anticoagulation, high agreement with EHRA guidelines emerged. CONCLUSION: CRT represents a valid therapeutic option in heart failure treatment. Nowadays, CRT implantations remain stable and are mainly performed in patients with class I indications. ECG remains the preferred tool for patient selection, whereas imaging is increasingly used to determine the left pacing target area. In most patients, the left ventricular lead can be successfully positioned in the target vein, but in some cases, the result can be unsatisfactory; however, the decision to explore alternative resynchronization approaches is rarely pursued.
Assuntos
Terapia de Ressincronização Cardíaca , Insuficiência Cardíaca , Seleção de Pacientes , Humanos , Terapia de Ressincronização Cardíaca/efeitos adversos , Itália/epidemiologia , Insuficiência Cardíaca/terapia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/fisiopatologia , Pesquisas sobre Atenção à Saúde , Dispositivos de Terapia de Ressincronização Cardíaca , Padrões de Prática Médica/tendências , Padrões de Prática Médica/estatística & dados numéricos , Eletrocardiografia , Hospitais com Alto Volume de Atendimentos/estatística & dados numéricos , Resultado do Tratamento , Inquéritos e QuestionáriosRESUMO
Adeno-associated viral vectors (AAVs) have proved a mainstay in gene therapy, owing to their remarkable transduction efficiency and safety profile. Their production, however, remains challenging in terms of yield, the cost-effectiveness of manufacturing procedures and large-scale production. In this work, we present nanogels produced by microfluidics as a novel alternative to standard transfection reagents such as polyethylenimine-MAX (PEI-MAX) for the production of AAV vectors with comparable yields. Nanogels were formed at pDNA weight ratios of 1 : 1 : 2 and 1 : 1 : 3, of pAAV cis-plasmid, pDG9 capsid trans-plasmid and pHGTI helper plasmid respectively, where vector yields at a small scale showed no significant difference to those of PEI-MAX. Weight ratios of 1 : 1 : 2 showed overall higher titers than 1 : 1 : 3, where nanogels with nitrogen/phosphate ratios of 5 and 10 produced yields of ≈8.8 × 108 vg mL-1 and ≈8.1 × 108 vg mL-1 respectively compared to ≈1.1 × 109 vg mL-1 for PEI-MAX. In larger scale production, optimised nanogels produced AAV at a titer of ≈7.4 × 1011 vg mL-1, showing no statistical difference from that of PEI-MAX at ≈1.2 × 1012 vg mL-1, indicating that equivalent titers can be achieved with easy-to-implement microfluidic technology at comparably lower costs than traditional reagents.