Liver Gene Therapy: Employing Surgery and Radiology for Translational Research

Gene therapy is a therapeutic strategy that aims to employ nucleic acids as drugs for the transient or permanent treatment of inherited or acquired pathologies. Based on the type of vector employed for the gene transfer, gene therapy can be classified as viral gene therapy and nonviral gene therapy. Nonviral gene therapy is less efficient but safer than viral gene therapy. Hydrodynamic naked DNA transfer has shown great translational potential, achieving therapeutic levels of a human protein in the murine model. The translational process of the procedure has already been performed. Different radiologic and surgical approaches permitted pressurizing the liver in vivo by excluding its vascularization partially or totally. These approaches mediated a tissue rate of human alpha-1-antitrypsin protein translation (100–1000 copies per cell) close to those obtained with the mouse gold standard model in a safe mode that could be translated to human settings

Electrochemical POC device for fast malaria quantitative diagnosis in whole blood by using magnetic beads, Poly-HRP and microfluidic paper electrodes

© 2019 Elsevier B.V. Malaria, a parasitic infection caused by Plasmodium parasites and transmitted through the bite of infected female Anopheles mosquitos, is one of the main causes of mortality in many developing countries. Over 200 million new infections and nearly half a million deaths are reported each year, and more than three billion people are at risk of acquiring malaria worldwide. Nevertheless, most malaria cases could be cured if detected early. Malaria eradication is a top priority of the World Health Organisation. However, achieving this goal will require mass population screening and treatment, which will be hard to accomplish with current diagnostic tools. We report an electrochemical point-of-care device for the fast, simple and quantitative detection of Plasmodium falciparum lactate dehydrogenase (PfLDH) in whole blood samples. Sample analysis includes 5-min lysis to release intracellular parasites, and stirring for 5 more min with immuno-modified magnetic beads (MB) along with an immuno-modified signal amplifier. The rest of the magneto-immunoassay, including sample filtration, MB washing and electrochemical detection, is performed at a disposable paper electrode microfluidic device. The sensor provides PfLDH quantitation down to 2.47 ng mL−1 in spiked samples and for 0.006–1.5% parasitemias in Plasmodium-infected cultured red blood cells, and discrimination between healthy individuals and malaria patients presenting parasitemias >0.3%. Quantitative malaria diagnosis is attained with little user intervention, which is not achieved by other diagnostic methods

Magnetic Bead Handling Using a Paper-Based Device for Quantitative Point-of-Care Testing

Low-cost assay automation; Malaria quantitative diagnosis; Smartphone colorimetric detectionAutomatització d'assaigs de baix cost; Diagnòstic quantitatiu de la malària; Detecció colorimètrica del telèfon intel·ligentAutomatización de ensayos de bajo costo; Diagnóstico cuantitativo de la malaria; Detección colorimétrica de teléfono inteligenteMicrofluidic paper-based analytical devices (μPADs) have been extensively proposed as ideal tools for point-of-care (POC) testing with minimal user training and technical requirements. However, most μPADs use dried bioreagents, which complicate production, reduce device reproducibility and stability, and require transport and storage under temperature and humidity-controlled conditions. In this work, we propose a μPAD produced using an affordable craft-cutter and stored at room temperature, which is used to partially automate a single-step colorimetric magneto-immunoassay. As a proof-of-concept, the μPAD has been applied to the quantitative detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection. In this system, detection is based on a single-step magneto-immunoassay that consists of a single 5-min incubation of the lysed blood sample with immuno-modified magnetic beads (MB), detection antibody, and an enzymatic signal amplifier (Poly-HRP). This mixture is then transferred to a single-piece paper device where, after on-chip MB magnetic concentration and washing, signal generation is achieved by adding a chromogenic enzyme substrate. The colorimetric readout is achieved by the naked eye or using a smartphone camera and free software for image analysis. This μPAD afforded quantitative Pf-LDH detection in <15 min, with a detection limit of 6.25 ng mL−1 when the result was interpreted by the naked eye and 1.4 ng mL−1 when analysed using the smartphone imaging system. Moreover, the study of a battery of clinical samples revealed concentrations of Pf-LDH that correlated with those provided by the reference ELISA and with better sensitivity than a commercial rapid diagnostic test (RDT). These results demonstrate that magneto-immunoassays can be partly automated by employing a μPAD, achieving a level of handling that approaches the requirements of POC testing.This research was funded by Fondo de Investigaciones Sanitarias of Instituto de Salud Carlos III (co-funded by the European Regional Development Fund; grants CPII18/00025, IFI18/00020, JR18/00022 and QUPID EuroNanoMed AC21_2/00021) and La Caixa Foundation (ID 100010434, fellowship LCF/BQ/DI18/11660061). Diagnostic Nanotools is a consolidated group supported by Secretaria d’Universitats i Recerca, Generalitat de Catalunya (2017 SGR 240)

Consensus Statement on Hemostatic Management, Anticoagulation, and Antiplatelet Therapy in Liver Transplantation

Anticoagulation and antiplatelet therapies are increasingly used in liver transplant (LT) candidates and recipients due to cardiovascular comorbidities, portal vein thrombosis, or to manage posttransplant complications. The implementation of the new direct-acting oral anticoagulants and the recently developed antiplatelet drugs is a great challenge for transplant teams worldwide, as their activity must be monitored and their complications managed, in the absence of robust scientific evidence. In this changing and clinically heterogeneous scenario, the Spanish Society of Liver Transplantation and the Spanish Society of Thrombosis and Haemostasis aimed to achieve consensus regarding the indications, drugs, dosing, and timing of anticoagulation and antiplatelet therapies initiated from the inclusion of the patient on the waiting list to post-LT surveillance. A multidisciplinary group of experts composed by transplant hepatologists, surgeons, hematologists, transplant-specialized anesthesiologists, and intensivists performed a comprehensive review of the literature and identified 21 clinically relevant questions using the patient-intervention-comparison-outcome format. A preliminary list of recommendations was drafted and further validated using a modified Delphi approach by a panel of 24 transplant delegates, each representing a LT institution in Spain. The present consensus statement contains the key recommendations together with the core supporting scientific evidence, which will provide guidance for improved and more homogeneous clinical decision making

Waiting time dictates impact of frailty: A Spanish multicenter prospective study

Background & aims: Frailty is prevalent in liver transplant (LT) candidates. It is considered an independent predictor of adverse outcomes pre- and post-transplant according to data obtained in the United States. We aimed to externally validate the liver frailty index (LFI) in a multicenter cohort of LT candidates. Methods: Outpatients with cirrhosis were prospectively recruited from five Spanish centers (2018-2020). Patients were defined as "frail" by an optimal cut-off of LFI ≥4.5. Patients were followed for at least 6 months to study associations of pre-LT frailty with pre- and post-transplant mortality, length of hospital and intensive care unit (ICU) stays, risk of early (<30 days) and late (30-90 days) post-transplant complications, retransplantation and cardiovascular events. Results: Of 212 patients included, 45 patients (21%) were frail pre-LT, and the median LFI was 3.9 (IQR 3.5-4.4). After a median waiting time of 78 days, 2% died or were delisted for clinical worsening. The LFI at baseline was not predictive of mortality/delisting in LT candidates in univariable or multivariable analyses after adjusting for age and MELD-Na score (hazard ratio 1.48; p = 0.586). In contrast, compared to non-frail patients, frail LT candidates had a significantly higher length of hospital stay (9 vs. 13 days; p = 0.001) and rate of early (<30 days) post-transplant complications (55% vs. 100%; p = 0.021). Conclusions: In the context of a short LT waiting time, frailty does not impact pretransplant mortality and/or delisting. In contrast, LT frailty is predictive of higher post-transplant complication rates and length of hospital stay. Whether strategies aimed at pre- and/or re-habilitation are beneficial in settings with short waiting times needs to be confirmed in prospective studies. Impact and implications: Literature is scarce on the actual impact of physical frailty on adverse outcomes in the liver transplant scenario outside North America. Evidence-based justification to extend the use of objective frailty tools in the decision-making processes in other liver transplant settings is needed. This study is the first to evaluate the predictive value of the liver frailty index in outpatients in the European liver transplant setting, showing that in a low MELD, high access system, frailty does not impact pretransplant mortality and/or delisting but is predictive of higher complication rates and longer post-transplant length of stay. In practical ways, physicians should consider physical frailty as a vital sign to be measured systematically and routinely during clinic visits; researchers are encouraged to initiate prospective studies to evaluate the benefit of applying strategies aimed at pre- and or re-habilitation in liver transplant settings with short waiting times

Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters; A Retrospective Cohort Analysis and Pilot Study

BackgroundTwo years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. ObjectivesTo measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. ConclusionsLaboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible

Management of pancreatic gastrinoma associated with Von Hippel-Lindau disease: a case report

Background: Pancreatic neuroendocrine tumors (PNET) are a heterogeneous group and constitute 1.3% of all pancreatic tumors. Approximately 10% of these occur in the context of hereditary syndromes, such as VHL disease. Case report: We report a case of a female patient of 37 years diagnosed VHL and intervened on several occasions by cerebral hemangioblastoma and renal carcinomas. During its follow-up she was diagnosed 2 gastrinomas functioning under 2 cm were enucleated. Later developed new PNET and underwent a total duodenopancreatectomy without pyloric preservation. Discussion: The management of PNET in VHL is difficult due to the association of multiple tumors in different organs and the morbidity and mortality associated with the surgery of the pancreas. Management must be individualized for each patient, based on the ability to produce hormones and present symptoms, the size and location, and in the context of other tumors that usually present in these patients

Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.

Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest.A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated.Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases.Hydrofection of hAAT DNA to "in vivo" isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma is observed and might be species-related. This study reinforces the potential application of closed liver hydrofection for therapeutic purposes, provided protein secretion improves