Expresión génica simultánea y regulada del péptido tímico timulina y la green fluorescent protein utilizando un sistema adenoviral Tet-Off

Introducción: Una de las cuestiones clave en la implementación exitosa de terapias génicas radica en la capacidad de regular la expresión consistentemente. Con este propósito, y a fin de ampliar nuestros estudios en timulina y su papel en el desarrollo del eje hipotálamo-hipófiso-ovárico, construimos un sistema bivectorial regulable Tet-Off que expresa, simultáneamente, los transgenes para green fluorescent protein (GFP) y metFTS (análogo de timulina). Objetivos: Estudiar la funcionalidad y regulabilidad de nuestro sistema in vitro y evaluar la expresión in vivo mediante su inyección en músculos y ventrículos cerebrales de ratas. Materiales y métodos: El sistema experimental consta de dos adenovectores recombinantes (RAd): el RAd-(GFPTRE- FTS)bidir, unidad de respuesta en la cual la transcripción de los transgenes se encuentra controlada por un promotor bidireccional inducible por doxiciclina, y el RAd-tTA, unidad transactivadora que expresa la proteína regulatoria tTA. El sistema control no expresa metFTS. La expresión de GFP se determinó por microscopía y fluorometría. La timulina bioactiva fue medida por un bioensayo de rosetas. Resultados y conclusión: El sistema construido mostró ser activo tanto en experimentos in vitro, donde células CHOK1 incubadas con ambos RAds mostraron una eficiente expresión de los transgenes, pudiendo inhibirse por el agregado de doxiciclina al medio de cultivo; como in vivo, donde se dosaron niveles significativamente aumentados de timulina sérica y en LCR en ratas inyectadas intramuscular e intracerebrovectricularmente con ambos RAds, respectivamente. La utilización del gen de la GFP en nuestro sistema sirvió para comprobar, sencilla y rápidamente, la transcripción del gen de la timulina.Instituto de Investigaciones Bioquímicas de La Plat

Construcción y caracterización <i>in vitro</i> e <i>in vivo</i> de un sistema bivectorial bidireccional regulable Tet-Off para el gen sintético del péptido tímico timulina y el gen reportero de la GFP

La timulina es una hormona tímica involucrada en diversos aspectos de la diferenciación intra- y extratímica de las células T. Esta hormona, que consiste en un nonapéptido (FTS) coordinado al ión Zn+2 (FTS-Zn) en una relación equimolecular, posee una acción inmunomodulatoria, actividad hipofisotrófica y actividad antiinflamatoria y analgésica en el sistema nervioso central. Además, se ha demostrado que es clave en la acción del timo sobre el eje hipotálamo-hipófiso-ovárico. En estudios previos hemos implementado terapia génica neuroendocrina para timulina utilizando nuestro vector adenoviral recombinante (RAd) que expresa constitutivamente un gen sintético para un análogo de timulina (el decapéptido metFTS). El control de la expresión de transgenes en células de mamíferos es deseable para el estudio de la función de los genes y para la terapia génica. La utilización de sistemas regulables de expresión génica permitiría la manipulación de la expresión de la timulina.Facultad de Ciencias Médica

Thymulin-based gene therapy and pituitary function in animal models of aging

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. After its discovery and initial characterization in the 1970s, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, a growing core of information, to be reviewed here, points to thymulin as a hypophysiotropic peptide. Additionally, thymulin was shown to possess anti-inflammatory and analgesic properties in the brain. In recent years, a synthetic DNA sequence coding for a biologically active analog of thymulin, metFTS, was constructed and cloned in different adenoviral vectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be downregulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies suggest that thymulin gene therapy may be a suitable therapeutic strategy to prevent some of the endocrine and reproductive alterations that typically appear in congenitally athymic (nude) mice, taken as a suitable model of neuroendocrine and reproductive aging. The present article briefly reviews the literature on the physiology of the thymulin-pituitary axis as well as on the new molecular tools available to exploit the therapeutic potential of thymulin.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Thymulin-based gene therapy and pituitary function in animal models of aging

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. After its discovery and initial characterization in the 1970s, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, a growing core of information, to be reviewed here, points to thymulin as a hypophysiotropic peptide. Additionally, thymulin was shown to possess anti-inflammatory and analgesic properties in the brain. In recent years, a synthetic DNA sequence coding for a biologically active analog of thymulin, metFTS, was constructed and cloned in different adenoviral vectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be downregulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies suggest that thymulin gene therapy may be a suitable therapeutic strategy to prevent some of the endocrine and reproductive alterations that typically appear in congenitally athymic (nude) mice, taken as a suitable model of neuroendocrine and reproductive aging. The present article briefly reviews the literature on the physiology of the thymulin-pituitary axis as well as on the new molecular tools available to exploit the therapeutic potential of thymulin.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Role of thymulin on the somatotropic axis in vivo

Aims: There is clear evidence for the existence of a bi-directional thymus-somatotropic axis and several studies suggest that the thymic peptide thymulin may be involved in this communication. We undertook to assess the impact of serum thymulin immunoneutralization in C57BL/6 mice and that of neonatal thymulin gene therapy (NTGT) in nude mice on body weight (BW) gain and on the histomorphometric profile of the somatotrope population. Main methods: Immunoneutralization of thymulin was done from postnatal day 1 to 35 by i.p. injections of rabbit anti-thymulin serum (α-FTS) and normal rabbit serum (NRS) in controls. NTGT was implemented in nudes using an adenoviral vector expressing a synthetic gene for thymulin (RAd-FTS). On postnatal day 1, heterozygous (nu/+) and homozygous (nu/nu) pups received a single bilateral i.m. injection either RAd-FTS or RAd-GFP (a control vector expressing green fluorescent protein). BW gain was recorded and at the end of the study the pituitaries were immunostained for growth hormone (GH). Serum GH and thymulin were determined by radioimmunoassay and bioassay, respectively. Key findings: Thymulin immunoneutralization induced a significant decrease in BW gain, serum GH and somatotrope cell density as well as an increase in somatotrope cell size. NTGT markedly increased BW gain, serum thymulin (P < 0.01) and somatotrope cell and volume density in nu/nu mice. Significance: Our results suggest that thymulin plays a relevant physiological role on the thymus-somatotropic axis in mice.Fil: Reggiani, Paula Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Martines, Eliana V.. Universidad Adventista del Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Camihort, Gisela A.. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Poch, Brenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Goya, Rodolfo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Cónsole, Gloria M.. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentin

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research