RESUMO
BACKGROUND: Chagas disease (ChD) is endemic in many parts of the world and can be transmitted through organ transplantation or reactivated by immunosuppression. Organs from infected donors are occasionally used for transplantation, and the best way of managing the recipients remains a subject of debate. METHODS: We present a single-center cohort study describing a 10-year experience of kidney transplantation in patients at risk of donor-derived ChD and or reactivation. Patients received prophylactic treatment with Benznidazole and were monitored for transmission or reactivation. Monitoring included assessing direct parasitemia, serology, and polymerase chain reaction (PCR). RESULTS: Fifty-seven kidney transplant recipients (KTRs) were enrolled in the study. Forty-four patients (77.2%) were at risk of primary ChD infection, nine patients (15.8%) were at risk of disease reactivation, and four patients (7.0%) were at risk of both. All patients received Benznidazole prophylaxis, starting on the first day after transplantation. Parasitemia was assessed in 51 patients (89.5%), serology also in 51 patients (89.5%), and PCR in 40 patients (70.2%). None of the patients exhibited clinically or laboratory-detectable signs of disease. A single patient experienced a significant side effect, a cutaneous rash with intense pruritus. At 1-year post-transplantation, the patient and graft survival rates were 96.5% and 93%, respectively. CONCLUSION: In this study, no donor-derived or reactivation of Trypanosoma cruzi infection occurred in KTRs receiving Benznidazole prophylaxis.
RESUMO
We provide novel genomic resources to help understand the genomic traits involved in elephant health and to aid conservation efforts. We sequence 11 elephant genomes (5 African savannah, 6 Asian) from North American zoos, including 9 de novo assemblies. We estimate elephant germline mutation rates and reconstruct demographic histories. Finally, we provide an in-solution capture assay to genotype Asian elephants. This assay is suitable for analyzing degraded museum and noninvasive samples, such as feces and hair. The elephant genomic resources we present here should allow for more detailed and uniform studies in the future to aid elephant conservation efforts and disease research.
Assuntos
Elefantes , Animais , Elefantes/genética , Genômica , Genoma , Mapeamento Cromossômico , Animais de Zoológico , Mutação em Linhagem GerminativaRESUMO
This paper presents the concept of a novel adaptable sensing solution currently being developed under the EU Commission-founded PHOTONGATE project. This concept will allow for the quantification of multiple analytes of the same or different nature (chemicals, metals, bacteria, etc.) in a single test with levels of sensitivity and selectivity at/or over those offered by current solutions. PHOTONGATE relies on two core technologies: a biochemical technology (molecular gates), which will confer the specificity and, therefore, the capability to be adaptable to the analyte of interest, and which, combined with porous substrates, will increase the sensitivity, and a photonic technology based on localized surface plasmonic resonance (LSPR) structures that serve as transducers for light interaction. Both technologies are in the micron range, facilitating the integration of multiple sensors within a small area (mm2). The concept will be developed for its application in health diagnosis and food safety sectors. It is thought of as an easy-to-use modular concept, which will consist of the sensing module, mainly of a microfluidics cartridge that will house the photonic sensor, and a platform for fluidic handling, optical interrogation, and signal processing. The platform will include a new optical concept, which is fully European Union Made, avoiding optical fibers and expensive optical components.
Assuntos
Metais , Ressonância de Plasmônio de Superfície , Metais/química , Óptica e Fotônica , Bactérias , Fibras ÓpticasRESUMO
Melatonin has been reported to cause myocardial electrophysiological changes and prevent ventricular tachycardia or fibrillation (VT/VF) in ischemia and reperfusion. We sought to identify electrophysiological targets responsible for the melatonin antiarrhythmic action and to explore whether melatonin receptor-dependent pathways or its antioxidative properties are essential for these effects. Ischemia was induced in anesthetized rats given a placebo, melatonin, and/or luzindole (MT1/MT2 melatonin receptor blocker), and epicardial mapping with reperfusion VT/VFs assessment was performed. The oxidative stress assessment and Western blotting analysis were performed in the explanted hearts. Transmembrane potentials and ionic currents were recorded in cardiomyocytes with melatonin and/or luzindole application. Melatonin reduced reperfusion VT/VF incidence associated with local activation time in logistic regression analysis. Melatonin prevented ischemia-related conduction slowing and did not change the total connexin43 (Cx43) level or oxidative stress markers, but it increased the content of a phosphorylated Cx43 variant (P-Cx43368). Luzindole abolished the melatonin antiarrhythmic effect, slowed conduction, decreased total Cx43, protein kinase Cε and P-Cx43368 levels, and the IK1 current, and caused resting membrane potential (RMP) depolarization. Neither melatonin nor luzindole modified INa current. Thus, the antiarrhythmic effect of melatonin was mediated by the receptor-dependent enhancement of impulse conduction, which was associated with Cx43 phosphorylation and maintaining the RMP level.
Assuntos
Conexina 43 , Melatonina , Ratos , Animais , Conexina 43/metabolismo , Receptores de Melatonina/metabolismo , Melatonina/farmacologia , Melatonina/uso terapêutico , Antiarrítmicos/farmacologia , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/prevenção & controle , Miócitos Cardíacos/metabolismoRESUMO
Hyperprolactinemia is an endocrine disorder associated with infertility in many species, including elephants. In a recent survey of zoos accredited by the Association of Zoos and Aquariums (AZA), over half of African elephant females (N = 101) were not cycling normally, 30% of which exhibited hyperprolactinemia. We examined whether life experience and temperament predict ovarian cyclicity and circulating prolactin status in individual African elephant females. We hypothesized that, similar to humans, acyclicity and hyperprolactinemia in elephants will be associated with an apprehensive or fearful, anxious temperament, and an increased number of potentially challenging life events (transfers, deaths and births). Ninety-five adult African elephant females housed at 37 AZA institutions were included in this study. Blood samples were collected twice a month for 1 year to determine ovarian cycle (cycling, n = 44; irregular, n = 13; non-cycling, n = 38) and prolactin (normal, n = 44; low; n = 23; high; n = 28) status. Keeper ratings on a 6-point scale were obtained on 32 temperament traits in 85 of these elephants. We determined that giving birth and being exposed to herd mates entering the facility were positively associated with normal ovarian cycle and prolactin profiles. By contrast, age, serum cortisol, and an increased number of herd mates leaving a facility were negatively associated with both. Contrary to our hypothesis, hyperprolactinemia was associated with a popular and caring temperament rating, whereas consistently low prolactin was associated with a fearful, apprehensive temperament. These findings indicate that pituitary-ovarian function may be impacted by life history (cyclicity) and temperament (prolactin), which should be taken into consideration when making management decisions.
Assuntos
Animais de Zoológico/fisiologia , Elefantes/fisiologia , Ciclo Estral/fisiologia , Características de História de Vida , Prolactina/sangue , Temperamento/fisiologia , Animais , Animais de Zoológico/sangue , Elefantes/sangue , Ciclo Estral/sangue , Feminino , Humanos , Hiperprolactinemia/sangue , Hiperprolactinemia/veterinária , Masculino , América do Norte , Ovário/fisiologia , Periodicidade , Hipófise/fisiologia , GravidezRESUMO
BACKGROUND: This study aims to compare the characteristics between patients who underwent aortic valve replacement (AVR) through a J-shaped upper mini-sternotomy (UMS) and patients who underwent full sternotomy (FS) in the basis of clinical care and hospital outcomes. METHODS: A retrospective, cross-sectional study was conducted on adult patients who were subjected to AVR by UMS from 2014 to 2017, compared with a historical control of patients who had undergone UMS by FS from 2011 to 2014. Patients, who received combined valve replacement or aortic surgery, as well as heart valve reinterventions due to endocarditis, were excluded. Sociodemographic characteristics, medical history, hospital and intensive care stay, blood transfusions, complications, and mortality of both procedures were compared. RESULTS: There were 57 patients under UMS and 99 patients under FS included in this study. The median age was 67 years, and 56.77% of the patients were male. No differences were observed in the past medical history and the type of valve implanted between the groups. During surgery, patients under UMS received a lower percentage of red blood cell and platelet transfusions compared with FS. However, UMS had a higher percentage of cryoprecipitate transfusion. Intensive care stay was shorter in UMS compared with FS (three days; interquartile range [IQR], 2-4; and four days; IQR, 2-6, respectively) without differences in overall hospital stay, postoperative complications, in-hospital mortality, and 30-day mortality. CONCLUSIONS: The J-shaped upper mini-sternotomy is a feasible surgical technique that does not increase in-hospital or 30-day mortality, neither hospital stay nor infectious complications.
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Valva Aórtica/cirurgia , Doenças das Valvas Cardíacas/cirurgia , Implante de Prótese de Valva Cardíaca/métodos , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Esternotomia/métodos , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Cardiac connexin-43 (Cx43) creates gap junction channels (GJCs) at intercellular contacts and hemi-channels (HCs) at the peri-junctional plasma membrane and sarcolemmal caveolae/rafts compartments. GJCs are fundamental for the direct cardiac cell-to-cell transmission of electrical and molecular signals which ensures synchronous myocardial contraction. The HCs and structurally similar pannexin1 (Panx1) channels are active in stressful conditions. These channels are essential for paracrine and autocrine communication through the release of ions and signaling molecules to the extracellular environment, or for uptake from it. The HCs and Panx1 channel-opening profoundly affects intracellular ionic homeostasis and redox status and facilitates via purinergic signaling pro-inflammatory and pro-fibrotic processes. These conditions promote cardiac arrhythmogenesis due to the impairment of the GJCs and selective ion channel function. Crosstalk between GJCs and HCs/Panx1 channels could be crucial in the development of arrhythmogenic substrates, including fibrosis. Despite the knowledge gap in the regulation of these channels, current evidence indicates that HCs and Panx1 channel activation can enhance the risk of cardiac arrhythmias. It is extremely challenging to target HCs and Panx1 channels by inhibitory agents to hamper development of cardiac rhythm disorders. Progress in this field may contribute to novel therapeutic approaches for patients prone to develop atrial or ventricular fibrillation.
Assuntos
Conexina 43/metabolismo , Conexinas/metabolismo , Ativação do Canal Iônico , Miocárdio/metabolismo , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Conexina 43/ultraestrutura , Conexinas/ultraestrutura , Suscetibilidade a Doenças , Humanos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , Miocárdio/ultraestruturaRESUMO
Pharmacological concentrations of melatonin reduce reperfusion arrhythmias, but less is known about the antiarrhythmic protection of the physiological circadian rhythm of melatonin. Bilateral surgical removal of the superior cervical ganglia irreversibly suppresses melatonin rhythmicity. This study aimed to analyze the cardiac electrophysiological effects of the loss of melatonin circadian oscillation and the role played by myocardial melatonin membrane receptors, SERCA2A, TNFα, nitrotyrosine, TGFß, KATP channels, and connexin 43. Three weeks after bilateral removal of the superior cervical ganglia or sham surgery, the hearts were isolated and submitted to ten minutes of regional ischemia followed by ten minutes of reperfusion. Arrhythmias, mainly ventricular tachycardia, increased during reperfusion in the ganglionectomy group. These hearts also suffered an epicardial electrical activation delay that increased during ischemia, action potential alternants, triggered activity, and dispersion of action potential duration. Hearts from ganglionectomized rats showed a reduction of the cardioprotective MT2 receptors, the MT1 receptors, and SERCA2A. Markers of nitroxidative stress (nitrotyrosine), inflammation (TNFα), and fibrosis (TGFß and vimentin) did not change between groups. Connexin 43 lateralization and the pore-forming subunit (Kir6.1) of KATP channels increased in the experimental group. We conclude that the loss of the circadian rhythm of melatonin predisposes the heart to suffer cardiac arrhythmias, mainly ventricular tachycardia, due to conduction disorders and changes in repolarization.
Assuntos
Arritmias Cardíacas/patologia , Ganglionectomia/efeitos adversos , Coração/fisiopatologia , Traumatismo por Reperfusão Miocárdica/cirurgia , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Ritmo Circadiano , Conexina 43/genética , Conexina 43/metabolismo , Masculino , Melatonina/metabolismo , Ratos , Ratos Wistar , Receptores de Melatonina/genética , Receptores de Melatonina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Conservation strategies for crocodilians often include captive breeding to create stable assurance populations. Evaluating adrenal and gonadal hormone patterns can provide animal managers with data to more effectively monitor animal welfare and reproductive status. This study evaluated the effects of season (breeding, nesting, or off), sex (male and female), and reproductive status of females (egg-laying/housed with a male or non-laying/housed solo) on concentrations of fecal glucocorticoid metabolite (FGM), fecal androgen metabolite (FAM), and fecal progestogen metabolite (FPM) in seven Cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park. Overall, seasonal changes in FGM and FPM concentrations were only observed in egg-laying females; FGM and FPM concentrations were both higher during the nesting season compared to the breeding and off seasons. Seasonal changes in FAM concentrations were only observed in males; males had higher FAM concentrations during the breeding and nesting seasons compared to the off season. Future studies investigating the use of fecal hormone metabolites in crocodilians are necessary to understand differences between individuals and species, to further elucidate the interactions between hormones and environmental factors, such as social housing, and to develop long-term datasets for the management of this species.
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Córtex Suprarrenal/metabolismo , Jacarés e Crocodilos/fisiologia , Androgênios/metabolismo , Fezes/química , Glucocorticoides/metabolismo , Reprodução/fisiologia , Córtex Suprarrenal/fisiologia , Androgênios/química , Animais , Animais de Zoológico , Feminino , Glucocorticoides/química , Masculino , Estações do AnoRESUMO
Many zoo elephants do not cycle normally, and for African elephants, it is often associated with hyperprolactinemia. Dopamine agonists successfully treat hyperprolactinemia-induced ovarian dysfunction in women, but not elephants. The objective of this study was to determine how longitudinal dopamine, serotonin, and oxytocin patterns in African elephants are related to ovarian cycle function. We hypothesized that dopamine concentrations are decreased, while oxytocin and serotonin are increased in non-cycling, hyperprolactinemic African elephants. Weekly urine and serum samples were collected for eight consecutive months from 28 female African elephants. Females were categorized as follows: (1) non-cycling with average prolactin concentrations of 15 ng/ml or greater (HIGH; n = 7); (2) non-cycling with average prolactin concentrations below 15 ng/ml (LOW; n = 13); and (3) cycling with normal progestagen and prolactin patterns (CYCLING; n = 8). Both oxytocin and serotonin were elevated in hyperprolactinemic elephants. Thus, we propose that stimulatory factors may play a role in the observed hyperprolactinemia in this species. Interestingly, rather than being reduced as hypothesized, urinary dopamine was elevated in hyperprolactinemic elephants compared to CYCLING and LOW prolactin groups. Despite its apparent lack of regulatory control over prolactin, this new evidence suggests that dopamine synthesis and secretion are not impaired in these elephants, and perhaps are augmented.
Assuntos
Dopamina/sangue , Elefantes/fisiologia , Ciclo Estral/fisiologia , Hiperprolactinemia/sangue , Ocitocina/sangue , Prolactina/sangue , Serotonina/sangue , Doenças dos Animais/sangue , Doenças dos Animais/fisiopatologia , Animais , Animais de Zoológico , Estudos de Casos e Controles , Dopamina/urina , Elefantes/sangue , Elefantes/urina , Ciclo Estral/sangue , Feminino , Hiperprolactinemia/fisiopatologia , Hiperprolactinemia/urina , Hiperprolactinemia/veterinária , Doenças Ovarianas/sangue , Doenças Ovarianas/fisiopatologia , Doenças Ovarianas/urina , Ovário/fisiologiaRESUMO
Hypokalemia prolongs the QRS and QT intervals, deteriorates intercellular coupling, and increases the risk for arrhythmia. Melatonin preserves gap junctions and shortens action potential as potential antiarrhythmic mechanisms, but its properties under hypokalemia remain unknown. We hypothesized that melatonin protects against low potassium-induced arrhythmias through the activation of its receptors, resulting in action potential shortening and connexin-43 preservation. After stabilization in Krebs-Henseleit solution (4.5 mEq/L K+ ), isolated hearts from Wistar rats underwent perfusion with low-potassium (1 mEq/L) solution and melatonin (100 µmol/L), a melatonin receptor blocker (luzindole, 5 µmol/L), melatonin + luzindole or vehicle. The primary endpoint of the study was the prevention of ventricular fibrillation. Electrocardiography was used, and epicardial action potentials and heart function were measured and analyzed. The ventricular expression, dephosphorylation, and distribution of connexin-43 were examined. Melatonin reduced the incidence of low potassium-induced ventricular fibrillation from 100% to 59%, delayed the occurrence of ventricular fibrillation and induced a faster recovery of sinus rhythm during potassium restitution. Melatonin prevented QRS widening, action potential activation delay, and the prolongation of action potential duration at 50% of repolarization. Other ECG and action potential parameters, the left ventricular developed pressure, and nonsustained ventricular arrhythmias did not differ among groups. Melatonin prevented connexin-43 dephosphorylation and its abnormal topology (lateralization). Luzindole abrogated the protective effects of melatonin on electrophysiological properties and connexin-43 misdistribution. Our results indicate that melatonin receptor activation protects against low potassium-induced ventricular fibrillation, shortens action potential duration, preserves ventricular electrical activation, and prevents acute changes in connexin-43 distribution. All of these properties make melatonin a remarkable antifibrillatory agent.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Conexina 43/metabolismo , Melatonina/farmacologia , Miocárdio/metabolismo , Potássio/efeitos adversos , Receptores de Melatonina/metabolismo , Fibrilação Ventricular/metabolismo , Animais , Masculino , Miocárdio/patologia , Potássio/farmacologia , Ratos , Ratos Wistar , Fibrilação Ventricular/induzido quimicamente , Fibrilação Ventricular/patologia , Fibrilação Ventricular/fisiopatologiaRESUMO
Longitudinal analyses of serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, glucose, insulin, triglycerides, cholesterol, total and free thyroxine (T4), total triiodothyronine (T3), thyroid stimulating hormone (TSH), and cortisol were conducted to investigate pituitary, metabolic, and adrenal changes related to testicular function and musth status in zoo-housed elephant bulls. Blood samples were collected twice a month for 12â¯months from 14 African and 12 Asian bulls at 17 facilities in North America. Building on previous studies, our results show that musth is associated with increased testosterone, LH, FSH, and cortisol secretion, and a decrease in thyroid hormone (total and free T4) production. In addition, glucose and triglycerides were higher during musth than non-musth periods, indicative of altered sugar and fat metabolism. There were significant differences associated with age for LH, FSH and testosterone, all increasing, whereas the glucose-to-insulin ratio (G:I) decreased with age. A species comparison found African and Asian elephants differed in measures of insulin, prolactin, cholesterol and the G:I. Across all hormones, high inter-individual variability was observed, making it difficult to define a general musth endocrine profile or to assess musth status from single samples. These results highlight the need for facilities hosting bulls to closely and consistently monitor each individual from an early age and throughout musth and non-musth periods to determine the pattern for each male.
Assuntos
Glândulas Suprarrenais/metabolismo , Comportamento Animal , Elefantes/metabolismo , Gônadas/metabolismo , Hormônios/metabolismo , Hipófise/metabolismo , Glândula Tireoide/metabolismo , África , Agressão , Animais , Ásia , Glicemia/metabolismo , Colesterol/sangue , Elefantes/sangue , Insulina/sangue , Modelos Lineares , Masculino , Triglicerídeos/sangueRESUMO
Ischemic postconditioning (IPoC) reduces reperfusion arrhythmias but the antiarrhythmic mechanisms remain unknown. The aim of this study was to analyze IPoC electrophysiological effects and the role played by adenosine A1, A2A and A3 receptors, protein kinase C, ATP-dependent potassium (KATP) channels, and connexin 43. IPoC reduced reperfusion arrhythmias (mainly sustained ventricular fibrillation) in isolated rat hearts, an effect associated with a transient delay in epicardial electrical activation, and with action potential shortening. Electrical impedance measurements and Lucifer-Yellow diffusion assays agreed with such activation delay. However, this delay persisted during IPoC in isolated mouse hearts in which connexin 43 was replaced by connexin 32 and in mice with conditional deletion of connexin 43. Adenosine A1, A2A and A3 receptor blockade antagonized the antiarrhythmic effect of IPoC and the associated action potential shortening, whereas exogenous adenosine reduced reperfusion arrhythmias and shortened action potential duration. Protein kinase C inhibition by chelerythrine abolished the protective effect of IPoC but did not modify the effects on action potential duration. On the other hand, glibenclamide, a KATP inhibitor, antagonized the action potential shortening but did not interfere with the antiarrhythmic effect. The antiarrhythmic mechanisms of IPoC involve adenosine receptor activation and are associated with action potential shortening. However, this action potential shortening is not essential for protection, as it persisted during protein kinase C inhibition, a maneuver that abolished IPoC protection. Furthermore, glibenclamide induced the opposite effects. In addition, IPoC delays electrical activation and electrical impedance recovery during reperfusion, but these effects are independent of connexin 43.
Assuntos
Arritmias Cardíacas/prevenção & controle , Conexina 43/fisiologia , Pós-Condicionamento Isquêmico/métodos , Canais KATP/metabolismo , Isquemia Miocárdica/complicações , Proteína Quinase C/metabolismo , Receptores Purinérgicos P1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Canais KATP/genética , Camundongos , Camundongos Transgênicos , Proteína Quinase C/genética , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P1/genéticaRESUMO
Hyperprolactinemia is a common disorder of the hypothalamic-pituitary axis, and a cause of ovarian dysfunction in women. Currently, over half of non-cycling African elephant females in North America also are hyperprolactinemic, suggesting a similar link between these two conditions may exist. The objective of this study was to determine the relationship between acyclicity and prolactin status by comparing mean prolactin concentrations of bi-weekly samples collected over a 1-year period in 2012 with 20 years of historical weekly progestagen data to assess cyclicity. Females were categorized as: 1) non-cycling with an average prolactin concentration of 15 ng/ml or greater (HIGH; n = 17); 2) non-cycling with an average prolactin concentration below 15 ng/ml (LOW; n = 16); and 3) typical temporal patterns of progestagen and prolactin secretion (NORMAL; n = 45), and evaluated based on length of time (in years) they had experienced ovarian inactivity. Results showed that the majority of HIGH prolactin elephants had been acyclic for at least 5 years, and in a number of cases (n = 9) for over 10 years. By contrast, most of the LOW prolactin elephants had experienced acyclicity for less than 5 years. Finally, there was a positive association between duration of acyclicity and mean prolactin concentrations, with an increase in the likelihood of having higher prolactin concentrations the longer an individual was acyclic. This study highlights the importance of longitudinal hormonal datasets to examine temporal changes in biological functioning and better understand the etiology of infertility problems.
Assuntos
Animais de Zoológico , Elefantes/fisiologia , Ciclo Estral/fisiologia , Hiperprolactinemia/veterinária , Ovário/fisiologia , Animais , Feminino , Hiperprolactinemia/etiologiaRESUMO
The recruitment of T-cells by bispecific antibodies secreted from adoptively transferred, gene-modified autologous cells has shown satisfactory results in preclinical cancer models. Even so, the approach's translation into the clinic will require incremental improvements to its efficacy and reduction of its toxicity. Here, we characterized a tandem T-cell recruiting bispecific antibody intended to benefit gene-based immunotherapy approaches, which we call the light T-cell engager (LiTE), consisting of an EGFR-specific single-domain VHH antibody fused to a CD3-specific scFv. We generated two LiTEs with the anti-EGFR VHH and the anti-CD3 scFv arranged in both possible orders. Both constructs were well expressed in mammalian cells as highly homogenous monomers in solution with molecular weights of 43 and 41 kDa, respectively. In situ secreted LiTEs bound the cognate antigens of both parental antibodies and triggered the specific cytolysis of EGFR-expressing cancer cells without inducing T-cell activation and cytotoxicity spontaneously or against EGFR-negative cells. Light T-cell engagers are, therefore, suitable for future applications in gene-based immunotherapy approaches.
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Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Receptores ErbB/imunologia , Imunoterapia , Neoplasias/terapia , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Ativação Linfocitária , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
PURPOSE OF REVIEW: Here, we review the known relations between hypertension and obesity to inflammation and postulate the endogenous protective effect of melatonin and its potential as a therapeutic agent. We will describe the multiple effects of melatonin on blood pressure, adiposity, body weight, and focus on mitochondrial-related anti-inflammatory and antioxidant protective effects. RECENT FINDINGS: Hypertension and obesity are usually associated with systemic and tissular inflammation. The progressive affection of target-organs involves multiple mediators of inflammation, most of them redundant, which make anti-inflammatory strategies ineffective. Melatonin reduces blood pressure, body weight, and inflammation. The mechanisms of action of this ancient molecule of protection involve multiple levels of action, from subcellular to intercellular. Mitochondria is a key inflammatory element in vascular and adipose tissue and a potential pharmacological target. Melatonin protects against mitochondrial dysfunction. Melatonin reduces blood pressure and adipose tissue dysfunction by multiple anti-inflammatory/antioxidant actions and provides potent protection against mitochondria-mediated injury in hypertension and obesity. This inexpensive and multitarget molecule has great therapeutic potential against both epidemic diseases.
Assuntos
Anti-Inflamatórios/uso terapêutico , Hipertensão/tratamento farmacológico , Inflamação/tratamento farmacológico , Melatonina/uso terapêutico , Obesidade/tratamento farmacológico , Animais , Antioxidantes/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Inflamação/metabolismo , Inflamação/fisiopatologia , Mitocôndrias/metabolismo , Obesidade/complicações , Obesidade/fisiopatologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
Lethal ventricular arrhythmias increase in patients with chronic kidney disease that suffer an acute coronary event. Chronic kidney disease induces myocardial remodeling, oxidative stress, and arrhythmogenesis. A manifestation of the relationship between kidney and heart is the concomitant reduction in vitamin D receptor (VDR) and the increase in angiotensin II receptor type 1 (AT1 ). Melatonin has renal and cardiac protective actions. One potential mechanism is the increase in the heat shock protein 70 (Hsp70)-an antioxidant factor. We aim to determine the mechanisms involved in melatonin (Mel) prevention of kidney damage and arrhythmogenic heart remodeling. Unilateral ureteral-obstruction (UUO) and sham-operated rats were treated with either melatonin (4 mg/kg/day) or vehicle for 15 days. Hearts and kidneys from obstructed rats showed a reduction in VDR and Hsp70. Associated with AT1 up-regulation in the kidneys and the heart of UUO rats also increased oxidative stress, fibrosis, apoptosis, mitochondrial edema, and dilated crests. Melatonin prevented these changes and ventricular fibrillation during reperfusion. The action potential lengthened and hyperpolarized in melatonin-treated rats throughout the experiment. We conclude that melatonin prevents renal damage and arrhythmogenic myocardial remodeling during unilateral ureteral obstruction due to a decrease in oxidative stress/fibrosis/apoptosis associated with AT1 reduction and Hsp70-VDR increase.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Melatonina/uso terapêutico , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Calcitriol/metabolismo , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/metabolismo , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Fibrose/metabolismo , Proteínas de Choque Térmico HSP70/genética , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Rim/metabolismo , Masculino , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , NADPH Oxidases/metabolismo , Ratos , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/genética , Receptores de Calcitriol/genéticaRESUMO
We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management.
Assuntos
Lipopolissacarídeos/farmacologia , Proteína Adaptadora de Sinalização NOD1/genética , Peptidoglicano/farmacologia , Pericitos/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/metabolismo , Artérias Cerebrais/citologia , Artérias Cerebrais/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação , Interleucina-6/biossíntese , Interleucina-6/metabolismo , Interleucina-8/biossíntese , Interleucina-8/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Pericitos/citologia , Pericitos/metabolismo , Cultura Primária de Células , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Recombinant antibodies are highly successful in many different pathological conditions and currently enjoy overwhelming recognition of their potential. There are a wide variety of protein expression systems available, but almost all therapeutic antibodies are produced in mammalian cell lines, which mimic human glycosylation. The production of clinical-grade antibodies in mammalian cells is, however, extremely expensive. Compared to mammalian systems, protein production in yeast strains such as Pichia pastoris, is simpler, faster and usually results in higher yields. RESULTS: In this work, a trivalent single-chain fragment variable (scFv)-based N-terminal trimerbody, specific for the human carcinoembryonic antigen (CEA), was expressed in human embryonic kidney 293 cells and in Pichia pastoris. Mammalian- and yeast-produced anti-CEA trimerbody molecules display similar functional and structural properties, yet, the yield of trimerbody expressed in P. pastoris is about 20-fold higher than in human cells. CONCLUSIONS: P. pastoris is an efficient expression system for multivalent trimerbody molecules, suitable for their commercial production.
Assuntos
Biotecnologia/métodos , Pichia/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Antígeno Carcinoembrionário/imunologia , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Células HEK293 , Humanos , Proteínas Imobilizadas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Soro/metabolismo , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
Reperfusion arrhythmias are currently attributed to ionic imbalance and oxidative stress. Tamoxifen is a potent antioxidant that also modulates some ionic transport pathways. In this work, we tried to correlate the electrophysiological effects of 1, 2, and 5 µM of tamoxifen with the incidence and severity of arrhythmias appearing on reperfusion after 10 minutes of coronary occlusion in isolated hearts from female rats. All tamoxifen concentrations inhibited the action potential shortening observed in the control hearts during late ischemia (6-10 minutes), whereas 2 and 5 µM also reduced the resting membrane potential depolarization. The incidence of sustained ventricular tachycardia and/or ventricular fibrillation on reperfusion decreased from 10 of 12 (control group) to 5 of 10 (1 µM, P = 0.1718), 4 of 12 (2 µM, P = 0.0361), and 2 of 10 (5 µM, P = 0.0083). The possible role of chloride currents activated by cell swelling in these effects was explored in hearts submitted to a 10-minute hypotonic challenge, where tamoxifen (5 µM) blocked the action potential shortening and the late resting membrane potential depolarization produced by hypotonicity, mimicking its action in late ischemia. Tamoxifen produced a similar increase of the total antioxidant capacity of myocardial samples at all the concentration tested. In conclusion, our data strongly suggest that the antiarrhythmic action of this agent is mediated by its electrophysiological effect derived from modulation of chloride currents activated by cell swelling.