Prevalence of Neutralizing Antibodies against Adeno-Associated Virus Serotypes 1, 2, and 9 in Non-Injected Latin American Patients with Heart Failure-ANVIAS Study.

Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55-75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.

Potential Role of Antioxidants as Adjunctive Therapy in Chagas Disease.

Chagas disease (CD) is one of the most important neglected tropical diseases in the American continent. Host-derived nitroxidative stress in response to Trypanosoma cruzi infection can induce tissue damage contributing to the progression of Chagas disease. Antioxidant supplementation has been suggested as adjuvant therapy to current treatment. In this article, we synthesize and discuss the current evidence regarding the use of antioxidants as adjunctive compounds to fight harmful reactive oxygen species and lower the tissue oxidative damage during progression of chronic Chagas disease. Several antioxidants evaluated in recent studies have shown potential benefits for the control of oxidative stress in the host's tissues. Melatonin, resveratrol, the combination of vitamin C/vitamin E (vitC/vitE) or curcumin/benznidazole, and mitochondria-targeted antioxidants seem to be beneficial in reducing plasma and cardiac levels of lipid peroxidation products. Nevertheless, further research is needed to validate beneficial effects of antioxidant therapies in Chagas disease.

[Immune response and gene therapy with adenoassociated viral vectors]. / Respuesta inmune y terapia génica con vectores virales adenoasociados.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient's immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

En los últimos años la terapia génica se ha posicionado como una opción real y segura en el desarrollo de alternativas terapéuticas para la cura y la prevención de diferentes enfermedades. Consiste en la inserción de material genético en un tejido o célula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unión y entrada a la célula diana, la capacidad de transferencia del material genético al núcleo, la habilidad de expresión del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluación como opción terapéutica en un amplio número de enfermedades monogénicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducción del vector e impiden la expresión del gen que transporta, limitando su aplicación clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia génica con vectores AAV. La presencia de NAb depende principalmente de la exposición al virus en la naturaleza y varía drásticamente según edad, localización geográfica y estado de salud de la persona evaluada.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Measuring mitochondrial respiration in adherent cells infected with Trypanosoma cruzi Chagas, 1909 using Seahorse extracellular flux analyser.

Infection with Trypanosoma cruzi Chagas, 1909 is reported to increase the production of reactive oxygen species in patients with Chagas disease. Mitochondria dysfunction, host inflammatory response and inadequate antioxidant response are described as the main factors leading to oxidative stress during acute and chronic stages of the disease. The Seahorse XFe24 extracellular flux platform allows energy metabolism determination through mitochondrial respiration and glycolysis measurements. XFe24 platform can be used in in vitro models of T. cruzi-infected cells, which allow the assessment and even modulation of endogenous conditions of infected cells, generating readouts of real-time cellular bioenergetics changes. In this protocol, we standardised the use of XFe24 technology in T. cruzi infected AC16 cardiomyocytes and SGHPL-5 trophoblasts. In addition, we provide a list of optimised assay specifications, advantages and critical steps to be considered during the process. Cardiomyocytes and trophoblasts are attractive target cells to evaluate the metabolic environment in acute, chronic and congenital Chagas transmission scenarios.

Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol.

BACKGROUND: Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. OBJECTIVE: The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. METHODS: We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. RESULTS: The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype, and the general validation of the assay, at the end of 2016. The final results are expected mid-2017. CONCLUSIONS: This project is the first effort to evaluate NAb levels against AAV1, AAV2, and AAV9 serotypes in patients with HF in Latin America. Our results will allow us to check the cross-reactivity response between the serotypes assessed, to describe the epidemiological characteristics of the participant population, and to set up a link with earlier reports of NAb prevalence in the literature.

Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality.

Hyperinsulinemia is a predictor of new cardiovascular events in Colombian patients with a first myocardial infarction.

BACKGROUND: Acute Myocardial Infarction (AMI) is one of the main causes of mortality and disability in Colombia. The factors associated to a new event in surviving subjects to a first AMI in our population have not yet been fully identified. METHODS: Two hundred and ninety five surviving subjects to a first AMI (58.8±12.6 years) were included in a prospective cohort study between 2000 and 2006. Lipid profile, glycemia and plasma insulin levels were measured. Deaths of cardiovascular origin, a new AMI, unstable angina, heart failure, stroke, new myocardial revascularization or angioplasty were considered new cardiovascular events. RESULTS: The study included 61 (20.6%) women and 234 (79.4%) men. The mean follow up time was 50±30 months with a 38.9% incidence of new events. Fifty five patients (18.6%) were diabetic. Bi-varied analysis identified as risk factors for a new cardiovascular event the presence of: hypertension, anterior descending coronary artery stenosis, intrahospital cardiac failure, age over 55, low income, lack of education, Killip III-IV, heart rate over 76 bpm, pulse pressure over 80 mmHg, total cholesterol over 200 mg/dl and insulin over 10 IU/ml. After logistic regression analysis, the log values of insulin remained as the only significant predictor for new cardiovascular events. CONCLUSIONS: Hyperinsulinism was the most important factor associated to the occurrence of new cardiovascular events in Colombian patients with AMI, which emphasizes the pivotal role of insulin resistance in the physiopathologic mechanisms of atherosclerosis, especially in undeveloped countries.

A controlled, randomized-blinded clinical trial to assess the efficacy of a nitric oxide releasing patch in the treatment of cutaneous leishmaniasis by Leishmania (V.) panamensis.

A topical nanofiber nitric oxide (NO) releasing patch ( approximately 3.5 mumol NO/cm(2)/day for 20 days, NOP) was compared with intramuscular meglumine antimoniate (Glucantime, 20 mg/kg/day for 20 days) for the treatment of cutaneous leishmaniasis (CL) caused by Leishmania (V.) panamensis in Santander and Tolima, Colombia. A double-blind, randomized, placebo-controlled, clinical trial was conducted to determine whether the NOP is as effective as Glucantime for the treatment of CL. Patients were randomly assigned to Glucantime and placebo patches or NOP and placebo of Glucantime. The cure rates after a 3-month follow-up were 94.8% for the group that received Glucantime compared with 37.1% in the NOP group. Despite the lower efficacy of the NOP versus Glucantime, a significantly lower frequency of non-serious adverse events and a reduced variation in serum markers were observed in patients treated with NOP. Treatment of CL with NOP resulted in a lower effectiveness compared with Glucantime; however, the low frequency of adverse events and the facility of topic administration justify the development of new generations of NOP systems for the treatment of CL.

[A report of two cases of disseminated cutaneous leishmaniasis in Santander, Colombia]. / Leishmaniasis Cutánea Diseminada: Reporte de Dos Casos en Santander, Colombia.

Leishmaniasis is a zoonosis produced by the transmission of the protozoan leishmania caused by the bite of sand-flies from the Lutzomya specie. Several clinical manifestations present themselves, depending on the infecting strain and the host's immune response; the most frequent variety is localised cutaneous leishmaniasis. Atypical forms sometimes develop, such as the diffuse variety, in which the number of ulcers is greater than 10, thereby affecting several body areas requiring special considerations in its diagnosis and management. This article reports two cases of patients from endemic areas of Santander suffering from diffuse cutaneous leishmaniasis produced by the L. panamensis strain. Protocols for the active search of this type of case in endemic areas throughout Colombia should be implemented.

Leishmaniasis cutánea diseminada: reporte de dos casos en Santander, Colombia / A report of two cases of disseminated cutaneous leishmaniasis in Santander, Colombia

La leishmaniasis es una zoonosis ocasionada por la trasmisión del protozoo leishmania a causa de la picadura de mosquitos flebótomos de la especie Lutzomya. Se presentan diversas manifestaciones clínicas dependiendo de la cepa infectante y de la respuesta inmune del hospedero, la más frecuente la Leismaniasis Cutánea Localizada. En ocasiones se presentan formas atípicas como la forma difusa, en la cual el número de ulceras es mayor de 10, afectando diversas regiones corporales, requiriendo consideraciones especiales en su diagnóstico y manejo. En el presente artículo reportamos dos casos de pacientes con Leishmaniasis Cutánea Difusa generados por la cepa L. panamensis procedentes de zonas endémicas de Santander, destacando la necesidad de implementar protocolos de búsqueda activa para este tipo de casos en las zonas endémicas de todo el país.

Leishmaniasis is a zoonosis produced by the transmission of the protozoan leishmania caused by the bite of sand-flies from the Lutzomya specie. Several clinical manifestations present themselves, depending on the infecting strain and the host's immune response; the most frequent variety is localised cutaneous leishmaniasis. Atypical forms sometimes develop, such as the diffuse variety, in which the number of ulcers is greater than 10, thereby affecting several body areas requiring special considerations in its diagnosis and management. This article reports two cases of patients from endemic areas of Santander suffering from diffuse cutaneous leishmaniasis produced by the L. panamensis strain. Protocols for the active search of this type of case in endemic areas throughout Colombia should be implemented.

El sobrepeso es el factor determinante en la presentación de síndrome coronario agudo en adultos jóvenes colombianos / Overweight is a determinant factor in the presentation of acute coronary syndrome in colombian young adults

Antecedentes: la obesidad es un factor de riesgo para un primer infarto agudo del miocardio. La enfermedad coronaria prematura genera gran impacto socioeconómico por los años productivos perdidos, lo que hace importante su prevención y tratamiento. Objetivo: evaluar el impacto de la obesidad y otros factores de riesgo convencionales en la presentación de un primer evento coronario agudo en sujetos menores de 50 años.Métodos: estudio transversal que incluyó pacientes con diagnóstico de síndrome coronario agudo que ingresaron a la institución entre febrero de 2002 y febrero de 2007. La población se dividió en: sujetos menores y mayores de 50 años, estos últimos seleccionados de manera aleatoria en relación 1:1. La información demográfica, la historia cardiovascular y los factores de riesgo se identificaron en la historia clínica electrónica de la institución y y se corroboraron por vía telefónica. Se empleó la prueba t de student o Wilcoxon rank-sum, según la distribución de las variables. Se realizó un análisis multivariado para determinar los factores de riesgo independientes. Un valor de p < 0,05 se consideró significativo. Resultados: en el período de estudio ingresaron 942 sujetos con diagnóstico de síndrome coronario agudo. 16,1 porciento (152) correspondió a sujetos menores de 50 años y 90,79 porciento (132) presentaban un primer evento. El promedio de edad en el grupo joven fue 44,3 más/menos 5,1 y en el grupo mayor (n=132) fue 65,6 más/menos 8,3 años. El sobrepeso (OR 1.095; IC 1,01-1,18 p=0,019) y el recuento leucocitario (OR 1,00; IC 1.001-1.005 p=0,001) fueron los dos criterios independientes que predijeron el síndrome coronario agudo en adultos menores de 50 años, luego de realizar el análisis multivariado no condicional. Los otros factores de riesgo convencionales no mostraron diferencia significativa.Conclusión: el sobrepeso y la inflamación estimada por el conteo leucocitario, fueron los factores de riesgo independientes para la presentación de un primer episodio de síndrome coronario agudo en adultos colombianos menores de 50 años. Estos resultados confirman la importancia del sobrepeso y la inflamación en los mecanismos fisiopatológicos de la enfermedad cardiovascular en nuestra población.

The role of the L-arginine-nitric oxide pathway in preeclampsia.

Preeclampsia (PE) is a major cause of maternal and perinatal mortality, especially in developing countries. Its etiology involves multiple factors, but no specific cause has been identified. Evidence suggests that clinical manifestations are caused by endothelial dysfunction. Nitric oxide (NO), which is synthesized from L-arginine in endothelial cells by the endothelial nitric oxide synthase (eNOS), provides a tonic dilator tone and regulates the adhesion of white blood cells and platelet aggregation. Alterations in the L-arginine-NO pathway have been associated with the development of PE. Various studies, reporting decreased, elevated or unchanged levels of nitrite (NO(2)) and nitrate (NO(3)), two end products of NO metabolism, have been published. Our group contributed to those contradictory reports describing cases of PE with both elevated and decreased levels of NO(2) and NO(3). Apparently, diminished levels of NO could be related to deficiencies in the ingestion of dietary calcium associated to low levels of plasma ionic calcium, which is crucial to the eNOS' activity. Also, low levels of NO could be associated with the presence of eNOS polymorphisms or the presence of increased levels of ADMA, the endogenous inhibitor of NO. High levels of NO associated to low levels of cGMP suggest a decreased bioactivity of NO, which is probably related to an increased degradation of NO caused by a high production of superoxide in states of infection and inflammation. The present article analyses and reviews the reported paradoxical roles of the L-arginine-NO pathway in PE and gives a possible explanation for these results.