Biotecnología Aplicada

El proceso angiogénico y el cáncer

Angiogenesis, the growth of new capillaries from pre-existing vessels, is closely related to processes essential for the organism, which are entirely physiological, for example embryogenesis, reproductive cycle, and wound healing, and is also associated with pathological conditions such as tumor progression, metastasis, diabetic retinopathy, hemangioma, arthritis, psoriasis and atherosclerosis, among other chronic diseases. The development of specific anti-angiogenic agents arises as an attractive therapeutic approach to treat cancer and other angiogenesis-dependent diseases. Many of these agents are being evaluated in clinical trials and have shown promising antitumor activity. This review attempts to provide a comprehensive overview of key knowledge accumulated on angiogenesis and its role in cancer, including the components of signal transduction pathways that have been explored in this process. Additionally, this review focuses on the current approaches for the discovery of new compounds that inhibit angiogenesis, emphasizing on the clinical developmental status of antiangiogenic drugs.

La angiogénesis, el crecimiento de nuevos capilares a partir de los vasos pre-existentes, se encuentra estrechamente relacionado con procesos esenciales para el organismo, que son puramente fisiológicos; por ejemplo la embriogénesis, el ciclo reproductivo y la cicatrización de heridas. También se encuentra asociado a condiciones patológicas como el desarrollo de tumores, metástasis, retinopatía diabética, hemangioma, artritis, psoriasis y arterosclerosis, entre otras enfermedades crónicas. El desarrollo de compuestos anti-angiogénicos específicos se plantea como un atractivo enfoque terapéutico para el tratamiento del cáncer y otras enfermedades dependientes de la angiogénesis. Muchos de estos agentes están siendo evaluados en ensayos clínicos y han mostrado actividad antitumoral prometedora. Esta revisión pretende dar una visión general de los principales conocimientos acumulados acerca de la angiogénesis y su papel en el cáncer, incluyendo también los componentes de las vías de transducción de señales que se han investigado de este proceso. Además esta revisión se centra en los enfoques actuales para el descubrimiento de nuevos compuestos que inhiben la angiogénesis, con énfasis en el estado de desarrollo clínico del producto antiangiogénico.

Metodología para la elaboración de las pautas reguladoras para productos biofarmacéuticos obtenidos de plantas transgénicas en Cuba

Molecular farming is an evolving field in Cuba; therefore, establishing the guidelines guaranteeing the quality, safety and efficacy of biopharmaceutical products is a challenge for the National Regulatory Agency (Center for State Control on the Quality of Drugs; CECMED). The methodology to elaborate guidelines for this type of products was mainly based on the revision of the legal and regulatory international framework, the evaluation of the current and prospective developmental stage of the technology in Cuba and the comparative analysis among the manufacturing process steps of a biotechnological product obtained in traditional expression systems and that derived from transgenic plants. A first draft of the regulatory document was compared to guidance drafts issued by other regulatory agencies (such as FDA and EMEA) and extensively reviewed by CECMED specialists as well as by experts of the local biotech industry. As a result, a regulation project was elaborated, and its applicability tested while the assessment of an application for scientific advisory, related to product obtained by this technology. Finally, the regulation was approved, being applicable for products generated in genetically modified plants. It is also valid for edible vaccines and other compounds which do not constitute active pharmaceutical ingredients.

La obtención de productos biofarmacéuticos a partir de plantas transgénicas es un campo en evolución en Cuba, por lo que la elaboración de pautas reguladoras que garanticen su calidad, seguridad y eficacia constituye un reto para la Autoridad Reguladora de Medicamentos, el Centro para el Control Estatal de la Calidad de los Medicamentos, CECMED. La metodología para establecer las pautas reguladoras para estos productos se basó fundamentalmente en la revisión del entorno legal y regulador internacional, la evaluación del estado de desarrollo actual y prospectivo de la tecnología en Cuba, y en la comparación de las diferentes etapas del proceso de obtención de un producto biotecnológico en sistemas tradicionales de expresión y a partir de plantas transgénicas. La primera versión del documento normativo se comparó con las guías preliminares emitidas por otras autoridades reguladoras, como la Administración de Alimentos y Medicamentos (FDA) y la Agencia Europea para la Evaluación de Medicamentos (EMEA) y la revisaron especialistas del CECMED y de la industria. Como resultado, se elaboró un proyecto de regulación, cuya aplicabilidad se comprobó mediante la evaluación de un trámite de registro de un producto cubano obtenido utilizando esta tecnología. Finalmente, se aprobó un documento normativo aplicable a proteínas obtenidas de plantas genéticamente modificadas, válido también para la obtención de vacunas comestibles y de otros compuestos que no constituyan el principio activo.

Células de tabaco transgénico: sistema permisivo para la evaluación de estrategias de resistencia al virus del enrollamiento foliar amarillo del tomate

Tomato yellow leaf curl virus (TYLCV) is a major threat to tomato production in the tropics and sub-tropics around the world. The application of genetic engineering and pathogen derived resistance mechanisms to obtain tomatoes that are resistant to this pathogen is considered a promising alternative to the current protective practice against the virus. However, the development of transgenic tomato plants that are resistant to the virus is a resourceconsuming and time-consuming procedure, often with unpredictable efficiency, which hinders the evaluation of genetic designs. For this reason an assessment of the strategies against TYLCV replication preceding transgenic tomato development would ensure the protective potential of the candidate transgene. Attempting to circumvent this issue, the present study demonstrated the feasibility of using tobacco cell lines to study the consequences of c1 antisense expression on TYLCV replication. As a result, the transgenic tobacco cell lines were able to produce siRNA that is complementary to the c1 sequence and inhibited TYLCV multiplication, forecasting what would happen in transgenic plants harboring this antiviral strategy.

Tomato yellow leaf curl virus (TYLCV) constituye el mayor peligro para la producción de tomate en el mundo, principalmente en las regiones tropicales y subtropicales. La aplicación de la ingeniería genética y los mecanismos de resistencia derivada del patógeno para obtener tomates resistentes a este patógeno es considerada una alternativa promisoria en la práctica corriente de protección contra este virus. Sin embargo, el desarrollo de plantas transgénicas de tomate resistentes a virus es un procedimiento que consume mucho tiempo y recursos, y en la mayoría de las veces resulta impredecible su eficiencia, lo cual dificulta la evaluación de los diseños antivirales. Por ello la evaluación de las estrategias que inhiban la replicación de TYLCV en un sistema previo a la obtención de plantas de tomate transgénicas aseguraría el potencial protectivo del transgén candidato. En un intento por evadir el proceso de transformación de tomate en el presente trabajo se demostró la factibilidad de utilizar líneas celulares de tabaco para estudiar las consecuencias de la expresión del gen c1 en antisentido sobre la replicación de TYLCV. Como resultado las líneas celulares de tabaco fueron capaces de producir ARN de interferencia, complementarios a la secuencia del gen c1, e inhibieron la multiplicación de TYLCV, previendo lo que sucedería en las plantas transgénicas portadoras de esta estrategia antiviral.

Influencia del número de animales en la producción del anticuerpo monoclonal CB.Hep-1 mediante el método de producción ascitis

The ascites production method of inoculating hybridoma B cells in histocompatible mice can successfully produce monoclonal antibodies (MAb). The aim of this paper is to study the influence of the number of animals on MAb CB.Hep-1 production by the ascites production method. CB.Hep-1 mouse hybridoma cells were inoculated in different groups of animals (I, 750 mice; II, 1000 mice, III, 3500 mice; IV, 4000 mice and V, 6000 mice) previously irritated with mineral oil. The ascitic fluid was harvested through abdominal paracentesis and the results showed a marked influence of the number of animals on the amount of MAb CB.Hep-1 produced by the mice (I, 15.70 mg; II, 19.74 mg; III, 9.91 mg; IV, 8.46 mg; V, 5.30 mg). In conclusion, the concept of Reduction (3R concepts) was rigorously studied in this paper, determining the lowest number of inoculated animals needed for the highest amount of the MAb CB.Hep-1. Results indicate that the number of mice with tumors and the MAb CB.Hep-1 production decrease with the increase in the number of animals inoculated. Therefore between 50 000 and 250 000 animals could be spared each year if the number of animals was of ≤ 3500 per inoculation under the conditions studied here.

El método de producción de ascitis mediante la inoculación de hibridomas histocompatibles con los ratones puede producir anticuerpos monoclonales (AcM). El objetivo de este trabajo fue estudiar la influencia del número de animales sobre la producción del AcM CB.Hep-1 mediante la producción de ascitis. Las células del hibridoma de ratón CB.Hep-1 se inocularon en diferentes grupos de animales (I, 750; II, 1000; III, 3500; IV, 4000 and V, 6000) previamente irritados con aceite mineral. El fluido ascítico se cosechó mediante paracentesis abdominal y los resultados demostraron una marcada influencia del número de animales sobre la cantidad de AcM CB.Hep-1 por ratón (I, 15.70 mg; II, 19.74 mg; III, 9.91 mg; IV, 8.46 mg; V, 5.30 mg). En conclusión, el concepto de Reducción (conceptos 3R) fue rigurosamente estudiado en este trabajo, determinándose el mínimo número de animales que permite obtener la máxima cantidad del AcM CB.Hep-1. Los resultados indican que el número de animales con tumor y la producción del AcM CB.Hep-1 disminuyen con el incremento del número de animales inoculados y por lo tanto entre 50 000 y 250 000 animales por año podrían ser salvados si el número de animales inoculados fueran ≤ 3500 por cada inoculación bajos las condiciones estudiadas.

Cuantificación del EGF contenido en formulaciones semisólidas. Determinación de la liberación tópica del Factor de crecimiento epidérmico

In order to obtain a procedure to quantify EGF by ELISA from semisolid formulations different buffers and organic solvents were used, having different polarities and miscibility with water. We studied it from oil-in-water (o/w) cream, water-in-oil (w/o), polyethylene glycol (PEG) ointment, and a jelly. Precision, accuracy and selectivity were determined and variation coefficients were less than 10% after different extraction method with a recovery of 95.54 -108.98%. According to results, it is possible to estimate the EGF concentration with high precision and reproducibility. No statistically significant differences were detected, when compared a placebo extraction solution with the ELISA standard solution demonstrating that placebo does not produce interferences in the quantification of the molecule. To determine the delivery of the active ingredient from different preparations we used a static diffusion cell system to evaluate the release and penetrability of the active ingredient throughout abdominal pig skin. Results showed that release and penetrability of EGF increased proportionally with the hydrosolubility of the vehicle. Therefore, the rate was obtained in jelly, PEG > o/w > w/o. Additionally, PEG ointment and o/w cream allowed the highest distribution of labeled 125I EGF in the epidermis and dermis and receptor medium.

Con el objetivo de obtener un procedimiento para cuantificar el EGF mediante ELISA a partir de formulaciones semisólidas, diferentes tampones y solventes orgánicos con diferentes polaridades y miscibilidad en agua fueron usados. En este estudio las formulaciones utilizadas fueron una crema de aceite-en-agua (o/w), una agua-en-aceite (w/o), un ungüento de polietilenglicol (PEG) y una jalea. La precisión, la exactitud y la selectividad fueron determinadas y los coeficientes de variación fueron menores del 10 % luego de emplear diferentes sistemas de extracción, con una recuperación de 95.54-108.98 %. De acuerdo con estos resultados, es posible calcular la concentración de EGF con alta precisión y reproducibilidad. No se observaron diferencias significativas en la cuantificación del placebo comparada con la solución estándar del ELISA, por lo que se demostró que el placebo no produce interferencias en la cuantificación de la molécula. Para determinar la liberación y la penetrabilidad del ingrediente farmacéutico activo a partir de diferentes formulaciones se utilizó un sistema de celda de difusión estática y piel abdominal de cerdo. Los resultados mostraron que la liberación y la penetrabilidad del EGF aumentaron proporcionalmente con la hidrosolubilidad del vehículo. Por lo tanto, se obtuvo el siguiente orden de liberación jalea, PEG > o/w > w/o. Además, el ungüento PEG y la crema o/w permitieron una distribución mayor del EGF125I en la epidermis, la dermis y el medio receptor.

Comparación entre una prueba inmunocromatográfica con un ELISA amplificado para la detección de antigeno e y de anticuerpos anti-antígeno e en la Hepatitis B crónica

The disappearance of the e antigen and the appearance of anti-e antigen antibodies are two biomarkers that indicate favorable prognosis in Hepatitis B. In this study the Advanced QualityTM immunochromatographic test for detecting those biomarkers was compared to the Vidas® semi-quantitative ELISA test. Our hypothesis was that it is possible to use these biomarkers measured in a rapid and simple Advanced QualityTM immunochromatographic test for evaluating the therapeutic response in clinical trials with chronic hepatitis B patients. The two methods were done following the manufacturers instructions. The sera were taken from 69 patients with chronic hepatitis B of the clinical trial of the CIGB 440 therapeutic candidate. The immunochromatographic test and ELISA for detecting e antigen and anti-e antigen antibodies presented from substantial to almost perfect agreement in the evaluation of the sera of chronic Hepatitis B patients in a clinical trial. The immunochromatographic test for detecting e antigen had a low positive average agreement and a high negative average agreement compared to the ELISA. Nevertheless, the immunochromatographic test for detecting anti-e antigen antibodies had a high negative and positive average agreement in comparison to the ELISA. The immunochromagraphic test for the e antigen had a lower positive average agreement compared to the ELISA and some patients infected with Hepatitis B virus could not be detected by the former assay. The immunochromatographic test for anti-e antigen antibodies showed a similar performance to that of ELISA and could therefore be used in clinical trials for chronic Hepatitis B in health institutions without the need of a highly qualified lab technician.

Evaluación integral de la calidad para redes de laboratorios de diagnóstico

The Center of Immunoassay of Cuba (CIE) has been dedicated for years to develop the SUMA (Ultra Micro Analytical System) technology, applied in pre- and post-natal screening programs, epidemiological surveillance and certification of blood, organs and placenta. CIE has developed and updated the quality control systems based on clinical laboratory theoretical background of this field, to guarantee the quality performance of this technology in the Cuban laboratory network and in other countries. These objectives have resulted in an informatics support or software named Modular System for Quality Control which comprises an informatics tool intended to evaluate the quality of laboratory networks by combining different types of internal control, such as internal, external and statistical data. They support an integral assessment of quality performance in these labs. The elements of design; development and evaluation used by the Modular System for Quality Control are described here. This system is already installed in the laboratory networks of the Cuban Health System, guaranteeing its safety, reliability and quality.

El Centro de Inmunoensayo es una institución cubana dedicada durante años al desarrollo de la tecnología SUMA (Sistema Ultra Micro Analítico), la cual se aplica en programas de pesquisas prenatal y neonatal, vigilancia epidemiológica y certificación de sangre, órganos y placenta. Con el objetivo de garantizar la calidad del funcionamiento de dicha tecnología en las redes de laboratorios cubanos y de otros países donde se encuentra instalada, el CIE ha desarrollado y perfeccionado sistemas de evaluación de la calidad sustentados en los fundamentos teóricos de esta rama del laboratorio clínico, los que se han materializado en un soporte informático o software de aplicación llamado Sistema Modular para el Control de Calidad, (consistente en una herramienta informática destinada a la evaluación de la calidad en las redes de laboratorios, que posee la particularidad de combinar los diferentes tipos de control de la calidad (interno, externo y resultados estadísticos) para realizar un análisis integral del comportamiento de la calidad de dichos laboratorios El presente trabajo describe elementos relativos a la concepción, desarrollo y evaluación del Sistema Modular para el Control de Calidad que se encuentra instalado en las redes de laboratorios de diagnóstico pertenecientes a los programas de salud cubanos y constituye un componente de extraordinaria importancia para su seguridad, fiabilidad y garantía de la calidad.

Interferón gamma recombinante en el tratamiento de enfermedades pulmonares y causadas por micobacterias.

**Protein hydrolysates from the alga Chlorella vulgaris 87/1 with potentialities in immunonutrition**

Chlorella vulgaris (Chlorophyta, Chlorophyceae) has received a particular attention in the programmes of microalgae utilisation in biotechnology. Enzymatic hydrolysis of cell proteins represents a very promising method to increase protein digestibility and thus, for obtaining hydrolysates with improved nutritional and functional properties. However, this technology has been little approached and the biological evaluation of hydrolysates has had a strictly nutritional nature. The design of hydrolysis conditions that combined for the first time, the use of C.vulgaris 87/1 treated with ethanol and pancreatin at pH values of 7.5-8.0, led to a product with a degree of hydrolysis of 20-22% and yields of 50-55%, characterised by a high digestibility (97.2%) and nitrogen solubility over a wide pH range (2.0- 10.0). Hydrolysis curves were fitted to an exponential model, common to many food proteins. The bulk of the product dry matter consists of soluble peptides and free amino acids (47.7%) with three main peptides of molecular masses between 2 and 5 kDa. The oral administration of Chlorella hydrolysate (500 mg/kg) to undernourished Balb/c mice provided benefits in terms of liver protein metabolism and the induction of anabolic processes in gut mucosa. The hydrolysate also enhanced the immunological recovery, as judged by the stimulation of haemopoiesis, monocyte-macrophage system activation, as well as humoral and cell mediated immune functions, like T-dependent antibody response and the reconstitution of delayed-type hypersensitivity (DTH) response. These results represent the first findings in the world concerning the immunomodulating effects of a microalgae protein hydrolysate.

**Physiological study in Saccharomyces cerevisiae for overproduction of a homogeneous human epidermal growth factor molecule**

The epidermal growth factor (EGF) is a molecule of potent mitogenic activity that accelerates the healing of wounds and ulcers of different tissues in the human body. Over the last years, some institutions of the Havana Western Scientific Pole in Cuba have been developing novel therapeutic products and projects using the human EGF (hEGF) like Heberprot-P® (CIGB) and Cimavax EGF® (CIM). Currently, the growing demand for this molecule has led to strategies being focused on a significant increase in the production capacity of the hEGF. The aim of this work was to increase productivity in the hEGF fermentation process by a combined study of physiological variables, cultivation media and bioreactor operation mode. The influence of culture medium components and the operation during fermentation on the homogeneous production of hEGF and the producer strain growth characteristics were evaluated. A 3.5-fold increase in EGF productivity was obtained by using high cell density fed-batch fermentation process. This fermentation process allowed reaching a cell concentration of 241.6 ± 18.8 g/L (wet mass) and hEGF production of 259.2 ± 37.2 mg/L. This is the first report of hEGF fermentation process expressed in yeasts with productivity of 4.82 ± 0.57 mg/L·h. As part of the analysis, we also discuss the impact of maintaining the highest hEGF expression on productivity in the fermentation process.

Engineering drought and salt tolerance in plants using SodERF3, a novel sugarcane ethylene responsive factor

The ability of plants to tolerate salt and drought conditions is crucial for agricultural production worldwide. The increased understanding of the regulatory networks controlling drought stress response has led to practical approaches for engineering salt and drought tolerance in plants. By a single-pass sequencing of randomly selected clones from a λ ZAP-cDNA library generated from ethephon-treated young sugarcane leaves, we identified an expressed sequence tag encoding a putative protein with a DNA-binding domain that is typically found in EREBP/ AP2-type transcription factors. The full-length cDNA clone, named SodERF3 (EMBL accession number AM493723) was further isolated from the excised library. SodERF3 encodes a 240 amino acid DNA-binding protein that acts as a transcriptional regulator of the Ethylene Responsive Factor (ERF) superfamily, but also contains a C-terminal short hydrophobic region resembling an ERF-associated amphiphilic repression-like motif, typical for class II ERFs. This protein binds to the GGC box, and its deduced amino acid sequence contains an N-terminal putative nuclear localization signal. SodERF3 is induced in sugar cane leaves by ethylene, abscisic acid, salt stress and wounding as judged by Northern and Western blots assays. Greenhouse grown transgenic tobacco plants (Nicotiana tabacum L. cv. SR1) expressing SodERF3 were found to display increased tolerance to drought and osmotic stress without any visible phenotypic change in growth and development. According to our results SodERF3 will be a valuable tool to assist the manipulation of plants to improve their stress tolerance.