Biotecnología Aplicada

Human neutrophil elastase and lung surfactant in acute respiratory distress syndrome

Human Neutrophil Elastase (HNE) is one of the main proteases secreted into the alveolar space by infiltrated neutrophils during several inflammatory lung diseases such as cystic fibrosis, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Consequently, a number of therapeutic approaches based on the specific inhibition of HNE are currently under investigation. The present work reviews the physiopathological role of HNE in ALI/ARDS and its relationship to the pulmonary surfactant system, as well as the clinical potential of protease inhibitors in this setting. In spite of the complex physiopathology of these diseases, the available evidence points to a direct link between HNE and ALI/ARDS, with increased local concentrations of this protease in animal models of ALI as well as in patients. Furthermore, the unbalanced ratio of protease/endogenous inhibitors characteristic of these disorders has led to the pharmacological and clinical evaluation of HNE inhibitors, examining their addition to currently available exogenous surfactant with promising results.

En el desarrollo de algunas enfermedades inflamatorias pulmonares, tales como la fibrosis cística, el daño agudo del pulmón (ALI, del inglés Acute Lung Injury) y el síndrome de distrés respiratorio agudo (SDRA), se liberan numerosas proteasas a partir de neutrófilos infiltrados en el espacio alveolar; entre ellas se distingue la elastasa de neutrófilos humana (ENH). Con el objetivo de inhibir la actividad elastolítica, se han desarrollado estrategias terapéuticas. Este artículo revisa algunas de las funciones fisiopatológicas de la ENH en el ALI/SDRA, y su relación con el sistema surfactante pulmonar, así como el potencial terapéutico de los inhibidores de proteasas. Los resultados postulan que, aunque la fisiopatología de estas enfermedades es compleja, muchos ensayos demuestran una relación directa entre la ENH y el ALI/SDRA: se ha observado un incremento de la concentración de esta proteasa en modelos animales con ALI, así como en pacientes. Al mismo tiempo, debido al desequilibrio proteasa/inhibidores endógenos, se ha efectuado la evaluación farmacológica y se investiga la aplicación clínica de inhibidores de la ENH, así como su posible asociación con las preparaciones de surfactante pulmonar exógeno, y se tienen resultados prometedores.

Micro-magnetic and molecular magnetic resonance imaging in modern biotechnology and pharmacy

Magnetic Resonance Imaging is an essential diagnostic technique for the non-invasive study of biological entities with high contrast as well as high spatial and temporal resolution. This work describes the current state of the art in Micro-Magnetic Resonance Imaging and Molecular Magnetic Resonance Imaging. These new technologies are transforming the modus operandi of the current research and applications being performed and developed in the biotechnological and medical-pharmaceutical industries; establishing metabolic and genotypic relationships to phenotypical traits and validating the design of active molecules, their structure-function relationship and their interaction with the intended target. The use of endogenous and exogenous contrast mechanisms is also described. Therefore, underlining the importance, a special emphasis is being placed on these methods capable of detecting, quantifying and visualizing dynamic molecular and cellular phenomena in tissues and organs at spatial resolutions of the order of dozens of microns and temporal resolutions of milliseconds. Examples of the application of these methods in the study of pathologies like cancer and brain stroke are shown. The development of recent advances in hardware, devices and contrast mechanisms is referred to increase productivity, specificity, reliability and impact in experimental work. In the near future, the study of these techniques will be an indispensable tool for the design, development and validation of new pharmaceuticals.

Las imágenes de resonancia magnética son esenciales para estudiar de modo no invasivo la naturaleza biológica, con elevado contraste y alta resolución temporal y espacial. Se describe el estado actual del arte de las microimágenes y de las imágenes moleculares de resonancia magnética, nuevas ideas transformadoras de las investigaciones y aplicaciones en la biotecnología y la industria medico-farmacéutica que constituyen una herra-mienta efectiva para el establecimiento de la relación metabólica y genotípica con la expresión fenotípica y para comprobar diseños de moléculas activas así como su relación estructura-función e interacción con blancos terapéuticos. Se expone el uso de mecanismos contrastantes endógenos y exógenos. Se destaca la importancia de estos métodos que detectan, cuantifican y visualizan fenómenos dinámicos moleculares y celulares en tejidos y órganos, con resoluciones espaciales de hasta decenas de micras y temporales, de milisegundos. Se detalla su utilización para el estudio de afecciones como la isquemia cerebral y el cáncer. Se hace referencia a los avances obtenidos en los equipos, dispositivos, metodologías y mecanismos de contraste para elevar la productividad, especificidad, fiabilidad e impacto de las investigaciones. En breve tiempo, los estudios por imágenes serán ineludibles para el diseño, desarrollo y validación de nuevas drogas.

A Pichia pastoris fermentation process to express Mucor pusillus rennin

Rennin from Mucor pusillus (MPR) is an efficient substitute of bovine rennin used as milk clotting enzyme for cheese manufacture. A new fermentation process in the laboratory scale was developed to produce MPR by means of the strain RP61 Muts from Pichia pastoris containing multiple copies of the gene for MPR expression. This process includes three phases, the first one in batch mode for growth on glycerol with MYPG medium supplemented with 10 g/L peptone and 20 g/L yeast extract, the second one a fed-batch phase with glycerol; and the last one with methanol for protein expression. Growth, yield of MPR and milk clotting activity were used to define the best conditions achieved with a methanol feed flow of 4.5 g/L·h, pH of 5.5 at 28 °C. As a result, the yield obtained for MPR was 0.95 g/L at 112 hours of fermentation with milk clotting activity of 565 IU/mL. On the other hand, the volumetric productivity obtained for rennin production was 8.5 mg/L·h, which means 1.7 times higher than those obtained in other works before.

La enzima renina de Mucor pusillus (MPR), constituye un sustituto eficiente de la renina bovina que se usa como coagulante de la leche en la fabricación de quesos. En este trabajo se desarrolló un nuevo proceso de fermentación en la escala de laboratorio para producir MPR por medio de la cepa Pichia pastoris RP61 Muts que contiene copias múltiples del casette de expresión con el gen MPR. El proceso incluye tres fases, una de crecimiento en glicerol en modo discontinuo con el medio MYPG suplementado con 10 g/L de peptona y 20 g/L de extracto de levadura, otra en modo incrementado con glicerol y una última fase de incremento con metanol para la expresión de la proteína. Las mejores condiciones para el crecimiento, el rendimiento de MPR y la actividad coagulante en leche se lograron con un flujo de alimentación de metanol de 4.5 g/L·h, un pH de 5.5 y 28 ºC. El rendimiento final obtenido de MPR fue 0.95 g/L en 112 horas de fermentación con una actividad coagulante en leche de 565 IU/mL. La productividad volumétrica alcanzada para la producción de renina fue de 8.5 mg/L·h, 1.7 veces mayor que la obtenida en trabajos anteriores.

A barley cystatin stably expressed in rice exhibits strong in vitro inhibitory activity against gut proteinases of rice water weevil

Rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), is one of the most important rice pests in Cuba. Here, we describe the production of transgenic rice plants expressing barley cystatin HvCPI-1 (Icy1 gene) to explore the potential of this protein for the control of rice water weevil. Rice plants (Oryza sativa L. cv IACuba-28) were transformed via Agrobacterium tumefaciens with a plasmid carrying the Icy1 gene fused to the 35S promoter and the first exon/intron/exon from rice actin-1 gene. From 65 independent transgenic lines, 62 were positive in the PCR-Southern blot analyses. The transgene was correctly translated as indicated by western- and dot-blot assays with level of expression in T¹ plants of up to 2% of the total extracted protein. The functional integrity of the protein was confirmed in vitro by a reduction of up to 90% of the cysteine-proteinase activity in the gut of rice water weevils exposed to rice leaf extracts. Moreover, proteins extracted from T² transgenic rice roots showed a significant inhibition of up to 70% at pH 4.5 and 45% at pH 6.0 of the cathepsin B-like activity in the L. brevirostris larvae gut. These results demonstrate the potential of barley cystatin as an effective compound that may be combined with other pest control methods as an alternative in the struggle against insect resistance.

El picudo acuático del arroz, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), es una de las principales plagas del cultivo en Cuba. En este trabajo describimos la obtención de plantas transgénicas de arroz que expresan la cistatina de cebada HvCPI-1 (gene Icy1) como un modelo para explorar la potencialidad de esta proteína en el control del picudo acuático. Se transformó arroz (Oryza sativa L. cv IACuba-28) vía Agrobacterium tumefaciens conteniendo un plásmido que porta el gen Icy1 fusionado al promotor 35S del CaMV y al primer exón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas T1. La integridad funcional fue confirmada por la reducción hasta un 90% de la actividad cisteino proteinasa en el extracto digestivo de larvas del picudo acuático por extractos de hojas de arroz. Además, extractos de raices de plantas transgénicas de generación T2 produjeron una inhibición significativa, 70% a pH 4.5 y 45% a pH 6.0, de la actividad tipo catepsina B en extractos del intestino de larvas de L. brevirostris. Estos resultados demuestran el potencial de la cistatina de cebada como un efectivo componente para ser usado en combinación con otras estrategias para el control de esta plaga como una alternativa contra el desarrollo de insecto resistencia

Influence of albumin content and freeze-drying on the immunogenicity of human recombinant Interferon alpha-2b in mice

Antibodies against interferon (IFN) could explain the lack of response to treatment or the development of autoimmune processes. The albumin content and the freeze-drying process of IFN alpha formulations could be involved in the generation of such antibodies in treated patients. To study this, the immunogenicity of four formulations of human recombinant IFN alfa-2b was compared in mice. One-hundred sixty Balb/c mice were randomly and evenly distributed among the following four formulations: Heberon Alpha R, lyophilized containing 1.5 mg albumin per 3 x 106 IU of IFN, lyophilized albumin-free, or liquid albumin-free, and Reaferon-ES, lyophilized with 5 mg albumin per 3 x 106 IU of IFN. They were immunized subcutaneously with 100 000 IU of IFN alpha-2b three times a week for four weeks. Mice immunized with Reaferon-ES produced the highest serum titers of IFN alpha-2b antibodies by ELISA in the last 2 weeks, while the Heberon Alpha R liquid albumin-free formulation was the least immunogenic. Intermediate titers were obtained with the Heberon Alpha R lyophilized formulations, although they were larger for the formulation with albumin. We can conclude that albumin content and the freeze-drying process seem to have an influence on the formation of antibodies in mice.

La formación de anticuerpos contra el interferón (IFN) puede ser causa de no respuesta al tratamiento o el desen-cadenamiento de procesos autoinmunes. El contenido de albúmina y el proceso de liofilización de las formulaciones de IFN alfa pueden ser factores involucrados en la generación de tales anticuerpos. Para investigar lo anterior se comparó la inmunogenicidad de cuatro formulaciones de IFN alfa-2b humano recombinante en ratones. Un total de 160 ratones Balb/c se distribuyeron al azar y uniformemente para recibir una de las siguientes cuatro formulaciones: Heberon Alfa R liofilizado conteniendo 1.5 mg de albúmina por 3 x 106 UI de IFN, liofilizado sin albúmina o líquido sin albúmina, y Reaferon-ES, liofilizado que contiene 5 mg de albúmina por 3 x 106 UI de IFN. Ellos se inmunizaron subcutáneamente con 100 000 UI de IFN alfa-2b tres veces por semana durante cuatro semanas. Los ratones inmunizados con Reaferon-ES produjeron los títulos de anticuerpos contra IFN alfa-2b más elevados por ELISA en las últimas 2 semanas, mientras que el Heberon Alfa R líquido sin albúmina resultó ser la formulación me-nos inmunogénica. Con las formulaciones liofilizadas de Heberon Alfa R se obtuvieron títulos intermedios, aunque superiores para la que contiene albúmina. Podemos concluir que el contenido de albúmina y el proceso de liofili-zación parecen tener influencia sobre la formación de anticuerpos en ratones.

Comparative immunogenicity and protective capacity of two Dengue-4 vaccine candidates based on P64k-envelope domain III

In this work we compared, in the same immunization schedule in mice, two vaccine candidates against Dengue-4 based on the envelope protein domain III and the carrier P64k. Their molecular and antigenic features, immunogenicity and protective capacity in the viral encephalitis murine model are assessed. Our study endorses the strategy of using both sites of inclusion within the same P64k molecule, in order to enhance the immunogenicity of protein fragments.

En este trabajo comparamos en el mismo esquema de inmunización en ratones, dos candidatos vacunales contra el virus Dengue-4 basados en el dominio III de la proteína de la envoltura y la proteína portadora P64k. Además caracterizamos antigénicamente y molecularmente ambas variantes proteicas y evaluamos la inmunogenicidad y capacidad protectora en el modelo murino de encefalitis viral. Este estudio avala la estrategia de emplear a la misma vez, dos sitios de inclusión en la P64k como proteína portadora, con el objetivo de potenciar la inmunogenicidad de fragmentos proteicos heterólogos.

Anhydrobiotic cells of the nematocidic agent Tsukamurella paurometabola C-924

Biological control of nematodes is a valuable alternative to the use of chemical nematicides in agriculture, because of the high negative impact generated by such chemicals in agroecosystems. From the Gram positive bacterium Tsukamurella paurometabola C-924, the bionematicide HeberNem-L™ has been developed, which is presented as a liquid formulation; nevertheless its stability is still low at 4 °C, with a shelf-life time of 6 months. One way to improve the stability of this bioproduct could be the desiccation of the cells, keeping the viability upon rehydration. Considering this aspect, the aim of our work was to obtain anhydrobiotic cells of the strain C-924 using freeze-drying and spray-drying technologies. One of the main results was the obtaining of anhydrobiotic cells of T. paurometabola C-924, with survival rates higher than 60%. In addition, it was shown that anhydrobiotic cells are more stable vacuum-stored at 4 °C, the rehydrated cells having nematicidal activity in field trials; therefore the powder formulation constitutes a good bionematocide candidate for agricultural use. Furthermore, a new methodology and also a mathematical model were developed to evaluate and predict the stability of desiccated bacterial cells.

HER1 Vaccine: An autologous EGFR vaccine candidate to treat epithelial tumors

The Epidermal Growth Factor Receptor (EGFR) plays an essential role in regulating neoplastic processes. EGFR over-expression in many human epithelial tumors has been correlated with disease progression and bad prognosis. Passive EGFR-directed immunotherapy has been introduced in medical oncology practice, but no active specific approaches have been used. We designed a vaccine candidate based on the extracellular domain (ECD) of human EGFR (HER1), and the homologous vaccine for mice based on murine EGFR. This vaccine candidate uses the Very Small-Sized Proteoliposomes from Neisseria meningitidis (VSSP) and Montanide ISA 51-VG as adjuvants. The autologous vaccine circumvents the tolerance to self EGFR by inducing a specific immune response with an anti-metastatic effect on EGFR+ tumors. The vaccine candidate based on HER1-ECD induced anti-EGFR polyclonal antibodies (PAb) that bind EGFR+ human tumor cell lines from different tissues. These anti-EGFR PAb abrogate ligand-dependent EGFR phosphorylation, provoking the inhibition of tumor cell growth and apoptosis. Preclinical results further encouraged the evaluation of the HER1 vaccine candidate in phase I clinical trials

CIMAvax-EGF: A novel therapeutic vaccine for advanced lung cancer

The results allowing the Cuban Regulatory Agency (CECMED) to grant the Sanitary Registration to the CIMAvax-EGF cancer vaccine for advanced non-small cell lung cancer (NSCLC) are shown. This was the first registration of a therapeutic vaccine in Cuba and also the first registration of a lung cancer vaccine in the world. Hence, a unique therapeutic vaccine is offered to lung cancer patients, which will increase survival and their quality of life. For this purpose, significant preclinical, clinical, regulatory, productive and negotiation challenges were to be faced. The results obtained in these fields led to 18 scientific papers published in high impact journals and 4 invention objects, generating several patents in Cuba and other countries. In the pre-clinical setting, immunogenicity, safety and anti-tumoral effects were demonstrated in different animal species. The clinical experience began in 1995. Up to now, five phase I-II clinical trials have concluded in Cuba, two phase II have also concluded, one in Cuba and another one in Canada and the UK, and a phase II-III trial with an optimized schedule as well as a phase III trial are currently in progress in Cuba. In the regulatory field, a fast-track registration strategy was designed and performed. It required novel regulatory conceptions to develop this unique product. A scalable, reproducible and controlled productive process was carried out, together with a quality system that ensured full GMP compliance. Funds for product development came from implementing a novel negotiation strategy: negotiation of intangibles.

Association between enterovirus infection and type 1 diabetes: new findings from Cuban studies

Enteroviruses have been examined for their possible role in the etiology of type 1 diabetes mellitus (T1DM) for nearly 40 years; however, the evidence remains inconclusive so far. This article provides new findings from Cuban studies suggesting the involvement of enteroviruses, either with the clinical T1DM onset or the pre-clinical stages of the β- cell autoimmunity in populations with low incidence of diabetes and high enteroviral circulation. Potential mechanisms of pathogenesis such as direct β- cell lysis and molecular mimicry are also discussed.