Biotecnología Aplicada

A therapeutic perspective of the immunological function of the liver

Therapeutic vaccination of chronic infectious diseases has been extensively explored because of its possible contribution to their eradication. In particular, therapeutic vaccination of hepatitis B virus chronic infections is especially interesting since this disease is characterized by a sustained necro-inflammatory process of the liver that may evolve into more severe conditions including cirrhosis and hepatocellular carcinoma. The basic role of the immune system in the healing process of this chronic infection suggests that it offers a favorable setting for immunotherapeutic treatments, either spontaneously or as a result of antiviral therapy. However, no vaccine has been able to cure this or any other chronic infection in spite of the large number of vaccine candidates tested. The knowledge of the liver as a lymphoid organ and the limited advances of therapeutic vaccination demand more thorough analyses within the rationale of current vaccine candidates. In the last ten years there has been an increased knowledge of innate immunity and intra- and extra-hepatic signaling mechanisms, to support a rational design of vaccine strategies. The high costs and low effectiveness of conventional treatments, and the large amount of chronic carrier patients for this virus, indicate a favorable setting for the development of immunotherapeutic products against chronic hepatitis B. It is possible to predict that adjuvant strategies that take into account the properties of the liver as a lymphoid organ would have an impact in the development of this new field of therapeutic vaccines.

La vacunación terapéutica contra las enfermedades infecciosas crónicas se ha explorado ampliamente porque se estima que podría contribuir en mucho a combatirlas. Resulta interesante la vacunación contra la infección crónica por el virus de la hepatitis B, enfermedad que se caracteriza por un proceso necroinflamatorio sostenido del hígado, que puede evolucionar a formas severas de la enfermedad, entre ellas la cirrosis y el carcinoma hepatocelular. La función esencial del sistema inmune en la curación de la infección crónica por ese virus, bien de modo espontáneo o como resultado de tratamientos antivirales, sugiere que es un escenario propicio para el tratamiento inmunoterapéutico. Sin embargo, aún no hay una vacuna que cure esta u otra infección crónica; aunque sí se ha ensayado un grupo elevado de candidatos vacunales. El conocimiento acerca del funcionamiento del hígado como órgano linfoide, y los limitados avances en la vacunación terapéutica, obligan a revisar la racionalidad de los candidatos vacunales actuales. En los últimos diez años ha habido un desarrollo impetuoso del conocimiento de la inmunidad innata y los mecanismos de señalización intra y extrahepáticos, que permiten un diseño racional de las estrategias vacunales. Los precios elevados, la baja efectividad de los tratamientos convencionales y la gran cantidad de pacientes portadores crónicos de este virus, indican que hay un nicho oportuno para el desarrollo de un producto inmunoterapéutico contra la hepatitis B crónica. Es posible predecir que las estrategias de adyuvación que estén en línea con las características del hígado como órgano linfoide, tendrán un impacto en el desarrollo de esta rama de la vacunología.

Mechanism of clogging by exchange flow of a spinfilter as filtrating centrifuge

Perfusion cultures usually reach high cell densities and, therefore, require a spinfilter for cellular retention. But spinfilters may clog prematurely so we decided to study the process by pumping a suspension culture through the filter, so that the cells are trapped inside and the clarified flow-through can be measured by collecting it in a measuring cylinder until partial or total obstruction of the filter, which in this case is used as a filtrating centrifuge. Filtrate volume with time was measured at several cellular concentrations and rotational speeds, calculating the behavior of specific resistance for the apparent cake, the resistance of the filtration medium, cake porosity and hydrodynamic regime. The results were used to adjust the filtration profile.

El filtro rotatorio (spinfilter) se emplea para la retención celular en el cultivo en perfusión, con altas densidades celulares. Debido a su obstrucción precoz, se precisó el estudio de este sistema, introduciéndole el caldo de cultivo (suspensión) por el interior del filtro, para obtener la suspensión clarificada por el exterior de este, y que las células queden retenidas en su interior. El líquido claro se recogió en una probeta, hasta que el filtro se obstruyó parcial o totalmente. Por lo tanto, el filtro rotatorio se usó como una centrífuga filtrante, en la que se fue midiendo el volumen de filtrado con respecto al tiempo, para varias concentraciones de suspensión y de velocidad de giro del dispositivo. Se le ajustó el mecanismo de filtración que controla el proceso. Para el ajuste del perfil de filtración, se determinó la influencia de la concentración de la suspensión y de la velocidad de giro del filtro rotatorio, y se obtuvo el comportamiento de la resistencia específica (de la torta aparente), de la resistencia del medio filtrante, la porosidad de la torta y el régimen en que ocurre el proceso hidrodinámico de separación.

Gamma Interferon and prednisone decreasing-dose therapy in patients with Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disease to which no treatment has demonstrated to be a modifier of the disease pattern. The aim of this study was to obtain evidences of the efficacy and safety of a 6 month-course gamma interferon (IFNγ) treatment and prednisone decreasing-dose in patients with IPF. A pilot, open label, non-controlled clinical trial was carried out. Twelve patients with histophatologically-confirmed IPF were treated with 1 000 000 IU of recombinant human intramuscularl IFNγ, three times per week. Oral daily prednisone was concomitantly administered, once a day for a year, whose dose was gradually reduced from 60 to 20 mg. Clinical, functional, and imagenological evaluations were done before and at 3, 6 and 12 months after treatment. Most of the patients had clinical improvement; dyspnea, dry cough and crepitations were notably reduced. Forced vital capacity increased by over 12% in three patients while only one reduced such magnitude. Alterations in arterial gases were less frequent in the last evaluations. Fibrotic lesions were reduced in around half of the treated patients. Two of them died, but just one due to the disease. Considering withdrawals as failures, a 75% of patients was considered as responders (improvement + stable) at the end of IFNγ treatment (month 6), while 58.3% of response was obtained after follow-up (month 12). Treatment with IFNγ was well tolerated, since mild to moderate flu-like adverse reactions predominated. Two severe, unexpected events (deaths) occurred but no related to treatment. These results suggest that in IPF a rapid clinical response could be obtained with a therapeutic schedule with the well-tolerated IFNγ combined with prednisone decreasing-dose. Further, extensive controlled studies are encouraged.

La Fibrosis Pulmonar Idiopática (FPI) es una enfermedad letal para la cual no existen tratamientos que modifique el curso de la enfermedad. El objetivo del presente estudio fue obtener evidencias de la eficacia y seguridad de un ciclo de 6 meses de tratamiento con interferón (IFNγ) y dosis decrecientes de prednisona en pacientes con FPI. Se llevó a cabo un ensayo clínico piloto, abierto, no controlado. Doce pacientes con diagnóstico histopatológico confirmado de FPI se trataron con 1 000 000 UI de IFNγ humano recombinante por vía intramuscular, 3 veces por semana. Concomitantemente se administró prednisona oral, diaria durante un año, reduciendo gradualmente la dosis de 60 a 20 mg. Se realizaron evaluaciones clínicas, funcionales e imagenológicas antes del tratamiento y en los meses 3, 6 y 12. La mayoría de los pacientes presentó mejoría clínica; la disnea, la tos seca y las crepitaciones se redujeron notablemente. La Capacidad Vital Forzada aumentó más de 12% en 3 pacientes, mientras que uno solo redujo esta magnitud. Las alteraciones hemogasométricas fueron menos frecuentes en las últimas evaluaciones. Las lesiones fibróticas se redujeron en aproximadamente la mitad de los pacientes. Fallecieron 2 de ellos, pero solo uno a causa de la enfermedad. Al final del tratamiento con IFNγ (mes 6), el 75% de los pacientes fueron considerados respondedores (mejoría + estabilización), por intención de tratar, respuesta que se redujo a un 58.3%.posterior al seguimiento (mes 12). El tratamiento con IFNγ fue bien tolerado, ocurriendo reacciones típicas pseudogripales leves a moderadas. Los dos eventos serios inesperados (muertes) presentados no se asociaron al tratamiento. Estos resultados sugieren que en esta enfermedad puede obtenerse una respuesta clínica rápida al utilizar un tratamiento con IFNγ y dosis decrecientes de prednisona. Ello alienta la realización de estudios controlados extensos.

Preliminary modeling of the perfusion culture of mammalian cells with a spinfilter as retention device

Specific equations describing the behavior of cell growth and the filtration mechanism of a perfusion culture in stirred tank fermentors at 30 L scale were derived from basic equations for mass balance and mechanical energy in a spinfilter. These equations, when used for modeling the operation process in the Matlab package together with previously reported experimental data, yielded results similar to those of culture kinetics. The operational variables with the highest influence on the process were analyzed with the Matlab module, comparing them to a basal case using the spin rate of the spinfilter and the filtration area usually employed in the production runs as comparison parameters. Two additional comparisons were also performed, using cases with different filtration areas (smaller or higher than that of the basal case) in which the stirring rate was varied to analyze the behavior of the perfusion flow capacity during the run. The influence of the filtration area on the fermentation life was corroborated, with higher values for the latter as filtration area and spin rate of the filter increased.

Partiendo de las ecuaciones básicas del balance de masa y de energía mecánica aplicado en el spinfilter, se llegó a las ecuaciones particulares que describen el comportamiento del crecimiento celular y al mecanismo de filtración en el cultivo en perfusión en los fermentadores de tanque agitado a escala de banco (30 L). Con las ecuaciones desarrolladas, los datos experimentales reportados en trabajos anteriores y utilizando el programa Matlab, se simuló el proceso de operación y los resultados fueron semejantes a los de la cinética del cultivo. Se analizaron las variables de operación que tienen una marcada influencia durante el proceso, utilizando el módulo del Matlab. Estos se compararon con un caso base, tomando como parámetros: la velocidad de agitación del spinfilter y el área de filtración normalmente empleados en producción. También se compararon con dos casos a dos niveles de áreas de filtración (menor y mayor que la del caso base) en los que se varió la velocidad de agitación para observar el comportamiento de la capacidad de flujo de perfusión durante la corrida. Se comprobó la influencia sobre el tiempo de vida de la fermentación (de la filtración), cuyo valor ascendió para la mayor área de filtración y la mayor velocidad de giro del spinfilter.

Effects of the bacterial-fungal interaction between Tsukamurella paurometabola C 924 and Glomus fasciculatum and Glomus clarum fungi on lettuce microrrizal colonization and foliar weight

The study of fungal-bacterial interactions in soils is not only interesting from a basic point of view but has also yielded findings of societal and economical relevance, such as in the application of biological controls of plant diseases. This study evaluated the effect of Tsukamurella paurometabola C 924, a bacterium with nematocidal action isolated from banana rhizosphere, as single inoculant or combined with arbuscular mycorrhizal fungi (AMF) Glomus fasciculatum and Glomus clarum in lettuce (Lactuca sativa L.). Controls included non-bacteria non-AMF, and each AMF species alone. Five replicates were used. AMF did not show any influence, neither in T. paurometabola C 924 c.f.u. counts in soil, nor over its phenotypic nematocidal characters. On the other hand, the bacterium stimulated AMF colonization for both fungi species as well as an early infection. Combined inoculation improved significantly fresh weight of plants as compared with the microorganisms separately or the non-inoculated control.

El estudio de las interacciones entre bacterias y hongos en suelos no solo es interesante desde el punto de vista científico, también tiene relevancia económica y social, como en la aplicación de controles biológicos a enfermedades de plantas. Este trabajo evalúa el efecto de Tsukamurella paurometabola C 924, bacteria con actividad nematicida aislada de la rizosfera de banano, como inoculante único o combinado con los hongos micorrízicos Glomus fasciculatum y G. clarum en lechuga (Lactuca sativa L.). Se incluyeron controles sin inoculantes microbianos, así como cada especie de hongo micorrízico por separado. Se emplearon cinco réplicas por tratamiento. Los hongos micorrízicos no mostraron influencia sobre las concentraciones de T. paurometabola C 924 en suelos, ni sobre sus caracteres fenotípicos nematicidas. Por otra parte, la bacteria estimuló la colonización fúngica del sistema radicular de las plantas con ambas especies de hongos, así como la colonización temprana de las raíces. La inoculación conjunta de la bacteria y los hongos incrementó de forma significativa el peso fresco de las plantas al compararlos con las plantas inoculadas con cada microorganismo de forma separada o con el control sin inocular.

Emerging oral therapies for Multiple Sclerosis. Report of the 25th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS)

Report of the Fifth Nimotuzumab Global Meeting

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

New evidence of the contribution of apoptosis to dengue hemorrhagic fever pathophysiology

Dengue is considered the most important arthropod-borne viral disease in humans and is caused by any of the four dengue virus (DENV-1-4) serotypes. DENV may cause the potentially fatal disease named dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Although apoptosis has been implicated in DHF/DSS pathogenesis, the in vivo mechanisms have not been largely explored yet. In this study, formalin fixed and paraffin embedded tissues of eight fatal DHF/DSS cases from two Cuban dengue epidemics were examined, in 1997 by DENV-2 and 2001 by DENV-3. We detected DENV antigens by an immunohistochemistry assay and apoptotic cells by the TUNEL, (Terminal deoxynucleotydil Transferase-mediated dUTP nick-end labelling) technique in sections from different organs. Apoptosis was demonstrated in six out of the eight studied fatal cases. Apoptotic cells were observed in brain, intestine, liver and lung. This is the first report in literature demonstrating apoptosis in white blood cells, brain cells, and endothelial cells from the intestinal and pulmonary microvasculature from DENV infected individuals. Interestingly, it is likely that apoptotic microvascular endothelial cells were associated to plasma leakage manifested by the studied subjects. These results suggest that apoptosis may contribute to DHF/DSS pathophysiology. More studies are necessary to clarify the apoptotic cell death implications in DHF/DSS pathogenesis.

Neurochemical changes in the pedunculopontine nucleus of hemiparkinsonian rats and effect of different treatments

The present work focuses on the physiopathology of Parkinson’s disease (PD), analyzing the alterations in neurotransmitter profile of the basal ganglia triggered by the degeneration of cells from the substantia nigra compacta (SNpc). For this purpose, the neurochemical, morphological and molecular changes of the pedunculopontine nucleus (PPN) of rats upon the induction of hemiparkinsonism were evaluated with surgical, immunochemical and molecular biology techniques together with cerebral microdialysis and behavioral tests, also examining the effect of systemic treatments with MK-801 or (-) nicotine or the results of subthalamic injury on these changes. The results evidenced an increase in the extracellular concentration (EC) of glutamate (Glu) (p < 0.001) and GABA (p < 0.001) as well as a higher density of muscarinic receptors, together with a statistically significant decrease in the density of BDZ gabaergic receptors (p < 0.001) and mu opioid receptors (p < 0.01) in the PPN of hemiparkinsonian rats. Our results also constitute the first published description of cell death in the PPN of hemiparkinsonian rats. All the treatments achieved a statistically significant decrease in the Glu (p < 0.01) and GABA (p < 0.001) EC in the PPN, with a neuroprotective effect on the dopaminergic cells of the ventral tegmental area and the SNpc itself. The administration of (-) nicotine improved the striatal expression of brain-derived neurotrophic factor (p < 0.01). We conclude that the PPN of hemiparkinsonian rats displays neurochemical changes which can be modified and/or reverted by the treatments described in this work. Our results had the added benefit of requiring the local manufacture of cerebral microdialysis cannulae, resulting in significant cost savings.