Constant light suppresses production of Met-enkephalin-containing peptides in cultured splenic macrophages and impairs primary immune response in rats.

The light-dark cycle is an environmental factor that influences immune physiology, and so, variations of the photoperiod length result in altered immune responsivity. Macrophage physiology comprises a spectrum of functions that goes from host defense to immune down-regulation, in addition to their homeostatic activities. Macrophages also play a key role in the transition from innate to adaptive immune responses. Met-enkephalin (MEnk) has been recognized as a modulator of macrophage physiology acting in an autocrine or paracrine fashion to influence macrophage activation, phenotype polarization and production of cytokines that would enhance lymphocyte activation at early stages of an immune response. Previously it was shown that splenic MEnk tissue content is reduced in rats exposed to constant light. In this work, we explored whether production of Met-enkephalin-containing peptides (MECPs) in cultured splenic macrophages is affected by exposure of rats to a constant light regime. In addition, we explored whether primary immune response was impaired under this condition. We found that in rats, 15 days in constant light was sufficient to disrupt their general activity rhythm. Splenic MEnk content oscillations and levels were also blunted throughout a 24-h period in animals subjected to constant light. In agreement, de novo synthesis of MECPs evaluated through incorporation of (35)S-methionine was reduced in splenic macrophages from rats exposed to constant light. Moreover, MECPs immunocytochemistry showed a decrease in the intracellular content and lack of granule-like deposits in this condition. Furthermore, we found that primary T-dependent antibody response was compromised in rats exposed to constant light. In those animals, pharmacologic treatment with MEnk increased IFN-γ-secreting cells. Also, IL-2 secretion from antigen-stimulated splenocytes was reduced after incubation with naloxone, suggesting that immune-derived opioid peptides and stimulation of opioid receptors are involved in this process. Thus, the immune impairment observed from early stages of the response in constant light-subjected rats, could be associated with reduced production of macrophage-derived enkephalins, leading to a sub-optimal interaction between macrophages and lymphocytes in the spleen and the subsequent deficiency in antibody production.

La melatonina induce la síntesis y liberación de las encefalinas en el cerebro de la rata / Melatonin induces enkephalins synthesis and release in the rat brain

Since enkephalins discovery in 1975, several opioid peptides have been included in neuroscience research. Enkephalins have been involved in the homeostasis maintenance of the organism, mostly with cellular and molecular mechanisms implicated in antinociception and narcotic responses. Moreover, enkephalins have been shown to be involved in the control of stress, regulation of cardiovascular functions, modulating primary immune responses, in addition to cellular differentiation processes. As opioid peptides appear to modulate several bioactivities and physiological responses in organisms, this posits that several modifications should occur during their synthesis, cell release, and receptor binding in target cells. At present, it has been demonstrated that the endogenous opioid system (EOS), displays a circadian rhythm, in which its tissue content, presynaptic release, and receptor's number reaches its maximal concentration during the dark phase (24:00h) and the minimal during the early morning (05:00 h). Recently, our group reported that functional pinealectomy disrupts the enkephalin circadian rhythm and significantly reduces the tissue content of opioid peptides in the rat brain. However, the effect was shown to be specific to the hour along the 24h daytime. There were no significant changes during the light period, only during the dark period (01:00h), when the enkephalin tissue content decreased in the experimental group. The effect was reverted when pinealectomized rats were injected with single doses of melatonin (MEL) (150µg/kg i.p.). If the lack of melatonin in the rat brain significantly reduced the enkephalin tissue content, and its exogenous administration re-established the enkephalin tissue levels, it is possible that the hormone is involved in the enkephalin synthesis. In this paper we provide further evidence that supports the relation between melatonin and opioid peptides synthesis and release. In addition, we studied the effect of darkness and melatonin administration in enkephalin tissue levels. Finally, we analyzed the luzindole effect as a melatonin receptor antagonist in the pinealectomized rat brain. Material and methods Subjects: Male Wistar rats were housed in a light and temperature controlled room. Water and pellet food were available ad libitum. This group was subdivided in: 1. Functional pinealectomy group (FP). Rats were housed individually during 15 days in a room with continuous light (<50lux). 2. FP rats were housed in a dark room during four or six hours. 3. FP rats were injected with melatonin (150, 300, 600µg/kg s.c.). 4. FP rats were injected with Luzindole (187.5, 375, 750µg/kg i.p.). After 30 min, the animals were injected with melatonin (150µg/kg). 5. FP rats were injected with melatonin (800µg/ kg) and subjected to the in vitro release processes. The rats were sacrificed by decapitation and the blood collected for melatonin serum determination. The brains were removed and processed for an analytical preparative procedure for the enkephalin determination by radioimmunoassay technique. The in vitro release methodology was performed as follows: tissue samples were homogenized by applying 8 strokes with a Thomas grinder system. The homogenates were centrifuged at 4,000rpm, 4°C during 10 min. Supernatants were recovered and centrifuged at 12,000rpm at 4°C for 20 min. Supernatants were discarded and pellets were resuspended in the homogeneization buffer (1:9 w/v). Samples were placed on top of a Percoll gradient density (23%, 15%, and 10%) and centrifuged at 20,000 rpm at 4°C for 25min. The synaptosomal enriched fraction (15-23%) was obtained and diluted in 1mL of Krebs buffer (mM: NaCl 119, KCl 4.6, CaCl2·2H2O 1.25, KH2PO4 0.85 MgSO4 0.84, NaHCO3 24.8, sucrose 10). Buffer was gasified with a mixture of C0(2) 95% and O2 5%, pH 7.4. 800µL aliquots were placed into plastic chambers. After 20 min of stabilization with Krebs buffer, three different superfusates were collected: 1. basal, 2. potassium [50mM], and 3. post-stimulus (Krebs buffer without potassium). Samples were collected into HCl 0.1N, boiled and subsequently loaded into Amberlite XAD-2 columns (8 × 0.7cm) for solid-phase peptide extraction. The flow rate was held constant at 0.5 mL min-1 and elution of the whole peptide fraction was carried out using a continuous gradient with absolute methanol. Eluted samples were lyophilized and resuspended in 2mL of distilled water and finally stored at -20°C for further quantification of IR-Enkephalin using standard radioimmunoassay procedures. The results showed that functional pinealectomy reduced the opioid tissue content in the different brain structures assayed. The lack of melatonin significantly decreased the enkephalin tissue content when compared to the control group. However, tissue levels of enkephalin material were completely restored after four and six hours of administration of different doses of exogenous melatonin administration to the rats. As continuous light decreases the melatonin content in the brain, darkness should be able to counteract the aforementioned effect. Our results showed that tissue levels of enkephalin material were increased over 200% and 300%, after exposing animals to a four or six-hour period of darkness, when compared to animals exposed to continuous light. Luzindole was used to abolish any melatonin activity via activation of its membrane brain receptors. Our experiments showed that different doses of the antagonist were not able to obliterate the increased content of opioid peptides induced with melatonin administration in the tested brain tissues.

En la actualidad se reconoce que el Sistema Endógeno Opioide (SEO) participa en la regulación y control de la homeostasis; por lo tanto se requiere que los procesos bioquímicos que dan lugar a su síntesis, liberación y unión a receptores se encuentren reguladas de manera endocrina. Diversas líneas de investigación han demostrado que la concentración y liberación presináptica de las encefalinas no permanece constante durante un ciclo de 24 horas. Por el contrario, su síntesis y liberación alcanzan su máxima concentración durante la fase de oscuridad y la mínima durante las primeras horas de la mañana. Recientemente, nuestro grupo de trabajo ha demostrado que la ausencia de melatonina (MEL), por efecto de la pinealectomía funcional, rompe el ritmo circádico y reduce significativamente la concentración de las encefalinas durante la fase de oscuridad. Si la ausencia de la hormona abate la concentración de opioides, es factible que la MEL se encuentre involucrada en la síntesis de las encefalinas. En el presente trabajo se muestran los resultados del efecto de la pinealectomía funcional sobre la concentración tisular y la liberación de los opioides, así como el efecto de la administración exógena de MEL y de su antagonista el luzindol (LZ). Diseño experimental Control Naive. Se utilizaron ratas macho de la cepa Wistar (200-250g), que permanecieron en un cuarto con un ciclo de luz y oscuridad controlada. La fase de luz duró 12 horas y comenzó a las 06:00h. A su vez, este grupo fue subdividido en: 1. Luz Continua (LC): Para abatir la concentración sérica de la hormona, se usó el modelo experimental conocido como pinealectomía funcional. 2. Luz Continua + Oscuridad: La luz abate la concentración de MEL y la exposición a la oscuridad revierte dicho efecto. Un grupo de ratas sometido a la luz continua se colocó en un cuarto con oscuridad durante cuatro y seis horas. 3. Luz continua+melatonina: Para analizar el efecto de la MEL sobre el contenido tisular de opioides, se administró una dosis de MEL (150, 300 y 600µg/Kg s.c.). 4. Luz continua+luzindol (LZ)+MEL: Si la melatonina ejerce su mecanismo de acción al unirse a sus receptores presentes en la membrana plasmática, entonces su efecto podría ser revertido por la presencia del LZ. Por lo tanto, a tres grupos de ratas sometidas a la pinealectomía funcional se les inyectó con LZ a una dosis de 187.5, 375, 750 µg/kg. s.c. Después de transcurridos 30min., los animales fueron inyectados con MEL (150µg/kg. s.c.). 5. Liberación: Se utilizaron tres grupos de ratas: a) control, b) luz continua y c) luz continua+MEL (800µg/kg. s.c.). Post mortem la amígdala fue sometida al proceso preparativo para la obtención de los sinaptosomas. Resultados Los resultados obtenidos en el presente trabajo nos muestran que la pinealectomía funcional reduce significativamente la concentración de encefalinas en todas las estructuras cerebrales analizadas. Sin embargo, el efecto es revertido tanto por la administración exógena de MEL, como por la exposición a la oscuridad. Las diferentes dosis administradas del LZ bloquearon parcialmente el efecto estimulante de la MEL sobre la síntesis de los opioides. Por último, la pinealectomía funcional redujo significativamente la liberación presináptica de encefalinas, misma que se pudo reestablecer con la administración exógena de la hormona. Discusión Las evidencias experimentales del presente trabajo sugieren que la MEL se encuentra relacionada con la síntesis y la liberación de las encefalinas. Por un lado, la pinealectomía funcional redujo significativamente el contenido tisular de encefalinas, pero el efecto fue revertido tanto con la administración exógena de la hormona como con la exposición a la oscuridad. La administración del LZ sólo fue capaz de bloquear parcialmente el efecto estimulante de la MEL sobre la síntesis de encefalinas. El efecto de la MEL sobre la liberación de opioides pone de manifiesto la relación funcional entre ambos sistemas. A menor cantidad de opioides por efecto de la pinealectomía, menor liberación presináptica y por el contrario, una vez administrada la hormona los valores de la síntesis-liberación se restablecen. Conclusión La MEL puede estar involucrada en la síntesis y la liberación de los péptidos opioides.

Programa de Protección Ambiental y Bioseguridad en el Instituto Nacional de Psiquiatría Ramón de la Fuente / Environmental protection and biosecurity program at the National Institute of Psichiatry Ramón de la Fuente

Since the beginning, mankind has been subjected to various natural disasters such as floods, hurricanes, earthquakes, droughts and wildfires. Therefore, societies have to organize themselves in order to cope with nature, provide support to each other and to protect the most vulnerable individuals and their properties. Depending on their social organization, some countries have developed their response strategies in the field of civil protection faster than others. After September 19th, 1985, when an earthquake of Richter magnitude of 8.1 affected Mexico City, the Mexican government decided to establish the guidelines of the National Civil Protection System (SINAPROC). After a few years, the Environmental Protection and Ecological Equilibrium Law was published. This environmental protection regulation is specific and is directed to minimize the negative impact over the environment that could be generated through industrial activities, as well as from research centers, hospitals, and others institutions. To ensure full compliance with the Mexican regulations, in 1998 the Instituto Nacional de Psiquiatría Ramón de la Fuente (INPRF) initiated the Environmental and Civil Protection Program (ECPP). The aim of this program is to meet the legal and operational requirements applicable to an institution which has biomedical research, administration and hospitalization areas. In addition, the ECPP is in charge of giving workers training and generating specific strategies for environmental management in all activities that take place at the Institute. By establishing compliance guidelines to meet the government regulations on environmental and civil protection, the INPRF would develop strategies for minimizing risks, as well as staff training to enhance the use of institutional resources and reduce the response time to deal with emergencies. Objective Since its foundation, the environmental and civil protection program at the INPRF has had two basic objectives: 1. to attend the legal and operative requirements in a third level institution conformed by hospitalization, teaching, research and administrative areas, and 2. to offer continuous training to the internal population of a hospital in order to minimize work risks, to make an optimum use of the institutional resources and to reduce the response time of the workers in emergency situations. To develop and implement such an environmental and civil protection program, the following areas have been considered: Biohazardous wastes, saving electricity, use and optimization of drinking water, control of air emissions from stationary combustion equipment, radiation safety and emergency response procedures. The purpose of this work is to show the results achieved by ECPP after nine years since its inception. Results Biohazardous wastes (BW), by definition, are those materials generated during medical care which contain biological agents that may cause adverse effects to human health and the environment. BW are generated frequently in biomedical research areas, clinical laboratories and hospitals. In order to minimize risks and prevent accidents, INPRF implemented since 1997 an operating procedure for handling biohazardous wastes in all generating areas. Some of those strategies are directed to training medical personnel (physicians, nurses and clinical laboratory technicians) about the identification and classification of biohazardous wastes. Moreover, we have designed and distributed different kinds of written ads in order to help people to recognize and separate wastes correctly. Besides the latter actions, the INPRF hired a registered and certificated company which provides the proper recollection, treatment and final disposal of biological wastes, according to current legal ordinances. As a result of those strategies in the handling of biohazardous wastes in the Institution, the amount of wastes and their disposal costs have gradually decreased. Through staff training, personnel have learned to do a proper segregation of wastes, reducing the quantities of BW and therefore reducing risks. Energy saving. As a way to optimize the institutional resources, as well as reducing costs and creating awareness among the people, the INPRF's Energy Saving program was implemented in 2003. Since 2003, when ECPP began implementing the saving strategies, accumulated energy consumption per year had a 9.6% reduction between 2004 and 2005. Use of potable water. In the search for strategies to optimize the use of potable water that is received through the municipal network, in 2003 the INPRF-ECPP implemented measures aimed not only at reducing the per capita consumption of potable water but also to give it a proper use on the institution areas. As a result of these saving measures, there was a gradual decrease in the use of drinking water. So, from the 170 liters/person-day used in 2003, during 2006 only 98 liters/person-day were used. Radiological safety. Due to the adverse effects to human health that could result from exposure to radioactive materials, an institution that uses this kind of material in its basic biomedical research area must implement the necessary actions to minimize the risk of contamination for users and work areas. For that reason, the INPRF began the Radiological Safety Program, which had as its main objectives to minimize the risks and to prevent accidents involving radioactive materials based on regulation compliance. As a result of these strategies, the maximum value of the total dose accumulated per year of ionizing radiation received by personnel exposed who has carried out research in this institution in recent years has been of 3.14 mSv. Therefore, it had not exceeded the annual limit dose of ionizing radiation allowed for the total body by the Comisión Nacional de Seguridad Nuclear y Salvaguardas (CNSNS) of 50 mSv = 5rem. To date, there has been no accidents or contamination with radioactive materials or wastes in the INPRF. Emergency simulations. Considering the need of being prepared for emergencies and to comply with the regulation that establishes the compulsory conducting simulations in INPRF, the Internal Committee for Civil Protection (ICPC) conducts periodic training of employees on risk prevention, through courses and simulations of situations of earthquake, attempt of fire, handling of hazardous substances spills and first aid. As a result of the earthquake simulations that have been carried out at the INPRF, the time of evacuation of workers, hospitalized patients and floating population from buildings has gradually reduced. An additional advantage of conducting emergency simulations has been the early detection of systematic security features that need to be repaired or installed. Emissions to air. In recent years, increasingly strict regulation and monitoring of gaseous emissions generated by stationary and mobile sources in Mexico City has been established. In order to comply with the current regulations, it became necessary to develop a program of annual preventive and corrective maintenance of combustion equipment that is used to provide hot water, and to make periodic analysis of gaseous emissions. Among the parameters that have to be reported are: temperature of combustion gases, monoxide and dioxide carbon content, sulfur dioxide, nitrogen dioxide percentages and generated soot.

A partir de 1998, en el Instituto Nacional de Psiquiatría Ramón de la Fuente (INPRF) se iniciaron las actividades del Programa de Protección Ambiental y Civil (PPAyC). Sus objetivos son dar cumplimiento a los requisitos legales, prevenir riesgos, generar estrategias de atención a emergencias, capacitar a la población interna y mejorar el uso y aprovechamiento de los recursos institucionales. El propósito del presente trabajo es presentar los resultados obtenidos después de nueve años de trabajo del PPAyC en las áreas de manejo de residuos biológico-infecciosos, ahorro de energía eléctrica, uso y optimización de agua potable, control de emisiones a la atmósfera de los equipos fijos de combustión, seguridad radiológica y simulacros de emergencia. Residuos peligrosos biológico-infecciosos (RPBI). Con el objeto de reducir riesgos y prevenir accidentes, en el INPRF se implementó un procedimiento operativo para el manejo de los residuos peligrosos biológico-infecciosos en todas las áreas generadoras. Los resultados de las estrategias aplicadas muestran que la cantidad de los residuos y sus costos de manejo han disminuido gradualmente y se han observado mejores prácticas de manejo de residuos por parte de los trabajadores involucrados, con la consecuente reducción de riesgos. Ahorro de energía. Ante la necesidad de optimizar el uso de la energía, disminuir costos y crear conciencia entre la población, se implementó en todo el país el programa de ahorro de energía. Los resultados obtenidos muestran que, a partir de la implementación de las estrategias de ahorro en el INPRF, se obtuvo una reducción en el consumo de energía de 9.6% anual acumulado entre 2004 y 2005. Uso y aprovechamiento de agua potable. A partir de 2003, en el INPRF se implementaron medidas destinadas a disminuir el consumo per capita de agua potable que se recibe por la red municipal y optimizar su uso en las instalaciones. Como resultado se observó una disminución gradual en el uso de agua potable, de tal manera que, de los 170 litros/persona que se utilizaban en 2003, en 2006 únicamente se utilizaron 98 litros/persona. Seguridad radiológica. El programa de seguridad radiológica en el INPRF se inició con el objeto de minimizar los riesgos y efectos adversos que pudieran generar los materiales radiactivos en la salud del personal expuesto ocupacionalmente (POE), prevenir accidentes y cumplir con la normatividad. Como resultado de estas acciones, ningún POE ha excedido el máximo valor de dosis total acumulada (DTA) de radiaciones ionizantes permitido por la Comisión Nacional de Seguridad Nuclear y Salvaguardas (CNSNS) para cuerpo total. Por otro lado, a la fecha no se han registrado accidentes o contaminación con materiales o residuos radiactivos. Simulacros. En el INPRF se implementó un programa de protección civil, cuya labor principal fue la de capacitar periódicamente a los trabajadores para responder ante situaciones de emergencia. Como resultado de los simulacros de sismo que se han realizado (40 parciales y siete generales), se han reducido gradualmente los tiempos de desalojo de los inmuebles y la detección oportuna y sistemática de elementos de seguridad que requieren ser reparados o instalados. Emisiones a la atmósfera. En cumplimiento con la normatividad vigente en la Ciudad de México, en el INPRF se elaboró el programa de mantenimiento anual preventivo y correctivo de las dos calderas que dotan de agua caliente a la institución. Como resultado, se detectó la necesidad de sustituir los equipos por otros que utilizaran tecnología más limpia. Esto favoreció el cumplimiento de los límites máximos permitidos para emisiones gaseosas contaminantes y los costos de mantenimiento se redujeron. Discusión La implementación de diversas estrategias en materia de protección ambiental y civil y la capacitación reducen los riesgos del personal y protegen los bienes inmuebles y el entorno de la ciudad que habitamos.

Liberación in vitro de encefalinas durante la depresión postictal y la actividad interictal / In vitro liberation of enkephalins during postical depression and interictal activity

Los péptidos opioides han sido propuestos como agentes inhibidores de la aparición, propagación y severidad de una crisis convulsiva. Su posible participación en los procesos epilépticos implica su liberación a partir de las terminales nerviosas que las contienen. El propósito del presente trabajo fue el de estudiar en diversas estructuras del cerebro de la rata, la liberación in vitro de encefalinas durante la fase ictal, depresión postictal y actividad interictal. Los experimentos se hicieron en ratas macho de la cepa wistar, las cuales fueron sometidas al kindling químico mediante la administración, por vía intraperitoneal, de una dosis diaria de 30mg/kg de pentilenetetrazol, y fueron sacrificadas durante la fase ictal y la depresión postictal. La actividad interictal se estudió al aplicar, en forma crónica, 50 UI de penicilina-G sódica en la amígdala del lóbulo temporal. La concentración de encefalinas se midió por medio de la técnica de radioinmunoensayo. Los resultados obtenidos muestran que la liberación in vitro de encefalinas, provocadas por potasio, aumenta significativamente durante la depresión postictal y la actividad interictal. El efecto es de naturaleza selectiva en cuanto a la estructura y el péptido analizado. Estos datos apoyan la hipótesis concerniente a la participación de las encefalinas en los mecanismos que subyacen en la depresión postictal y la actividad interictal

Neurobiología de los péptidos opioides / Neurobiology of opioid peptides

En los últimos 25 años hemos asistido a la formación de nuevas e importantes líneas de investigación en el dominio de las neurociencias, producido por el estudio de los neuropéptidos, los cuales constituyen virtualmente una nueva clase de transmisores químicos. Muchos de ellos pueden funcionar como hormonas endócrinas, parácrinas y amacrínas, además de poseer la cualidad de influir en funciones como las cognocistivas y en los facores de crecimineto. La diversidad bioquímica de las células nerviosas ha revelado la coexistencia, en la misma célula, de neuropéptidos con neurotransmisores clásicos, propiedad que ofrece nuevas perspectivas en la transmisión sináptica. Posiblemente, el grupo de neuropéptidos más grande hasta ahora identificado y estudiado, cuya influencia es decisiva en la homeostasis celular, es el de los péptidos opioides. En la presente revisión hacemos una breve reseña histórica del descubrimiento de las encefalinas, y ofrecemos un panorama general y actualizado sobre los procesos implicados en el metabolismo de los opioides, su síntesis, liberación, tipos de receptores y catabolismo. Para concluir, pasamos revista a las diferentes funciones en las cuales pueden estar implicados. El conocimiento acumulado en dos décadas de estudio sobre las propiedades y funciones de los péptidos opioides aunado a las técnicas modernas en la biología molécular y de diagnóstico clínico, como la tomografía por emisión de positrones y la resonancia magnética nuclear, nos permiten avisorar un panorama prometedor de intervención terapéutica