RESUMEN
Testing hybridoma supernatants for antibodies of interest involves extensive screening, particularly when the immunogen comprises whole cells. The number of different screening procedures is often large and unmanageable and depends on whether one is interested in, for example, cell surface or intracellular binding. This paper describes an initial screening technique using whole tissue homogenate rather than the individual tissue components. The tissue is fixed to the surface of 96-well microtitre plates by microwaves using a conventional microwave oven. This technique provides a rapid and cost-effective means of screening large numbers of monoclonal antibodies.
Asunto(s)
Anticuerpos Monoclonales/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Microondas , Nervios Periféricos/inmunología , Animales , Ratones , Ratones Endogámicos BALB CRESUMEN
The murine model of influenza viral infection was used to evaluate the effects of restraint stress on pathogenesis and survival in inbred strains of mice. We recently reported that restraint stress was associated with an enhanced probability of survival in one strain of inbred mouse, DBA/2, and not in another, C57BL/6. Those studies suggested that the protective mechanism(s) of stress on mortality in the DBA/2 mice might be attributable to elevated levels of circulating glucocorticoids. Therefore, daily levels of plasma glucocorticoids were measured during influenza viral infection in both these strains. The present studies demonstrated that influenza infection itself is perceived as a stressor in both C57BL/6 and DBA/2 mice as evidenced by elevated plasma glucocorticoid levels within 48 h of infection. However, augmentation of glucocorticoid levels was not seen in the DBA/2 mice that were also subjected to restraint stress during the course of infection. Thus, corticosterone levels alone did not account for the enhanced survival seen in this group of animals.
Asunto(s)
Corticosterona/sangre , Virus de la Influenza A , Infecciones por Orthomyxoviridae/sangre , Estrés Psicológico/sangre , Animales , Anticuerpos Antivirales/sangre , Privación de Alimentos , Inmunoglobulina G/sangre , Virus de la Influenza A/inmunología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Restricción Física , Especificidad de la EspecieRESUMEN
Genetic variation in the response to stress may play a critical role in susceptibility to inflammatory diseases and development of the immune response. Experimental influenza viral infection was used to study the effects of restraint stress (RST) on pathogenesis and development of the immune response. Three inbred strains of mice (C57BL/6, DBA/2, and C3H/HeN) were infected with influenza A/PR8 and subjected to repetitive cycles of RST during development of the immune response. RST diminished cellular immune and inflammatory responses in all three strains; yet only the DBA/2 strain demonstrated RST-associated reduction in influenza viral-induced mortality.
Asunto(s)
Infecciones por Orthomyxoviridae/inmunología , Estrés Fisiológico/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Células Cultivadas , Corticosterona/sangre , Susceptibilidad a Enfermedades , Inmunoglobulina G/biosíntesis , Virus de la Influenza A/inmunología , Interleucina-2/análisis , Pulmón/microbiología , Pulmón/patología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Infecciones por Orthomyxoviridae/mortalidad , Restricción Física , Especificidad de la Especie , Estrés Fisiológico/genéticaRESUMEN
The murine model of influenza viral infection was used to evaluate the effects of restraint stress on pathogenesis and survival in the DBA/2 inbred strain of mice. Restraint stress has been associated with an enhanced probability of survival during influenza infection in this strain of mouse. Previous studies suggested that the protective mechanism(s) of stress on mortality might be attributable to elevated levels of circulating glucocorticoids. Subsequent work demonstrated that corticosterone levels alone could not account for the enhanced survival seen in the DBA/2 mice. The present studies examined the role of catecholamines in behavioral stress during influenza infection. It appears that glucocorticoids may play a primary role in trafficking and restriction of inflammation, while catecholamines may play role in limiting activation of virus-specific effector cells. The studies presented here suggest that the interplay between these two physiological response mechanisms needs to be coordinated to optimize development of the immune response to an infection.
Asunto(s)
Catecolaminas/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/fisiopatología , Estrés Fisiológico/metabolismo , Estrés Fisiológico/fisiopatología , Sistema Nervioso Simpático/metabolismo , Animales , Corticosterona/sangre , Privación de Alimentos , Ratones , Ratones Endogámicos DBA , Infecciones por Orthomyxoviridae/inmunología , Oxidopamina , Restricción Física , Estrés Fisiológico/inmunología , Simpatectomía Química , Sistema Nervioso Simpático/fisiopatología , Privación de AguaRESUMEN
Changes in sensory function including chronic pain and allodynia are common sequelae of spinal cord injury (SCI) in humans. The present study documents the extent and time course of mechanical allodynia and cold hyperalgesia after contusion SCI in the rat using stimulation with graded von Frey filaments (4.97-50.45 g force) and ice probes. Fore- and hind-paw withdrawal thresholds to plantar skin stimulation were determined in rats with a range of SCI severities (10-g weight dropped from 6.25, 12.5, or 25 mm using the MASCIS injury device); animals with 25-mm injuries most consistently showed decreased hind-paw withdrawal thresholds to touch and cold, which developed over several weeks after surgery. Stimulation of the torso with graded von Frey hairs was performed at specified locations on the back and sides from the neck to the haunch. Suprasegmental responses (orientation, vocalization, or escape) to mechanical stimulation of these sites were elicited infrequently in the laminectomy control rats and only during the first 3 weeks after surgery, whereas in 25-mm SCI rats, such responses were obtained for the entire 10 weeks of the study. These data suggest that rats with contusion SCI may exhibit sensory alterations relevant to human spinal cord injuries.
Asunto(s)
Frío , Umbral Sensorial/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Sensación Térmica/fisiología , Tacto/fisiología , Animales , Femenino , Miembro Posterior/fisiología , Locomoción/fisiología , Ratas , Ratas Long-Evans , Traumatismos de la Médula Espinal/patologíaRESUMEN
Gastric stasis is frequently seen in conjunction with critical infectious illness, chronic inflammatory disorders, radiation sickness, and carcinogenesis. These conditions are associated with elevated circulating levels of the cytokine tumor necrosis factor-alpha (TNF-alpha). The present studies examined the relationship between endogenously produced TNF-alpha and the central neural mechanisms that augment gastric motility. Systemic lipopolysaccharide (LPS) was employed to induce TNF-alpha production in thiobutabarbital-anesthetized rats. Sixty minutes after intravenous LPS injection, gastric motility could not be stimulated by a potent centrally acting gastrokinetic stimulant, thyrotropin-releasing hormone (TRH). This failure to elicit gastric motility via central mechanisms coincided with high circulating levels of TNF-alpha. However, intravenous injections of bethanecol, a peripherally acting cholinergic agonist with direct gastrokinetic effects, were still able to elicit normal increases in gastric motility in the presence of TNF-alpha and LPS. Therefore, the inability to stimulate gastric motility via central TRH could not be attributed to the direct inhibitory effects of either LPS or TNF-alpha on the stomach. If the production of endogenous TNF-alpha was suppressed via the use of urethan as the anesthetic agent, then intravenous injections of LPS were no longer effective in suppressing gastric motility. Thus these effects on gastric motility are not directly attributable to LPS nor are they due to direct effects on the gastric smooth muscle. Our previous study demonstrated that microinjection of femtomole quantities of TNF-alpha in the brain stem dorsal vagal complex (DVC) can modulate gastric motility. This central TNF-alpha effect on gastric motility was dose dependent and required an intact vagal efferent pathway. The results from these two studies suggest that systemically produced TNF-alpha may gain access to the DVC to modulate gastric function.
Asunto(s)
Ventrículos Cerebrales/fisiología , Motilidad Gastrointestinal/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Betanecol/farmacología , Ventrículos Cerebrales/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Agonistas Muscarínicos/farmacología , Ratas , Ratas Long-Evans , Hormona Liberadora de Tirotropina/farmacología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The present study used activation of the c-Fos oncogene protein within neurons in the dorsal vagal complex (DVC) as a marker of neuronal excitation in response to systemic endotoxin challenge [i.e. , lipopolysaccharide (LPS)]. Specifically, we investigated whether vagal connections with the brain stem are necessary for LPS cytokine- induced activation of DVC neurons. Systemic exposure to LPS elicited a significant activation of c-Fos in neurons in the nucleus of the solitary tract (NST) and area postrema of all thiobutabarbital-anesthetized rats examined, regardless of the integrity of their vagal nerves. That is, rats with both vagi cervically transected were still able to respond with c-Fos activation of neurons in the DVC. Unilateral cervical vagotomy produced a consistent but small reduction in c-Fos activation in the ipsilateral NST of all animals within this experimental group. Given that afferent input to the NST is exclusively excitatory, it is not surprising that unilateral elimination of all vagal afferents would diminish NST responsiveness (on the vagotomized side). These data lead us to conclude that the NST itself is a primary central nervous system detector of cytokines.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/biosíntesis , Núcleo Solitario/metabolismo , Tiopental/análogos & derivados , Nervio Vago/metabolismo , Anestésicos , Animales , Lipopolisacáridos/farmacología , Masculino , Neuronas Aferentes/metabolismo , Neuronas Eferentes/metabolismo , Proteínas Proto-Oncogénicas c-fos/análisis , Ratas , Ratas Long-Evans , Núcleo Solitario/química , Núcleo Solitario/efectos de los fármacos , Factor de Necrosis Tumoral alfa/análisis , Vagotomía , Nervio Vago/citología , Nervio Vago/cirugíaRESUMEN
The stress of physical restraint has been shown to modulate the cellular immune response during a viral infection. We have studied the effects of stress on the humoral immune response during infection by influenza virus. Restraint stress altered the kinetics of the antibody response; seroconversion in the IgG and IgA isotypes was delayed in virus-infected C57BL/6 mice subjected to repeated cycles of physical restraint. However, the magnitude and isotype of the mature antibody response were unaffected during the plateau phase; no significant differences were observed between restrained/infected and nonrestrained/infected mice. Thus, the time during infection at which the antibody response was measured was a significant variable in the study of stress-induced alterations of the host's response to a replicating viral antigen. While restraint stress did not significantly affect the magnitude or class of the humoral response, it did alter the kinetics of response.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Virus de la Influenza A/inmunología , Estrés Fisiológico/inmunología , Animales , Isotipos de Inmunoglobulinas/biosíntesis , Cinética , Subgrupos Linfocitarios/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL/inmunología , Modelos Biológicos , Restricción FísicaRESUMEN
Contusion injuries of the rat thoracic spinal cord were made using a standardized device developed for the Multicenter Animal Spinal Cord Injury Study (MASCIS). Lesions of different severity were studied for signs of endogenous repair at times up to 6 weeks following injury. Contusion injuries produced a typical picture of secondary damage resulting in the destruction of the cord center and the chronic sparing of a peripheral rim of fibers which varied in amount depending upon the injury magnitude. It was noted that the cavities often developed a dense cellular matrix that became partially filled with nerve fibers and associated Schwann cells. The amount of fiber and Schwann cell ingrowth was inversely related to the severity of injury and amount of peripheral fiber sparing. The source of the ingrowing fibers was not determined, but many of them clearly originated in the dorsal roots. In addition to signs of regeneration, we noted evidence for the proliferation of cells located in the ependymal zone surrounding the central canal at early times following contusion injuries. These cells may contribute to the development of cellular trabeculae that provide a scaffolding within the lesion cavity that provides the substrates for cellular infiltration and regeneration of axons. Together, these observations suggest that the endogenous reparative response to spinal contusion injury is substantial. Understanding the regulation and restrictions on the repair processes might lead to better ways in which to encourage spontaneous recovery after CNS injury.