A novel method for the functionalization of gamma-irradiated single wall carbon nanotubes with DNA.

In this work we describe a novel method for highly efficient functionalization of single wall carbon nanotubes (SWCNTs) by DNA wrapping. Exposure of SWCNTs to gamma-irradiation (50 kGy) has lowered by one order of magnitude the amount of single stranded deoxyribonucleic acid (ssDNA) required for SWCNT modification. The resulting hybrids of gamma-irradiated SWCNTs and ssDNA were characterized by optical absorbance spectroscopy, Raman spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy was used to investigate the morphology of hybrids. While gamma-irradiation in three different media has significantly improved the process of SWCNT dispersion, irradiation in ammonia was the most efficient. The gamma-irradiated SWCNTs functionalized with ssDNA were stabilized by electrostatic forces. This preliminary study suggests that gamma-irradiation can significantly improve the functionalization of SWCNTs with DNA.

Stress-induced rise in serum anti-brain autoantibody levels in the rat.

Sera from Wistar rats subjected to different stress procedures were tested by ELISA for the presence of autoantibodies with specificity for neuron-specific enolase (NSE) and S100 protein that are preferentially localized in neurons and glia, respectively. Autoantibodies were present in sera of animals before exposure to stress, and raised with age. Anti-NSE and anti-S100 autoantibody levels were increased one day after termination of restraint (2 hours daily, 10 days) and electric tail shock (80 shocks daily, 19 days), and in fifth and tenth week of overcrowding stress. Differences between stressed and control animals were not present one month following restraint and electric tail shock and in twentieth week of overcrowding.

Suppression of adjuvant arthritis by kappa-opioid receptor agonist: effect of route of administration and strain differences.

It is well established that kappa-opioid receptor agonists exert antiinflammatory and antihyperalgesic effects during nonspecific inflammation as well as suppressive effects on the development of humoral and cell-mediated immune responses to foreign antigens. The aim of this study was to investigate the ability of the kappa-opioid receptor agonist MR 2034 to modulate adjuvant arthritis in the rat. In the first series of experiments, treatments of Wistar rats were performed using several routes of drug administration: intraperitoneal (ip), intracaudal (ic), intracerebroventricular (icv) and intraplantar (ipl). MR 2034 significantly suppressed joint swelling after ip and ic treatment, slightly reduced inflammation after ipl treatment, and did not produce any effect after icv treatment. In the second series of experiments, the suppressive effect of ip injected MR 2034 was investigated using Wistar, Dark August (DA) and Lewis rats. In Wistar rats, MR 2034 significantly decreased the incidence of adjuvant arthritis, and suppressed mean joint score and aggregate joint score. Similarly, in DA rats treated with MR 2034, mean arthritic score was significantly suppressed, but other clinical parameters were not affected. In Lewis rats, however, ip treatment with MR 2034 failed to produce any suppressive effect on joint disease and even potentiated the initial development of arthritis. These data suggest that immunosuppressive and antiinflammatory action of MR 2034 markedly depend on the route of drug administration and strain susceptibility to opioids.

Suppression of experimental allergic encephalomyelitis in offspring of DA and Wistar rats following immunization of mother with encephalitogen.

Immunization of female rats with encephalitogen before gestation, during gestation, and during lactation differentially decreased susceptibility to experimental allergic encephalomyelitis (EAE) in their offspring. The most pronounced suppression, revealed by lowered incidence and weaker clinical signs of the disease, was observed in offspring of mothers immunized before gestation and during lactation in both Dark August (EAE-susceptible), and Wistar (EAE-relatively resistant) rat strains. Induction of EAE in mothers during pregnancy only delayed the onset of the disease in DA progeny. The overall effect on EAE in offspring did not depend on the disease intensity in mothers. Our results suggest that anti-myelin basic protein (MBP) antibodies passively transferred from mothers are not responsible for the observed protection in offspring.

Experimental allergic encephalomyelitis in adult DA rats subjected to neonatal handling or gentling.

The present study investigated the effect of daily handling and gentling between postnatal days 1 and 28 on experimental allergic encephalomyelitis (EAE) in 8-week old DA rats. Handling consisted of removing pups from the mother, and placing them in the novel cage for 15 min. The gentling procedure included handling accompanied by 3 min of dorsal tactile stimulation before returning the pups to the nest cage. Adult rats of both sexes handled in infancy showed increased susceptibility to EAE, as revealed by higher incidence of the disease, and more severe clinical signs. Anti-myelin basic protein (MBP) autoantibodies were increased in handled males, and decreased in handled females, compared to controls. Gentling induced aggravation of clinical signs and histopathological lesions of EAE in males, while in gentled females suppression was observed. These results indicated that both neonatal handling and gentling aggravated EAE induced in adult male rats. In female rats handling exacerbated, and gentling suppressed clinical EAE. The overall effect of neonatal manipulations was more pronounced in males. Furthermore, in mothers separated from their offspring due to handling and gentling, and immunized for EAE at day 28 postpartum, earlier appearance of clinical signs, and increased frequency of relapses compared to control dams was recorded.

Maternal deprivation and early weaning modulate experimental allergic encephalomyelitis in the rat.

The present experiment deals with the effect of maternal deprivation (MD) and early weaning (EW) on the development and course of experimental allergic encephalomyelitis (EAE) in Dark August (DA) rats. Five litters (five to nine pups per liter) were subjected to MD (4 h daily) from Day 1 until Day 28. EW rats were weaned on Day 15 (EW-15, five litters) or Day 21 (EW-21, four litters). Control rats and MD rats were weaned on Day 28. At the age of 8 weeks, rats were immunized with guinea pig spinal cord in complete Freund's adjuvant and clinical signs of EAE were recorded daily. On Day 18 after immunization, rats were bled and sacrificed. Brain and spinal cord were examined histologically for EAE lesions. Serum anti-rat myelin basic protein (MBP) antibodies were detected by ELISA. MD female rats exhibited suppression of neurological and histological signs of EAE in comparison with control rats. MD and control females showed elevated anti-MBP antibody level compared to MD and control males. EW-15 female rats demonstrated potentiation of neurological signs of EAE compared to control females. EW-21 females developed more severe clinical signs and histological lesions compared to control females. These results show that neonatal experiences, such as maternal deprivation and early weaning, influence the development of EAE in adult DA rats.

Effect of Met-enkephalin and opioid antagonists on rat macrophages.

Effects of Met-enkephalin (Met-ENK) and opioid antagonists on H2O2 release by peritoneal macrophages from DA and AO rats were investigated. Met-ENK increased and decreased H2O2 production by macrophages of DA and AO rats, respectively. These effects were antagonized by low, but not high, concentrations of naloxone and ICI 174864. High concentrations of both antagonists directly modulated H2O2 release and retained the strain-related differences seen with Met-ENK. The results showed direct, strain- and dose-dependent, effects of Met-ENK, naloxone, and ICI 174864 on rat macrophage function.

Neonatal sound stress and development of experimental allergic encephalomyelitis in Lewis and DA rats.

This experiment deals with the effect of neonatal sound stress on the susceptibility of rats in adult life to the induction of experimental allergic encephalomyelitis (EAE). Two inbred strains of rats, Lewis and DA, highly susceptible to EAE were used. On postnatal days 15, 18 and 21, animals of both sexes were sound stressed in a sound attenuated chamber (90dB, 60 rings/5 sec during 1 h, on a variable interval schedule) in the presence or absence of the mother. Experimental groups were as follows: (a) pups stressed without the mother (SP); (b) pups stressed in the presence of the mother (SPM); (c) control nonstressed pups separated from the mother (CP), and (d) control nonstressed pups undisturbed in their nest cages (CPM). Rats were weaned on postnatal day 28. At the age of 8 weeks, all groups were immunized with guinea pig spinal cord in complete Freund's adjuvant. Signs of EAE were recorded daily until the day 20 after immunization when animals were bled and sacrificed. Serial sections of cerebrum, cerebellum and spinal cord were examined histologically for the presence of mononuclear cell infiltrates. Anti-myelin basic protein (MBP) antibodies were detected in serum samples using ELISA technique. Stressed Lewis rats (groups SP and SPM) compared to control groups CP and CPM, developed more severe EAE as revealed by a higher aggregate clinical score, more pronounced histological lesions and increased production of anti-MBP antibodies. The presence of the mother during stress session (group SPM) prolonged the disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of early experience on behavior and immune response in the rat.

The effect of maternal deprivation (MD) and preweaning handling on open field (OF) behavior, body and organ weights (spleen, thymus, and adrenals), and humoral immune response (plaque-forming cell response and antibody production) in adult male and female Wistar rats was studied. Maternal deprivation took place either for 28 postnatal days (2 h/day), or on days 15, 18, and 21 (2 h/day), whereas handling was performed daily during 28 postnatal days for 3 min. Sex differences were found both in behavior and immune response. The MD rats showed ambulatory hyperactivity in OF tests, females being more active than males, and a marked suppression of the PFC response. Handled rat's behavior was distinguishable from MD rats by an increased curiosity. Female handled rats were more active in the OF and their antibody production was higher. Male handled rats showed higher defecation scores and lower plaque-forming cell response. These results present evidence for a deprivation syndrome and immunosuppressive behavior in MD rats. Several mechanisms that may account for these immunobehavioral results are outlined.

Anaphylactic shock in neuropsychoimmunological research.

Anaphylaxis appears to be an excellent experimental model for investigating the interactions between central nervous system (CNS) and immune system. Both afferent and efferent regulatory pathways of anaphylactic response are well characterized. The potent mediators of anaphylactic shock, such as histamine and serotonin, are at the same time neurotransmitters, acting in the CNS, and regulators/modulators of the immune system, since receptors for these substances exist on the membrane of the cells of the immune system. In this article the results of studies on the relationship between anaphylaxis and CNS, performed by both pioneers and contemporary investigators, are briefly reviewed. Recent experiments done in our laboratory are presented, which showed that (a) anaphylactic shock can be induced by intracerebroventricular administration of the shocking dose of antigen; (b) rats can learn to associate the induction of anaphylactic shock with neutral stimuli from the environment; and (c) stress in the form of electric tail-shocks reduces the intensity of anaphylactic shock.

Stress-induced resistance to anaphylactic shock.

There have been many reports of the immunomodulatory effects of stress, but the influence of stress on anaphylaxis has been given little attention till now. In this study we investigated the influence of tail-shock stress on the course of anaphylactic shock (AS) in the rat. For this purpose, rats were sensitized to ovalbumin and subjected to stress procedure before the induction of AS. In the first series of experiments we used chronic (4 day) stress consisted of 80 inescapable tail shocks delivered at the same time each day. Anaphylactic shock was induced 24 hours later by intraperitoneal injection of 3 mg of ovalbumin. Results showed that stressed rats exhibited lower intensity of three investigated parameters of AS: clinical signs, hematocrit values, and drop of rectal temperature. In order to investigate whether acute stress procedure could also influence course of AS, rats were given various shock doses of ovalbumin immediately after the end of acute (1 day) tail-shock stress. Anti-anaphylactic effect of acute stress was demonstrated to be dose-dependent: the greatest protective effect was in animals that received the highest shocking dose of ovalbumin. Finally, we examined the duration of protective effect of acute inescapable tail shocks on AS, and these results showed that observed anti-AS phenomenon disappears 72 hours after the end of acute stress session.

Stress-induced suppression of experimental allergic encephalomyelitis in the rat.

Numerous experiments have demonstrated that physical stress can alter immunological parameters. However, little attention has been paid to the interrelationship between stress and autoimmune processes. The present study was designed to determine the influence of electric shock and sound stress on the development of experimental allergic encephalomyelitis (EAE). Ten-week-old male DA rats highly susceptible to EAE were used. Rats were subjected to the stress procedure during 19 days either before or after immunization with intradermal injection of 0.1 ml of an emulsion containing guinea pig spinal cord (20 mg/rat) in an equal volume of complete Freund's adjuvant (CFA). In addition, rats received subcutaneous injection of Bordetella pertussis in the dorsum of the same foot. Electric stress procedure consisted of 80 inescapable, unpredictable tail shocks (5 s, 1 mA) delivered at the same time each day. Sound stress procedure consisted of exposure of rats to a 90 dB fire alarm bell which rings 60 times for 5 s during one hour, at the same time of the day. Rats were observed daily for clinical signs of EAE and survived animals were sacrificed on day 20 after immunization. The brain and spinal cord sections were examined histologically for mononuclear cell infiltrates characteristics for EAE. The results clearly indicate that inescapable tail shocks suppressed the appearance and development of EAE when rats were subjected to stress procedure during 19 days after immunization, but not when rats were stressed during 19 day before the induction of EAE. On the other hand, in rats exposed to sound stress there was only delay in the onset of the disease.

Anaphylactic shock-induced conditioned taste aversion. I. Demonstration of the phenomenon by means of three modes of CS-US presentation.

A series of three experiments was conducted to investigate whether an anaphylactic response could induce a conditioned modification of behavior. Rats sensitized to ovalbumin were subjected to a conditioning trial in which the conditioned stimulus (CS; saccharin solution) signaled the presentation of the unconditioned stimulus (US; shocking dose of ovalbumin), eliciting the unconditioned response (UR; anaphylactic shock). In a subsequent two-bottle preference test, immunized rats given a CS-US pairing developed a conditioned taste aversion toward an otherwise preferred saccharin solution. The phenomenon of anaphylactic shock-induced conditioned taste aversion was found to be robust and resistant to extinction during the 6-day test period and was established employing three modes of CS-US presentation: (a) CS po, US ip; (b) CS po, US iv; and (c) CS iv, US iv. The most effective mode of CS-US presentation for producing anaphylactic shock-induced taste aversion was observed in Experiment 1 (CS po, US ip). Thus, aversive manifestations of anaphylactic shock can serve as afferent signals by which the immune system informs the central nervous system which in turn modulates behavior.

Anaphylactic shock-induced conditioned taste aversion. II. Correlation between taste aversion and indicators of anaphylactic shock.

Previous studies (V. J. Djuric, B. M. Markovic, M. Lazarevic, & B. D. Jankovic, 1987, in B. D. Jankovic, B. M. Markovic, & N. H. Spector (Eds.), Neuroimmune interactions, pp. 561-568, New York: New York Acad. Sci.; B. M. Markovic, V. J. Djuric, M. Lazarevic, & B. D. Jankovic, 1988, Brain Behav. Immun. 2, 11-23) have shown that rats learn to associate the taste of saccharin with the induction of anaphylactic shock, thus exhibiting conditioned taste aversion (CTA) toward an otherwise preferred saccharin solution. The present experiment investigates the effect of unconditioned stimulus intensity (the amount of antigen used for the induction of shock) on CTA. Rats were sensitized to ovalbumin and subjected to a conditioning trial in which the conditioned stimulus (CS; saccharin solution given orally) signaled the presentation of the unconditioned stimulus (US; shocking doses of ovalbumin ranging from 0.5 to 3 mg given intraperitoneally). Behavioral signs, hematocrit, and rectal temperature were used for evaluation of anaphylactic shock. Twenty-four hours after the conditioning trial, rats were subjected to a two-bottle preference test between saccharin solution and water. Multiple regression statistical analysis revealed significant correlations among saccharin preference ratio, dose of antigen used for the induction of shock, behavioral signs of shock, rise in hematocrit, and fall in rectal temperature. A dose-dependent relation among saccharin preference ratio and physiological indicators of shock suggests that conditioned anaphylactic shock-induced avoidance behavior is functionally related to homeostatic factors involved in immune reactivity.

Neuroimmunomodulation: potentiation of delayed hypersensitivity and antibody production by chronic electrical stimulation of the rat brain.

This study deals with the structural and functional correlates of the immunoneuroendocrine interconnections, and explores the effects of localized brain stimulation on immune response. Bilateral symmetrical electrodes were placed in the dorsomedial nucleus, ventromedial nucleus and posterior area of the hypothalamus, and in the sensorimotor cerebral cortex of the rat brain. Electrical stimulations were applied for 39 consecutive days, 10 days before and 29 days after single immunization. In sham-stimulated rats, electrodes were lowered into the hypothalamic areas and sensorimotor cortex without passing any current. Animals with electrodes were divided post mortem into groups according to the histological location of the electrode tips. Intact rats served as additional controls. All animals were immunized with bovine serum albumin in complete Freund's adjuvant and tested for immune reactions 7, 14, 21, and 28 days after immunization. The most significant finding was the potentiated delayed skin hypersensitivity and, to a lesser extent, antibody production to bovine serum albumin in rats with stimulated dorsomedial hypothalamus and sensorimotor cortex. On days 21 and 28, skin reactions increased in rats with stimulated posterior hypothalamus. These immune effects could not be obtained when the stimulation was applied to the hypothalamic ventromedial nucleus. Several pathways of the immunoneuroendocrine interplay are suggested.