Joan E. Cunnick

Aerobic exercise, but not flexibility/resistance exercise, reduces serum IL-18, CRP, and IL-6 independent of β-blockers, BMI, and psychosocial factors in older adults.

Increased serum levels of inflammatory mediators have been associated with numerous disease states including atherosclerosis, Type II diabetes, hypertension, depression, and overall mortality. We hypothesized that a long-term exercise intervention among older adults would reduce serum inflammatory cytokines, and this reduction would be mediated, in part, by improvements in psychosocial factors and/or by beta-adrenergic receptor mechanisms. Adults age 64 were randomly assigned to either an aerobic exercise treatment (CARDIO) or a flexibility/strength exercise treatment (FLEX) 3 days/week, 45 min/day for 10 months. A subgroup of subjects treated with non-selective beta(1)beta(2) adrenergic antagonists were included to evaluate the potential role of beta-adrenergic receptor adaptations as mediators of an exercise-induced change in inflammation. The inflammatory mediators [C-reactive protein (CRP), IL-6, tumor necrosis factor (TNF)-alpha, and IL-18] and the psychosocial factors (depression, perceived stress, optimism, sense of coherence, and social support) were measured pre- and post-intervention. The CARDIO treatment resulted in significant reductions in serum CRP, IL-6, and IL-18 compared to the FLEX treatment (significant treatment x time interaction, p<.05), whereas TNFalpha declined in both groups (main effect of time, p=.001). However, several psychosocial factors (depression, optimism, and sense of coherence) improved in both groups suggesting that the reduction of CRP, IL-6, and IL-18 in the CARDIO group was not mediated by improvements in psychosocial scores. With respect to the potential role of beta-adrenergic receptors, both CARDIO subjects treated with beta-adrenergic antagonists and those who were not treated with those medications demonstrated similar reductions in serum CRP, IL-6, IL-18, and TNFalpha. In summary, we have observed that an aerobic exercise intervention can significantly reduce serum inflammatory mediators, but beta-adrenergic receptors and psychosocial factors do not appear to be involved.

Microglial release of nitric oxide by the synergistic action of β-amyloid and IFN-γ

Abstract Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized hisotpathologically by a loss of neurons and an accumulation of β-amyloid plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. While most previous studies on the neurodegeneration of AD have focused on neuronal cells and direct β-amyloid-mediated neurotoxicity, few have focused on the role of reactive glial cells in β-amyloid-mediated neurotoxicity. In the present study nitric oxide release from cultured rat microglia was examined by exposing the cells to synthetic β-amyloid peptides (β25–35 and β1–40) alone and in combination with the cytokines IFN-α/β (100 U/ml), IL-1β (100 U/ml), TNF-α (100 U/ml), TNF-β (100 U/ml), or IFN-γ (10, 100, 500, or 1000 U/ml). Assessment of microglial release of nitric oxide was based on the colorimetric assay for nitrite in the culture medium and histochemistry for nitric oxide synthase. Of the cytokines tested, only IFN-γ (1000 U/ml) induced nitric oxide release from microglia. β25–35 did not stimulate nitric oxide release by itself, but it did induce nitric oxide release when co-exposed with IFN-γ (100, 500, and 1000 U/ml). In contrast, β1–40 did induce microglial release of nitric oxide by itself, and this effect was enhanced significantly by co-exposure with IFN-γ (100 U/ml). These findings warrant a further investigation into the role of microglia in the neurodegeneration of Alzheimers disease via nitric oxide toxicity induced by the synergistic action of β-amyloid and a costimulatory factor.

A Functional Antigen in a Practical Crop: LT-B Producing Maize Protects Mice against Escherichia coli Heat Labile Enterotoxin (LT) and Cholera Toxin (CT)

We have produced a functional heat labile enterotoxin (LT-) B subunit of Escherichia coli in maize. LT-B is a multimeric protein that presents an ideal model for an edible vaccine, displaying stability in the gut and inducing mucosal and systemic immune responses. Transgenic maize was engineered to synthesize the LT-B polypeptides, which assembled into oligomeric structures with affinity for GM1 gangliosides. We orally immunized BALB/c mice by feeding transgenic maize meal expressing LT-B or non-transgenic maize meal spiked with bacterial LT-B. Both treatments stimulated elevated IgA and IgG antibodies against LT-B and the closely related cholera toxin B subunit (CT-B) in serum, and elevated IgA in fecal pellets. The transgenic maize induced a higher anti-LT-B and anti-CT-B mucosal and serum IgA response compared to the equivalent amount of bacterial LT-B spiked into maize. Following challenge by oral administration of the diarrhea inducing toxins LT and CT, transgenic maize-fed mice displayed reduced fluid accumulation in the gut compared to non-immunized mice. Moreover, the gut to carcass ratio of immunized mice was not significantly different from the PBS (non-toxin) challenged control group. We concluded that maize-synthesized LT-B had features of the native bacterial LT-B such as molecular weight, GM1 binding ability, and induction of serum and mucosal immunity. We have demonstrated that maize, a major food and feed ingredient, can be efficiently transformed to produce, accumulate, and store a fully assembled and functional candidate vaccine antigen.

Immunosenescence in some but not all immune components in a free-living vertebrate, the tree swallow

A wide diversity of free-living organisms show increases in mortality rates and/or decreases in reproductive success with advancing age. However, the physiological mechanisms underlying these demographic patterns of senescence are poorly understood. Immunosenescence, the age-related deterioration of immune function, is well documented in humans and laboratory models, and often leads to increased morbidity and mortality due to disease. However, we know very little about immunosenescence in free-living organisms. Here, we studied immunosenescence in a free-living population of tree swallows, Tachycineta bicolor, assessing three components of the immune system and using both in vivo and in vitro immunological tests. Immune function in tree swallow females showed a complex pattern with age; acquired T-cell mediated immunity declined with age, but neither acquired nor innate humoral immunity did. In vitro lymphocyte proliferation stimulated by T-cell mitogens decreased with age, suggesting that reduced T-cell function might be one mechanism underlying the immunosenescence pattern of in vivo cell-mediated response recently described for this same population. Our results provide the most thorough description of immunosenescence patterns and mechanisms in a free-living vertebrate population to date. Future research should focus on the ecological implications of immunosenescence and the potential causes of variation in patterns among species.

Ontogeny of innate and adaptive immune defense components in free-living tree swallows, Tachycineta bicolor.

Little is known about the development of immune function in wild animals. We investigated the ontogeny of immune defense in a free-living bird, the tree swallow. We assessed total and differential leukocyte counts, natural antibodies, complement activity, in vivo skin swelling response, and in vitro lymphocyte proliferation and compared the levels of development between nestlings and young adults. We also assessed whether body condition explained variation in these immune components. We found some support for the prediction that innate defenses, which do not need to generate a broad repertoire of specific receptors, would reach adult levels earlier than adaptive defenses. In contrast, we found limited support for the prediction that adaptive defenses, which are thought to be more costly to develop, would be more related to body condition than innate defenses. We discuss our findings in the context of other studies on the ontogeny of immune function.

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells, indicative of alternative macrophage activation

ETHNOPHARMACOLOGICAL RELEVANCE The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages. AIM OF THE STUDY In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity. MATERIALS AND METHODS Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/- LPS. RESULTS Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression. CONCLUSIONS These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concomitantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.

Pharmacological manipulation of immune alterations induced by an aversive conditioned stimulus : evidence for a β-adrenergic receptor-mediated Pavlovian conditioning process

The effect of propranolol, a beta-adrenergic receptor antagonist, on the suppression of splenic mitogenic responsiveness induced by an aversive conditioned stimulus (CS) was evaluated in rats. Experiment 1 demonstrated that propranolol administration before presentation of the CS completely blocked the suppressive effect. In contrast, Experiment 2 showed that administration of propranolol during the training of the aversive CS had no effect on the suppressive effect of the CS in a subsequent test. These findings indicate that the release of catecholamines is responsible for the conditioned immune alteration of splenic lymphocytes. Taken further, these findings suggest that the learning of the conditioned immunomodulatory response to an aversive CS is a Pavlovian conditioning process that is not dependent on the performance of the conditioned response during training.

Interrelations among Immune Defense Indexes Reflect Major Components of the Immune System in a Free-Living Vertebrate

Understanding the relationships among immune components in free-living animals is a challenge in ecoimmunology, and it is important not only for selecting the immune assays to be used but also for more knowledgeable interpretation of results. In this study, we investigated the relationships among six immune defense indexes commonly used by ecoimmunologists and measured simultaneously in individual free-living tree swallows. Three main axes of variation in immune function were identified using a principal components analysis, representing variation in T-cell, B-cell, and innate immunity. Measures within each axis tended to be positively correlated among individuals, while measures in different axes were uncorrelated. A trade-off between T-cell function and B-cell function became apparent only when variation among individuals in body condition, age, and general quality was taken into account. Interestingly, the level of natural antibodies, a component of innate immunity, showed the strongest association with components of acquired B-cell function, possibly reflecting a common underlying genetic mechanism, as has been documented in poultry. Our results indicate that despite the complexity of the immune system, important insights can be gained by using the currently available assays but in a more comprehensive approach than has generally been used in the field of ecoimmunology.

Analysis of immune response in young and aged mice vaccinated with corn-derived antigen against Escherichia coli heat-labile enterotoxin.

Enterotoxigenic strains of Escherichia coli produce a heat-labile holotoxin (LT), which causes diarrhea. We engineered corn seeds to produce LT-B, the nontoxic subunit of LT, to serve as a plant-derived vaccine to travelers diarrhea and as an adjuvant for co-administered proteins. We previously demonstrated that a strong mucosal and systemic antibody response is elicited in young mice with oral administration of corn-derived LT-B. The present study examined systemic and mucosal antibody responses to LT-B in young and aged mice, and recall responses to oral administration and injection of LT-B in aged mice. Specific IgA and IgG antibodies were detectable during an 11-mo period, although the concentration of antigen-specific antibodies declined gradually. Booster by feeding or injection dramatically increased the concentration of specific IgA from that seen in young mice. Specific IgG levels were boosted to concentrations similar to those in young mice. This effect may be age-dependent and related to prior immunization exposure. Analysis of the antibody response of naive aged mice against corn-derived LT-B demonstrated an age-related suppression in specific IgG production, but not specific IgA. These results may provide important information for edible vaccine strategies for young and aged individuals.