Animal models for the study of the effects of spaceflight on the immune system.

Animal models have been used to determine the effects of spaceflight on the immune system. Rats and rhesus monkeys have been the primary animals used for actual space flight studies, but mice have also been utilized for studies in ground-based models. The primary ground based model used has been hindlimb unloading of rodents, which is similar to the chronic bed-rest model for humans. A variety of immune responses have been shown to be modified when animals are hindlimb unloaded. These results parallel those observed when animals are flown in space. In general, immune responses are depressed in animals maintained in the hindlimb unloading model or flown in space. These results raise the possibility that spaceflight could result in decreased resistance to infection in animals.

Influence of skinfold sum and peak VO(2) on immune function in children.

OBJECTIVE: To measure the relationship of skinfold sum and peak VO(2) power with immune function in children. DESIGN: Cross-sectional, with all children tested twice during a 2 month period for peak VO(2), sum of two skinfolds, and immune function, with data from the two measures averaged and then correlated (alpha level, < or = 0.01). Immune measures included leukocyte and lymphocyte subset counts, delayed-typed hypersensitivity (DTH), global IgG antibody response over 4 weeks to pneumococcal vaccination (pIgG), salivary IgA concentration (sIgA), PHA-stimulated lymphocyte proliferation (PHA-SLP), natural killer cell activity (NKCA), and granulocyte and monocyte phagocytosis and oxidative burst activity. SUBJECTS: Seventy-three children (n=42 males, n=31 females) ranging in age from 7 to 13 y (mean+/-s.d. age, 9.9+/-1.7 y). The mean skinfold sum was 28.9+/-17.1 mm, and peak VO(2) 45.8+/-8.1 ml/kg/min. RESULTS: Peak VO(2), skinfold sum, and immune measures did not differ significantly by age or gender. Therefore, correlations were made on combined indices for all subjects. Peak VO(2) and the skinfold sum were not significantly correlated with NKCA, oxidative burst activity, plgG or DTH. Peak VO(2) was negatively correlated with monocyte phagocytosis (r=-0.30, P=0.012) and positively correlated with PHA-SLP (6.25 microg/ml; r=0.35, P=0.004). The skinfold sum was positively correlated with the total leukocyte count (r=0.39, P<0.001), granulocyte count (r=0.36, P=0.002), monocyte count (r=0.38, P=0.001), monocyte phagocytosis (r=0.41, P<0.001), granulocyte phagocytosis (r=0.35, P=0.003), and sIgA (r=0.32, P=0.006), and negatively correlated with PHA-SLP (6.25 microg/ml; r=-0.39, P=0.001). CONCLUSIONS: Data from this study indicate that a high skinfold sum is related to elevated leukocyte subset counts and monocyte/granulocyte phagocytosis, and low PHA-SLP in children.

Efficacy and safety of orally/sublingually, intranasally, and intraperitoneally administered recombinant murine interferon in the treatment of murine encephalomyocarditis virus.

Interferons (IFN) have been shown to be effective in protecting animals against lethal viral infections when administered systemically in relatively high doses. Intraperitoneal (i.p.) injection of mice with encephalomyocarditis virus (EMCV) gives rise to a rapidly progressive fatal disease characterized by central nervous system involvement and encephalitis. IFN-alpha has been shown to be effective in protecting mice against lethal EMCV infection when given via parenteral and oral/sublingual routes. The current study was designed to explore the ability of orally/sublingually and intranasally (i.n.) administered IFN-alpha to treat mice infected with EMCV in support of a planned clinical trial to evaluate efficacy of oral IFN-alpha in human viral infections. The primary objective of the study was to determine the efficacy of recombinant murine IFN-alpha (rMuIFN-alpha) in the treatment of mice infected with 100 LD(50) EMCV following oral, i.n., and i.p. administration at doses of 20,000 and 100,000 IU. The results of the current experiment did not indicate protection from infection with EMCV in mice that received IFN by the i.n. or oral/sublingual routes. The negative controls, infection of mice with 100 LD(50) of EMCV followed by treatment with excipient via all three routes, resulted in death of nearly all mice, as expected. The positive control, treatment of EMCV-infected (100 LD(50)) mice with rMuIFN-alpha via the i.p. route, was successful in protecting a significant number of mice from death compared with matched controls. This study points out the need to determine the optimum conditions for administration of oral/sublingual or i.n. IFN to insure maximum efficacy against viral infections.

Norepinephrine as a growth stimulating factor in bacteria--mechanistic studies.

Catecholamines (norepinephrine, epinephrine, dopamine) enhance the growth of several species of gram-negative bacteria. Since catechol rings are known siderophores in bacteria, the administration of catecholamines may enhance growth by improving iron uptake in growth-limiting media, serving as auxiliary siderophores. We have tested the iron content in bacterial growth media which are known to support rapid growth and "slow growth" media. Additionally, we have examined the uptake of 3H-norepinephrine, to determine whether the catecholamine is actually taken into the bacteria or is merely adsorbed to the outside of the bacteria. Finally, we have been examining the supernatants produced by culturing bacteria with norepinephrine. These supernatants have been shown to have the capacity to enhance growth of naive cultures of bacteria, and are suggested to contain an "autoinducer of growth". We have found that both fast-growth and slow-growth media contain similar concentrations of iron, and that these levels do not change in most supernatants from NE-supplemented bacterial cultures. Examination of culture supernatants from NE-supplemented bacteria under different temperature conditions reveals some interesting differences. First, culture supernatant from NE-treated Escherichia coli, cultured at 37 degrees C, when examined by HPLC, exhibits a change in the norepinephrine content over time which is not seen in supernatant from 21 degrees C cultures or other media treatments. Second, the 37 degrees C culture NE-supplemented E. coli supernatant was significantly more effective in enhancing growth of three bacterial species than any other culture method other than NE-supplementation itself (this includes supernatant from NE-supplemented cultures of the other two species as well as supernatants from unsupplemented cultures of all three species).

Immune alterations in three mouse strains following 2-deoxy-D-glucose administration.

Using 2-deoxy-D-glucose (2-DG)-induced stress, our laboratory has developed studies to define stress effects on immune responses. Here, we report effects of increasing doses of 2-DG on the immune response of BALB/c, C57BL/6 and BDF(1) mice 2 h after three injections of 0 to 2000 mg/kg of 2-DG. Female 4- to 5-week-old mice were euthanized and blood and spleens were collected. A suspension of partially purified mature T splenocytes was obtained by negative selection using J11.d2 antibodies. Glucose and corticosterone levels were measured in the plasma of each mouse. Splenocyte and mature T splenocyte suspensions were tested in in vitro proliferation assays with or without concanavalin A. Splenocytes were analyzed for the following cell-surface markers: CD3, TCR alpha/beta, CD4, CD8 and major histocompatibility complex (MHC) Class II. Significant increases in blood glucose levels were observed in C57BL/6 and BALB/c strains with the highest 2-DG dose (p<0.05). Corticosterone levels were higher in BDF(1) mice and C57BL/6 mice following the administration of 1000 and 2000 mg/kg of 2-DG, respectively (p<0.01). In vitro proliferation of mature T splenocytes in the presence of concanavalin A was decreased in BDF(1) (p<0.05) but not in BALB/c and C57BL/6 mice. In addition, in BDF(1) mice the decrease was highly correlated with an increase of CD3+ and TCR alpha/beta+ cells in the spleen. These results demonstrated high variability in the response of different mouse strains to 2-DG-induced stress.

Inhibition of interferon, cytokine, and lymphocyte proliferative responses in elite swimmers with altitude exposure.

To determine the immunologic consequences of athletic training at altitude, blood samples were taken at rest from 10 swimmers and 8 control nontraining but altitude-exposed members of the 1996 Australian Olympic Swimming Team, near the start and completion of a 21-day training camp at 2102 m. Blood leukocyte numbers dropped in both groups (p < 0.05), with the decrease greater in the swimmers (-38% swimmers, -3% controls). Concanavalin A (ConA)-induced blastogenesis decreased in both groups (p < 0.01), but the drop was greater in the control group (-32% swimmers, -56% controls, p < 0.05). Lipopolysaccharide (LPS)-induced blastogenesis more than doubled in both groups (281% swimmers, 249% controls, p < 0.01). Increases in mitogen-induced interleukin-1beta (IL-1beta), IL-4, and interferon-gamma (IFN-gamma) production and a decrease in IL-2 levels were observed in both groups after altitude exposure (all p < 0.05). The percentage of cells expressing HLA-DR fell (-33% swimmers, -20% controls, p < 0.01), whereas those expressing CD-4 expression increased (16% swimmers only, p < 0.01). Although training at medium-level altitude alters some immunologic parameters, the training-induced changes may be secondary to those induced by altitude alone.

Effects of 2-deoxy-D-glucose administration on cytokine production in BDF1 mice.

Physical exercise and diet changes have been shown to affect immune parameters, and similar effects are also induced by the administration of a nonmetabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The present study was designed to characterize the effects of glucoprivation induced by 2-DG administration on concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the blood and interferon-gamma (IFN-gamma), IL-2, and IL-4 in vitro production by partially purified T splenocytes in BDF1 mice. Mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, or 1000 mg/kg of 2-DG, and blood and spleens were collected 2 h after the last injection. Partially purified T splenocytes were cultured 24 h in the presence of concanavalin A (ConA). A significant increase in the corticosterone levels with the amount of 2-DG injected was observed after one or three injections (p<0.05). The amount of 2-DG injected was associated with an increase in TNF-alpha, IL-1beta, and IL-6 concentrations in the blood of mice after one or three injections of 2-DG (p<0.05). A significant decrease in in vitro proliferation of partially purified splenocytes in the presence of ConA was associated with a decrease in IFN-gamma production in the culture supernatants and an increase in IL-1 receptor expression on the cell surface (p<0.05).

Effects of social conflict on immune responses and E. coli growth within closed chambers in mice.

Social conflict has been shown to affect the neuroendocrine stress response in rodents. The current study was designed to characterize the effects of social conflict on leukocyte subset distribution and function as well as in vivo bacterial growth. Male DBA/2 mice implanted or not implanted with a closed chamber containing Escherichia coli were repeatedly challenged by temporary placement in the territory of a dominant CF-1 mouse five times a day for 2 consecutive days. Nonstressed animals were similarly handled, but were not exposed to social conflict. Effects on immune responses and E. coli growth were analyzed 13 h after the last social conflict session. Social conflict alone was associated with an increase in plasma corticosterone concentration and decreases in thymocyte numbers and splenocyte ability to proliferate in vitro in the presence of lipopolysaccharide (p < 0.05). After social conflict, immature CD4+CD8+ thymocytes decreased, whereas mature T cells increased (p < 0.05). In the presence of E. coli, social conflict induced a significant increase in plasma concentration of interleukin-1beta, and a decrease in the number of thymocytes and the percentage of CD4+CD8+ T cells in the thymus (p < 0.05). In addition to the lymphocyte subpopulation changes observed with social conflict alone, the proportion of CD3+ and major histocompatibility complex (MHC) class II IAd+ cells were significantly higher in stressed mice implanted with a closed chamber containing E. coli (p < 0.05). Social conflict tended to favor E. coli growth in the closed chamber, indicating possible direct bacterial-neuroendocrine hormone interactions. Taken together, these results suggest that stress may modulate the host immune response by altering both bacterial growth and resistance to infection.

Influence of exercise mode and carbohydrate on the immune response to prolonged exercise.

The influence of exercise mode and 6% carbohydrate (C) versus placebo (P) beverage ingestion on lymphocyte proliferation, natural killer cell cytotoxicity (NKCA), Interleukin (IL)-1beta production, and hormonal responses to 2.5 hr of intense running and cycling (approximately 75% VO2max) was measured in 10 triathletes serving as their own controls. The C versus P condition (but not exercise mode) resulted in higher plasma glucose concentrations, lower plasma cortisol concentrations, reduced postexercise lymphocytosis and NKCA, and a lessened T-cell reduction during recovery, No condition or mode effects were observed for concanavalin A and phytohemagglutinin-induced lymphocyte proliferation. Significant mode (but not condition) effects were observed for lipopolysaccharide-induced IL-1beta production over time. However, when expressed per monocyte, the mode effect was abolished and a sustained suppression in IL-1beta/monocyte was observed in all sessions throughout recovery. These data indicate that carbohydrate ingestion significantly affects plasma glucose and cortisol concentrations, blood lymphocyte counts, and NKCA, whereas exercise mode has no effect on these parameters.

Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses.

Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.

Antiorthostatic suspension stimulates profiles of macrophage activation in mice.

The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

Immunological and ultrastructural disruptions of T lymphocytes following exposure to the glycopeptidolipid isolated from the Mycobacterium avium complex.

In this study, we examined the effects of the serovar-specific glycopeptidolipid (GPL) on the ultrastructure of purified T lymphocytes and the interleukin secretion by spleen and purified T lymphocytes. Electron microscopy indicated extensive disruption of the cytoplasmic compartment of T lymphocytes, which could result in altered function of immune cells. Despite the cellular damage as viewed by the electron microscopy, the expression of T-cell surface markers, Thy 1.2 and Lyt-2, were not affected. The data indicate that GPL is capable of inducing in-vitro interleukin (IL)-6 and IL-2 production by whole spleen or purified spleen T lymphocytes. The level of production of IL-6 and IL-2 following the exposure of the mycobacteria-infected cells to GPL was approximately the same as the uninfected control. A similar finding was also obtained with the total lipid extraction from the mycobacterium. The results suggest that the ability of the total lipid extraction, in inducing cytokine production, may be attributed to its GPL content.

Catecholamine enhancement of Aeromonas hydrophila growth.

Several species of bacteria have been shown to respond to the administration of norepinephrine and other catecholamines with increased growth (in culture) and virulence. In this study, we examined the effects of catecholamines on the growth of cultures of Aeromonas hydrophila, a Gram-negative bacillus found in brackish water. Bacterial cultures were maintained in tryptic soy both, then washed free of medium and transferred to a bovine serum-supplemented minimal salts medium. Treatment of A. hydrophila cultures with 10(-3)to 10(-5)M norepinephrine resulted in dramatic increases in growth at 24 h and longer, as assessed by spot plate analysis on tryptic soy agar plates. Norepinephrine-treated cultures had 4.5 log greater bacterial numbers than control cultures. Epinephrine, dopamine and isoproterenol were shown to be similarly effective in enhancing growth of A. hydrophila, over narrower concentration ranges. Acetylcholine supplementation of cultures did not alter the growth of A. hydrophila. Serotonin slightly enhanced Aeromonas growth when administered at very high concentrations (10(-3)M). The increased growth observed after catecholamine administration may alter the capacity to infect an animal under stressful conditions, and is another potential mechanism by which a stress response can affect susceptibility to disease.

Effects of space flight on surface marker expression.

Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.

Space flight, microgravity, stress, and immune responses.

Exposure of animals and humans to space flight conditions has resulted in numerous alterations in immunological parameters. Decreases in lymphocyte blastogenesis, cytokine production, and natural killer cell activity have all been reported after space flight. Alterations in leukocyte subset distribution have also been reported after flight of humans and animals in space. The relative contribution of microgravity conditions and stress to the observed results has not been established. Antiorthostatic, hypokinetic, hypodynamic, suspension of rodents and chronic head-down tilt bed-rest of humans have been used to model effects of microgravity on immune responses. After use of these models, some effects of space flight on immune responses, such as decreases in cytokine function, were observed, but others, such as alterations in leukocyte subset distribution, were not observed. These results suggest that stresses that occur during space flight could combine with microgravity conditions in inducing the changes seen in immune responses after space flight. The biological/biomedical significance of space flight induced changes in immune parameters remains to be established. Grant Numbers: NCC2-859, NAG2-933.

Effects of 2-deoxy-D-glucose administration on immune parameters in mice.

Physical exercise and diet alterations have been shown to affect immune parameters. Similar effects are also induced by the administration of the non-metabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The current study was designed to characterize the effects of glucoprivation induced by 2-DG administration on leukocyte subset distribution and function. BDF1 mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, 1000 or 1500 mg/kg of 2-DG. Two hours after the last injection of 2-DG, immunological parameters were analyzed. A dose-dependent increase in plasma glucose concentrations of mice injected once with up to 1500 mg/kg of 2-DG was observed (p < 0.001). After either one or three injections of up to 1500 mg/kg of 2-DG, corticosterone levels, leukocyte counts in the spleen, and CD3+ cells in the thymus increased. In vitro proliferation of partially purified lymphocytes from the spleen in the presence of both concanavalin-A and lipopolysaccharide decreased in a dose dependent manner (p < 0.05). In addition, after three injections, the proportion of both thymocytes and splenocytes bearing alphabeta-TCR increased as the concentration of 2-DG increased (p < 0.01). These results demonstrate that 2-DG administration induced dose-dependent changes in both thymus and spleen cell distribution and function.

Spaceflight and development of immune responses.

The NIH.R1 Space Shuttle experiment was designed to study the effects of spaceflight on rodent development. Pregnant rats were flown on the Space Shuttle for 11 days, and pregnant control rats were maintained in animal enclosure modules in a ground-based chamber under conditions approximating those in flight. Additional controls were in standard housing. The effects of the flight on immunological parameters of dams, fetuses, and pups were determined. Blastogenesis of spleen cells in response to mitogen was inhibited in flown dams but was not inhibited in cells from their pups. Interferon-gamma production by spleen cells showed a trend toward inhibition in flown dams but not in their pups. The response of bone marrow cells to colony-stimulating factor showed a trend toward inhibition after spaceflight in dams, but the response of fetus and pup liver cells was not inhibited. Total serum IgG was not affected by spaceflight. None of the examined immune parameters that were altered in rat dams after spaceflight was found to be altered in their offspring.