Rapid flow cytometric assay for the assessment of natural killer cell activity.

A new assay using flow cytometry has been established to assess natural killer (NK) lytic activity with common bench top instrumentation. This assay uses a cyanine membrane dye to stain live K562 target cells and an iodide nuclear dye to evaluate dead cells, and provides a method of reliably separating target and effector cell populations. Effector cells remain unstained (fluorescent negative) throughout the procedure. The damaged pre-labeled targets appear doubly stained as their membranes become permeable to the nuclear stain during incubation. Percent cytotoxicity of various effector:target cell ratios is discerned using flow cytometric analysis after a 2 h incubation in this new assay, as compared to 4 h with the 51Cr-release 'gold standard' assay for cell-mediated cytotoxicity. Comparisons of normal individuals tested in parallel with the fluorescent dyes and the 51Cr-release assay have shown direct correlations. This new two-color flow cytometric technique has proven to be uncomplicated and reproducible when used in the clinical setting.

The role of anti-pig antibody in pig-to-baboon cardiac xenotransplant rejection.

The role of naturally produced antibody in discordant xenograft rejection is still uncertain. Twelve orthotopic pig-to-baboon heart transplants (HTx) were performed. In 2 baboons, no antibody adsorption (AbA) was performed. In 5 baboons, AbA with a pig lung was performed during circulatory arrest. In 5 baboons, AbA and blood exsanguination at the beginning of cardiopulmonary bypass (CPB) were performed. Baboons were divided into 2 groups; group 1 (n = 4) died within 24 hr of HTx and group 2 (n = 8) survived more than 24 hr. Mean survival period was 9.8 +/- 3.0 hr in group 1 and 151 +/- 33 hr in group 2. Baboon anti-pig antibody (Ab) was measured before CPB, before circulatory arrest, during AbA, at the end of CPB, and daily after HTx. Anti-RBC Ab was measured by the titration method at temperatures of 4 degrees C and 37 degrees C (RAb-4 and RAb-37). Anti-endothelial cell Ab (EAb) and anti-white blood cell Ab (WAb) titers were measured with ELISA. RAb titration > or = 1/4 and EAB and WAb > or = 1/256 were determined to be seropositive (S(+)). S(+) rate of RAb-37 at the end of CPB (endCPB) in group 2 was significantly higher than that in group 1 (8/8 vs. 1/4; P < 0.05). The seronegative (S(-)) rates of RBC-4 and EAb (endCPB) in group 2 were higher than those in group 1 (7/8 vs. 1/4 and 6/8 vs. 1/4, respectively), but not significantly. There was no difference in S(-) rate of WAb (endCPB) between group 1 and group 2. More than 4-fold decrease in RAb-4 and RAb-37 by AbA with a pig lung was observed in 5 and 7 of 8 baboons, while EAb and WAb did not change by AbA. In all of group 2, RAb-4 reverted to S(+) within 3 days after HTx. One baboon had no rejection episode and died of infection 16 days after HTx (baboon 16); however, it also became S(+) for RAb-4 a day after HTx until death. In 4 of group 2, RAb-37 became S(+) 1 or 2 days before death by rejection. Baboon 16, however, became S(+) for RAb-37 7 days after HTx and S(-) again 9 days after HTx until death. EAb became S(+) in all of group 2, but 5 of them survived more than 5 days after seroconversion.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of moderate exercise training on immune response.

The relationship between moderate exercise training (ET) (five 45-min sessions per week, brisk walking at 60 heart rate reserve for 15 wk) and changes in immune system variables and function was investigated in a group of 36 sedentary, mildly obese women. The study was conducted using a two (exercise (EX) and nonexercise (NEX) groups) by three (baseline, 6 wk, and 15 wk testing sessions) factorial design, with data analyzed using repeated measures ANOVA. The pattern of change over time between groups for number of peripheral blood lymphocytes (total), T cells (CD5), B cells (CD20), and serum IgG, IgA, and IgM levels was significantly different. This was not the case for spontaneous blastogenesis or number of T helper/inducer cells (CD4) or T cytotoxic/suppressor cells (CD8). Within-EX-group changes were characterized by significant decreases in percentage and number of total lymphocytes, and in T cell number after 6 wk, and significant increases in each of the serum immunoglobulins after both 6 and 15 wk of training. B cell number increased significantly in NEX subjects relative to baseline values at both 6 and 15 wk, with no significant changes experienced in EX subjects. In summary, these data suggest that moderate ET is not associated with an improvement in lymphocyte function but is associated with a 20% increase in serum immunoglobulins and several small changes in circulating numbers of immune system variables, highlighted by significant decreases in circulating numbers of lymphocytes, particularly the T cell subpopulation. These changes were especially apparent after 6 wk of training, with some attenuation by 15 wk.

Physical activity and immune function in elderly women.

The relationship between cardiorespiratory exercise, immune function, and upper respiratory tract infection (URTI) was studied in elderly women utilizing a randomized controlled experimental design with a follow-up of 12 wk. Thirty-two sedentary, elderly Caucasian women, 67-85 yr of age, who met specific selection criteria, were randomized to either a walking or calisthenic group; 30 completed the study. Twelve highly conditioned elderly women, 65-84 yr of age, who were active in endurance competitions, were recruited at baseline for cross-sectional comparisons. Intervention groups exercised 30-40 min, 5 d.wk-1, for 12 wk, with the walking group training at 60% heart rate reserve and the calisthenic group engaging in mild range-of-motion and flexibility movements that kept their heart rates close to resting levels. At baseline, the highly conditioned subjects exhibited superior NK (119 +/- 13 vs 77 +/- 8 lytic units, P < 0.01) and T (33.3 +/- 4.9 vs 21.4 +/- 2.1 cpm x 10(-3) using PHA, P < 0.05) cell function, despite no differences in circulating levels of lymphocyte subpopulations. Twelve weeks of moderate cardiorespiratory exercise improved the VO2max of the sedentary subjects 12.6%, but did not result in any improvement in NK cell activity or T cell function. Incidence of URTI was lowest in the highly conditioned group and highest in the calisthenic control group during the 12-wk study, with the walkers in an intermediate position (chi-square = 6.36, P = 0.042). In conclusion, the highly conditioned elderly women in this study had superior NK and T cell function when compared with their sedentary counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of high- vs moderate-intensity exercise on natural killer cell activity.

The effect of 45 min of high- (80% VO2max) vs moderate- (50% VO2max) intensity treadmill exercise on natural killer cell cytotoxic activity (NKCA) was investigated in 10 well-conditioned (66.0 +/- 1.9 ml.kg-1.min-1), young males (22.1 +/- 1.3 yr). Blood samples were taken before and immediately after exercise, with three more samples taken during 3.5 h of recovery, and analyzed for proportion of NK cells (CD3-CD16+CD56+) and NKCA. Exercise at 80% vs 50% VO2max resulted in a greater immediate postexercise increase in proportion of NK cells, followed by a 1-h and 2-h decrease below preexercise levels for both intensity conditions. NKCA rose significantly above preexercise levels following high- but not moderate-intensity exercise. For both exercise intensity conditions, NKCA tended to drop below preexercise levels by 1 h postexercise, rising back to preexercise levels by 3.5 h postexercise. When NKCA was expressed on a per-NK cell basis, however, no change relative to preexercise levels occurred following moderate-intensity exercise, while a significant increase occurred after 2-h recovery from high-intensity exercise. These data demonstrate that both high- and moderate-intensity exercise are associated with significant shifts in circulating proportions of NK cells which significantly influence interpretation of NKCA data based on assays using separated mononuclear cells.

Apheresis enhances the selective removal of antinuclear antibodies in systemic lupus erythematosus.

Apheresis suppresses clinical manifestations of lupus and reduces levels of antinuclear antibodies implicated in the pathogenesis of systemic lupus erythematosus (SLE). It is not known, however, if reduced levels of antinuclear antibodies are due to nonspecific removal, or specific mechanisms associated with decreased production, or enhanced clearance from the circulation. In order to distinguish between specific and nonspecific effects of apheresis on antinuclear antibodies in SLE, we compared plasma levels of IgG antibodies to DNA and IgG antibodies to microbial antigens in 13 SLE patients before and after apheresis. Although apheresis lowered plasma levels of IgG (21% mean reduction), there was a disproportionate reduction in IgG antibodies to DNA (42% mean reduction, p less than 0.13). In marked contrast, reduction in antibodies to microbial antigens did not exceed those of plasma IgG. A rapid rebound of serum anti-DNA antibodies following apheresis in certain SLE patients suggests that the selective reduction in anti-DNA antibodies is due to enhanced clearance from the circulation rather than decreased production. These results indicate that apheresis enhances selective removal of antinuclear antibodies in some patients with SLE.

B-adrenergic receptor stimulation increases anti-DNA antibody production in MRL/1 pr mice.

To gain insight into a role for excessive B-adrenergic receptor stimulation in the pathogenesis of autoimmune diseases, MRL/1 pr mice were chronically injected with isoproterenol. The mean change of serum antibody levels after one month of treatment was higher in the isoproterenol treated mice than in controls (anti-ssDNA, p less than 0.05; anti-nDNA, p less than 0.04; and antiguanosine antibodies, p less than 0.03) without significant differences in serum IgG levels. Additionally, mean serum creatinine levels were higher and glomerulonephritis more severe in the isoproterenol treated group. Our findings provide the first evidence linking aberrant B-adrenergic receptor activities, serum anti-DNA antibodies, and end-organ pathology in an animal model of lupus nephritis.

The effects of acute moderate exercise on lymphocyte function and serum immunoglobulin levels.

The extent and duration of changes on lymphocyte function and serum immunoglobulin (Ig) levels were examined in 12 women who walked 45 min at 60% VO2 max in a laboratory setting. A 2-factor, 2 x 6 design with repeated measures on both factors was utilized. The first factor was condition (exercise and rest), and the second factor was time (six times of measurement over a 24-h period), with treatment order counterbalanced. The 45-min walk, in comparison to rest in a seated position, was not associated with significant changes in circulating numbers of interleukin-2-activated T cells (CD5 and CD25) or on spontaneous or concanavalin-A-stimulated lymphocyte proliferation. A trend for decreased phytohemagglutinin-stimulated lymphocyte proliferation in comparison to the rest condition, however, was seen 1.5 h following the exercise bout (p = 0.047). The patterns of change for serum IgG, IgA, and IgM were significantly different (p = 0.001, p less than 0.001, p = 0.010, respectively) between conditions. IgG rose 7.2% immediately following exercise, and then returned to baseline 1.5 h later, which contrasted significantly with changes in the rest condition. These same patterns of change occurred also with IgA and IgM, but increases immediately following exercise were not significant, although a trend was seen for IgA (p = 0.03). The 45-min walk had no effect on plasma cortisol and epinephrine levels relative to the rest condition, but was associated with a significant 89% increase in norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of high- versus moderate-intensity exercise on lymphocyte subpopulations and proliferative response.

The effect of 45 min of high- (80% VO2max) versus moderate- (50% VO2max) intensity treadmill exercise on circulating leukocyte and lymphocyte subpopulations, catecholamine and cortisol concentrations, and the mitogen-stimulated lymphocyte proliferative response was investigated in 10 well-conditioned (mean VO2max 66.0 +/- 1.9 ml/kg/min), young males (mean age 22.1 +/- 1.3 yrs). Blood samples were taken before and immediately after exercise, with three more samples taken during 3.5 h of recovery. Treatment order on the treadmill (graded walking at 7.3 +/- 0.1 km/h, 6.5 +/- 0.6% grade, versus level running at 16.1 +/- 0.3 km/h) was counterbalanced, with subjects acting as their own controls and results analyzed using a 2 x 5 repeated measures ANOVA. The concanavalin A- (Con A) stimulated lymphocyte proliferative response was decreased at 1 h and 2 h post-exercise relative to baseline levels following both exercise-intensity conditions. However, when adjusted on a per-T cell (CD3+) basis to account for the change in number of T cells in the in vivo assay, only the high-intensity exercise condition was associated with a 1-h post-exercise decrease (21%, p = 0.05) in the proliferative response relative to baseline. Exercise at 80% versus 50% VO2max resulted in significantly greater increases in cortisol and epinephrine concentrations, providing a physiological rationale for the immediate-post-exercise lymphocytosis, 1- to 3.5-h lymphocytopenia, and the decrease in Con A-stimulated lymphocyte proliferative response (per CD3+ cell) that occurred in greater measure following high-intensity exercise.