Effect of duration of a moderate exercise program on primary and secondary immune responses in mice. (Research Report).

Antibody production against a specific antigen is a hallmark of humoral immune function. (1) Immunoglobulins, found in serum and in fluids on the body's mucosal surfaces, are important soluble mediators of host defense against bacterial and viral infections. (1) Primary antibody response occurs following an initial exposure to an antigen. Subsequent exposure to the same antigen leads to a stronger secondary antibody response that results in longer-lasting immunity. There are reports on the effect of moderate exercise training (60%-80% of maximal oxygen consumption [V[o.sub.2]max]) on primary antibody response. (2-4) In all of the studies, (2-4) the primary antibody response to antigen was not affected by exercise. An increase in secondary antibody production, however, was evident between 4 and 8 weeks after exercises were begun. (2,5) Kaufman et al (6) used swim training as a moderate-intensity exercise (slowly increased to 2 hours a day for 5 days a week), with an exhaustive swim at the end of the 4-week training period, in rats. They found an enhancement of secondary immunoglobin M (IgM) and to a lesser extent immunoglobin G (IgG) antibody response to the antigen keyhole-limpet hemocyanin (KLH) 1 week after training began and at the completion of the 4-week period of training.

Endogenous opioids in moderate doses enhance antibody response. (7-9) The mechanism most likely involves opioid binding to receptors located on B and T lymphocytes, (10) which plays an important role in antibody formation. B lyrnphocytes differentiate into antibody-producing plasma cells with help from T lymphocytes. (1) Serum concentrations of endogenous opioids increase in response to some forms of exercise. (11) Recent work in our laboratory demonstrated a role of endogenous opioids in exercise-induced modulation of secondary antibody response. (12) In that study, we showed that secondary antibody response was suppressed in mice implanted with naltrexone pellets (an opioid antagonist) that exercised moderately for 8 weeks, suggesting to us that moderate exercise training-induced enhancement of secondary antibody response may be due, in part, to endogenous opioids. Because [beta]-endorphin, an endogenous opioid, increases during every moderate exercise session, (11) we hypothesized that a shorter exercise program of 2 weeks may be sufficient to enhance secondary antibody response.

Although the primary antibody response is not affected by moderate exercise training, (2-4) we sought to examine the effects of a moderate exercise program completed prior to the primary immunization on the secondary antibody response in mice. We believe knowledge of these effects could be useful for vaccinations where multiple doses have to be injected to obtain seroprotecrive levels of antibodies (eg, the hepatitis B vaccine). If exercise training prior to the first dose of a vaccine can have an enhancing effect on the antibody response following a second dose (secondary antibody response), then exercise prior to eliciting the primary antibody response p could decrease the number of doses required for achieving seroprotective levels of antibodies or alternatively could increase the seroprotective levels derived from the multiple-dose regimen.

Environmentals (13) and nutritional (14) factors influence immune function. Therefore, to conduct our study, we chose an animal model where housing in the same environment and feeding the same chow could be ensured. We chose to investigate the effects of a moderate exercise program of different durations on primary and secondary antibody responses. Antibody responses in mice are comparable to those in humans in terms of long-term immunity induced following vaccination and infection, and mice therefore serve as a good model to understand the mechanisms of long-term immunity. (15) Moreover, in our study the exercise intensity in mice was correlated to V[o.sub.2]max, (16-18) as can be done in designing human exercise programs. Using a mouse model immunized against human serum albumin (HSA), a potent protein antigen known to initiate antibody responses in mice, (19) the first purpose of our study was to examine the effectiveness of a 2-week versus an 8-week moderate exercise program in enhancing the secondary antibody response in mice. Shorter exercise programs may improve adherence to exercise programs in humans. The second purpose of our study was to examine the effects of moderate exercise prior to the primary immunization on the secondary antibody response.

Method

Design

A posttest control group design for repeated measures was used to study the effects of exercise duration on antibody response to the antigen HSA before and after primary immunization. The experimental designs addressing the 2 purposes of the study and defining the exercise and sedentary group protocols are presented in Tables 1 and 2. One group exercised for 2 weeks and another group exercised for 8 weeks prior to secondary immunization with HSA. A third group served as a control for the 2 exercise groups and was sedentary (Tab. 1). The secondary immunization was administered after either 2 weeks or 8 weeks of exercise, which resulted in an unequal interval between the primary and secondary immunizations in the 2 exercise groups. Following primary immunization, however, antibody levels rise rapidly and are maintained at a steady level for months. (15)

Furthermore, moderate exercise training has no meaningful impact on the level of total immunoglobulins in serum. (20) This allowed us to exercise mice for either 2 weeks or 8 weeks and immediately follow with secondary immunization after the exercise intervention. Because endogenous opioids in moderate doses enhance antibody response (7-9) and serum concentrations of endogenous opioids increase in response to exercise, (11) we continued to exercise the mice after secondary immunization to maintain the elevated levels of endogenous opioids in circulation as the secondary antibody response was evolving. To test the effect of exercise before the primary immunization on the secondary antibody response, one group exercised 3 weeks prior to the primary immunization and 2 weeks prior to the secondary immunization with HSA (exercise preprimary immunization group, Tab. 2). Another sedentary control group (for the exercise preprimary immunization group) rested 3 weeks prior to the primary immunization, as well as 2 weeks prior to the secondary immunization with HSA (Tab. 2).

Sample

The sample consisted of young (8- to 10-week-old), syngeneic, female C57BL/6 mice* (n=50). The mice were randomly distributed among the groups. Each of the 5 groups (Tabs. 1 and 2), consisting of 10 mice, was divided into 2 subgroups in order to house 5 mice to a cage. The mice were housed in micro-isolated shoebox cages. The cages were kept in a climate-controlled environment with 12-hour light and dark cycles. All mice had continuous access to food and water. The animals were selected by nonprobability, convenience sampling.

Measurements/Instrumentation

All antibody levels were measured (in micrograms per milliliter) using an enzyme-linked immunosorbent assay (ELISA). (21) Primary antibody response levels were measured in the exercise preprimary immunization group and its corresponding sedentary group 2 weeks following the primary immunization (Tab. 2). The secondary antibody response was measured in all groups of mice 10 days and 20 days following the secondary immunization (Tabs. 1 and 2).

Anti-HSA antibody levels were measured by assaying the serum using a microplate reader with software. ([dagger],21) The antibody level measurements included all immunoglobulins in the blood against HSA. The microplate reader was calibrated by the manufacturer.

Moderate-intensity exercise training consisted of running the mice on a Vitamaster Rhythm Walker Plus treadmill, ([double dagger]) which was modified for this experiment by the Medical Engineering Department of Emory University. The treadmill is a manual treadmill designed for humans that was motorized to drive the treadmill belt and control for speed accuracy. The treadmill consisted of 6 lanes, separated by aluminum partitions. The treadmill belt formed the floor of the lanes, and the roof of the lanes consisted of hinged Plexiglas. ([section])

Procedure

Transportation is a stressor of animals, and they require a period of adaptation and restoration to homeostasis after being transported. (22) Following random assignment of mice to groups, therefore, all mice experienced a period of acclimatization of at least 5 days before beginning the respective protocols.

Primary immunization was given subcutaneously in the nape of the neck with a 0.1-mL injection of antigen solution (HSA, 200 [micro]g/mL in the adjuvant 9% potassium aluminum sulfate). The secondary antibody response was initiated by a series of 4 booster injections (secondary immunization) of 0.05 mL of antigen solution subcutaneously in the dorsum of each foot (0.2 mL per animal).

Based on previous studies (16-18) in which the V[o.sub.2]max was calculated for C57BL/6 adult mice and correlated to treadmill speed, exercise intensity for this study was controlled by treadmill speed. Moderate-intensity exercise training (60%-80% of V[o.sub.2]max) consisted of mice running on a motorized treadmill for 30 minutes with a gradual increase in speed to 16 m/min with 0 degrees of slope, 5 days a week, for the required number of weeks as indicated in the experimental design. The treadmill speed corresponded to 76% to 77% of V[o.sub.2]max for the C57BL/6 mice in this study.

Exercising mice were placed on a stationary treadmill to acclimatize for 5 minutes. All exercise was performed on a calibrated, 0% grade Vitamaster Rhythm Walker Plus treadmill, a modified treadmill designed for humans that was converted into 6 lanes. The exercising mice were randomly assigned to a treadmill lane and began exercise with a warm-up at the speed of 6 m/min. After 5 minutes, the treadmill speed was turned up to 10 m/min. After 5 additional minutes, the treadmill speed was turned up to 13 m/min. After 5 more minutes, the treadmill speed was turned up to 16 m/min and continued at this speed for 15 additional minutes.

The sedentary protocol consisted of placing the mice in a Plexiglas cage and placing the cage on the lid over the treadmill lanes with the treadmill on to expose them to the noise and vibratory effects of the treadmill. The sedentary mice were so placed for 30 minutes, 5 days week, for the required number of weeks as indicated in the experimental design (Tabs. 1 and 2). Because mice are nocturnal animals, the exercise and sedentary protocol sessions were always conducted in the dark cycle of a 12-hour, light/dark cycle. For a given animal, an exercise session occurred only once in a 22-hour period. Two raters monitored the time of exercise duration for the mice on a single stopwatch at each exercise session.

The blood sampling was performed according to each group's protocol (Tabs. 1 and 2). The first and second blood samples were taken from the tail vein, and the final blood sample was drawn intracardially immediately after sacrifice. All blood samples were taken 24 to 36 hours postexercise to ensure that the changes detected in the antibody response reflected exercise training effects and not acute changes in response to the last exercise session. (20) After collection, the blood was allowed to clot and then centrifuged, and the serum was extracted and stored at -70[degrees]C in Eppendorf tubes for later ELISA analysis. For ELISA, HSA in carbonate buffer (0.05 M NaHC[O.sub.3], pH 9.6), at a concentration of 50 [micro]g/mL, was adsorbed to the surface of polystyrene 96-well, flat-bottom plates (50 [micro]L/well) by incubation overnight at 4[degrees]C.

The plates were decanted and washed 3 times with distilled water. Each well was blocked for nonspecific binding with 100 [micro]L of 1% bovine serum albumin (BSA), incubated for 30 minutes at room temperature, and removed. A standard curve was constructed for each plate using a series of 6 dilutions (1:250 to 1:32,000 in 1% BSA solution in phosphate-buffered saline [PBS]) of a pooled sample of mouse anti-HSA antiserum containing 1 mg/mL of anti-HSA as determined by quantitative precipitation. Similarly, serum samples from each mouse were diluted 6 times from 1:250 to 1:32,000 in 1% BSA solution in PBS. Diluted samples (50 [micro]L of an antibody standard and 50 [micro]L of each mouse serum sample) were tested in triplicate wells at each level of dilution.

The plates were incubated for 1 hour at room temperature and then washed 3 times in distilled water, as described previously. Polyclonal, affinity purified, alkaline phosphatase-conjugated rabbit F[(ab').sub.2] anti-mouse IgG (H+L) ([parallel]) was diluted in 1% BSA in PBS (1:5,000) and added to each well and then incubated for 1 hour at ambient temperature. The plates were washed 3 times with distilled water, and 100 [micro]L of substrate solution (1 mg of p-nitrophenyl phosphate per milliliter of substrate buffer, 48 mL of diethanolamine, 24.5 mg Mg[Cl.sub.2]x6[H.sub.2]O, 400 mL of distilled water, pH 9.8) was added to each well, resulting in a yellow-colored reaction. Thirty minutes after the reaction began, the optical density of each well was read at 405 nm, using a calibrated Vmax kinetic microplate reader with Softmax software. ([dagger])

The concentration of anti-HSA antibodies (Ig) was determined by comparing the optical density of the anti-HSA antibodies in each mouse serum sample with the optical density of the known anti-HSA antibody standard using a standard curve and expressed in micrograms per milliliter. The values from each set of triplicate wells from a given serum sample were averaged to give the value of anti-HSA antibodies for that particular mouse. Interrater reliability for exercise duration and for microplate reader of antibody levels was maintained as exact agreement of values obtained by concurrent, independent measurement of 2 investigators. Two raters recorded the time of exercise duration on a single stopwatch for each mouse at every exercise session. Similarly, 2 raters recorded ELISA readings from the computer printout. The variation in anti-HSA values by ELISA from repeated tests on the same serum sample is within 10% and is typical of ELISA. (23)

Data Analysis

Seven mice were excluded from the study because their blood sample volumes were insufficient to perform ELISA. One mouse from the sedentary group (3 weeks of sedentary protocol prior to primary immunization group) had to be euthanized before completion of the study due to development of extensive dermatitis (Tab. 3).

For each group of mice, the number of animals and the mean and standard deviation for the anti-HSA antibody levels were calculated at each time point of measurement (Tab. 3). Prior to statistical analysis, normality of distribution and homogeneity of variance were tested for each dependent variable within each group using the Shapiro-Wilk test and the Bartlett test, respectively. As a result of large variances in antibody values among animals within the 2-week exercise group and the 8-week exercise group, an analysis of variance across all groups could not be used. A Kruskal-Wallis test was used to compare antibody response on day 10 and day 20 separately between the sedentary versus 8-week exercise versus 2-week exercise groups followed by a Tukey post hoc test when necessary. This analysis was chosen due to the high degree of deviation from normality and lack of homogeneity among the sedentary, 2-week exercise, and 8-week exercise groups of mice (Tab. 1). The data for comparison of the effect of 3 weeks of exercise prior to the primary immunization on primary and secondary antibody responses were approximately normal and homogenous; therefore, a 2-way, repeated-measures analysis of variance (ANOVA) was used. The factors were the exercise condition (sedentary and exercise preprimary immunization groups) and time of measurement (primary antibody response and secondary antibody response on day 10 and day 20); time of measurement was the repeated measure. A Tukey honestly significant difference test was performed, as indicated, for significant ANOVA findings. All statistical analyses were 2-tailed, and a criterion significance level ([alpha]) of P[less than or equal to]-.05 was used. Power for the statistical tests was 0.80 using 8 to 10 mice per group.

Results

Effect of an 8-Week Exercise Program Versus a 2-Week Exercise Program on Secondary Antibody Response to HSA

There was an overall effect of exercise on secondary antibody production in the 2-week exercise and 8-week exercise groups compared with sedentary control mice. The secondary antibody response on day 10 (P=.0139) and day 20 (P=.0353) was different among the sedentary, 8-week, and 2-week exercise groups (Tab. 2, Fig. 1).

[FIGURE 1 OMITTED]

The secondary antibody response on day 10 increased 3.01-fold with moderate exercise in the 8-week exercise group compared with the sedentary group (P<.05, Tab. 3, Fig. 1). The secondary antibody response on day 10 increased 4.88-fold in the 2-week exercise group compared with the sedentary group (P<.05, Tab. 3, Fig. 1). There was no difference in secondary antibody response on day 10 between the 8-week exercise and 2-week exercise groups (P>.05, Tab. 3, Fig. 1).

The secondary antibody response on day 20 showed a 2.13-fold increase in the 8-week exercise group compared with the sedentary group (P<.05, Tab. 3, Fig. 1). The secondary response on day 20 increased 2.46-fold in the 2-week exercise group compared with the sedentary group (P>.05, Tab. 3, Fig. 1). There was no difference in secondary antibody response on day 20 between the 2-week exercise and 8-week exercise groups (P>.05, Tab. 3, Fig. 1).

Effect of 3 Weeks of Exercise Prior to the Primary Immunization on Primary and Secondary Antibody Responses

Mouse serum anti-HSA antibody levels among exercise conditions (sedentary and exercise preprimary immunization groups) and times of measurement (primary antibody response and secondary antibody response at day 10 and day 20) were statistically significant (P[less than or equal to].05) for the main effect of time of measurement and for the interaction effect of exercise condition and time of measurement (Tab. 4).

Neither the primary antibody response nor the secondary antibody response on day 10 was different (P>.05, Tab. 3, Fig. 2) between the exercise preprimary immunization group and the sedentary group. However, the secondary response on day 20 increased 1.92-fold with exercise in the exercise preprimary immunization group compared with the sedentary group (P=.0417, Tab. 3, Fig. 2). Furthermore, within the exercise preprimary immunization group (Fig. 2), the secondary antibody response on day 20 was greater than the secondary antibody response on day 10 (P)=.002) and the primary antibody response (P<.0001), and secondary antibody response on day 10 was greater than the primary antibody response (P=.0009). Finally, within the sedentary group (Fig. 2), the secondary antibody response on day 20 was not greater than secondary antibody response on day 10; however, the secondary antibody response on day 20 (P=.006) and day 10 (P=.03) was greater than primary antibody response.

[FIGURE 2 OMITTED]

Discussion

The results of our study indicate that a 2-week exercise program between the primary immunization and the secondary immunization produces an increase in antibody levels comparable to that produced during an 8-week exercise program. The increase in secondary antibody response in the 8-week exercise group was seen both early (day 10) and late (day 20) in the response when compared with the sedentary group. However, the increase in secondary antibody response in the 2-week exercise group was seen early (day 10) but not late (day 20) in the response when compared with the sedentary group. This finding was due to 2 mice (out of a total of 8 mice) whose response was not different from that of the sedentary group on day 20. Without data from these 2 mice, the P value produced by the Kruskal-Wallis test was .0169 and the secondary antibody responses in both the 2-week exercise and 8-week exercise groups were greater than the secondary antibody response in the sedentary group on day 20 (P<.05). These results suggest that a moderate exercise program of 2 weeks may be sufficient for most, but not all, mice to enhance their secondary antibody response. Additional findings indicate that an exercise program including 3 weeks of exercise prior to the primary immunization in addition to exercise following primary immunization, while having no effect on primary antibody response, may not provide additional enhancement of secondary antibody response.

We chose a duration of 2 weeks based on studies of human adherence to exercise programs. (24) In women post-myocardial infarction who were given a moderate exercise program, two thirds of the women were adherent to the program for approximately 10 exercise sessions (exercising 3 days per week for 12 weeks). (24) Therefore, in order to maximize similarities between studies of exercise adherence in humans and studies of exercise adherence in mice, 2 weeks of exercise, an equivalent of 10 exercise sessions for humans, was chosen. Because our data showed that a moderate exercise program of 2 weeks may be sufficient to improve secondary antibody production, a 2-week exercise program may encourage better exercise adherence in humans and serve as a useful strategy to enhance antibody response to vaccinations.

Three weeks of moderate exercise prior to the primary immunization had no effect on the primary antibody response and is in accordance with previous studies that showed no enhancement of the primary antibody response with moderate exercise training. (2-4) However, our data showed that exercise prior to the primary immunization did affect the secondary antibody response. Although a statistical test across all groups could not be made due to differences in experimental designs (Tabs. 1 and 2), a general comparison between sedentary and exercise groups was performed. Two groups, the 8-week exercise group and the exercise preprimary immunization group, showed enhancement in the secondary antibody response on day 20, compared with their respective sedentary groups.

The 2-fold increase in antibody levels on day 20 for the 8-week exercise group was equivalent to the 2-fold increase in antibody levels on day 20 in the exercise preprimary immunization group. Therefore, exercise before the primary immunization or exercise following the primary immunization results in similar increases in the secondary immune response. Based on this comparison, it is possible to conclude that moderate exercise training prior to primary immunization offers no advantage in enhancement of secondary antibody response compared with exercise after primary immunization and that exercise following primary immunization may be sufficient to enhance secondary antibody response. The implication of this finding, we believe, is relevant clinically because exercise can be used to enhance antibody production to seroprotective levels following vaccination in humans. For example, 3 doses of hepatitis B vaccine are required for achieving seroprotective levels in humans. (25) Therefore, an exercise program of moderate intensity following the first dose of vaccine may result in enhanced antibody levels that are seroprotective and that reduce the number of doses required to achieve seroprotective antibody levels. This may reduce the costs associated with vaccination.

Although mice have been used for numerous immunobiological studies and several models of infectious diseases that occur in animals are similar to human infectious disease in terms of infective agents, hosts responses, and pathogenicity, (26) the effects of exercise have to be confirmed in studies of humans before we can generalize our findings to humans. Humans are exposed to a variety of environmental and nutritional factors that also influence immune function, and studies of humans could clarify the effects of an moderate exercise program under differing environmental and nutritional conditions.

There are 2 studies of humans that have examined the effects of intense, in contrast to moderate, exercise training on antibody response in young elite athletes. Eskola and co-workers (27) gave tetanus toxoid booster immunization to 4 highly conditioned long-distance runners (25-30 years of age) after they finished running a complete marathon (42.195 km). Two weeks later, the antibody levels to tetanus toxoid were greater in runners than in controls (59 nonexercising subjects). Gleeson et al (28) investigated the ability of elite swimmers to mount an antibody response to the pneumococcal vaccine, Pneumovax 23, (#) at the end of an intensive 12-week training program, and these swimmers were capable of mounting an antibody response to pneumococcal antigens equivalent to that of age- and sex-matched sedentary control subjects. The ability of a moderate exercise program to enhance antibody response has not been examined in sedentary humans.

Finally, the question that remains to be answered is why moderate exercise training has an impact on secondary antibody response, but not on primary antibody response. We believe it is unlikely that the hormones produced in an exercise intervention prior to primary or secondary antibody response are different. Thus, one possible mechanism to explain the difference in the impact of exercise on primary and secondary antibody response may relate to the expression of opioid receptors on naive and memory B and T lymphocytes. Naive lymphocytes have not encountered antigen and are exposed to antigen for the first time during primary antibody response, whereas memory lymphocytes retain memory of antigen encountered during primary antibody response and play an important role in the secondary antibody response. (1) A differential expression of opioid receptors on naive and memory lymphocytes may play a role in the differential response to primary and secondary antibody responses following exercise. Although we are not aware of any studies that have examined the expression of opioid receptors in naive and memory lymphocytes, it appears that quiescent helper T lymphocytes do not express classic opioid receptors, but the delta-type receptor can be induced upon activation. (29) Moreover, in vivo administration of lower physiologic doses of [beta]-endorphin (an endogenous opioid) has no effect on primary antibody response to a protein antigen, KLH, but enhances the secondary antibody response. (9) Similarly, [alpha]- and [beta]-endorphin (endogenous opioids) enhance the secondary antibody response to tetanus toxoid at the nanomolar and picomolar levels. (9) Furthermore, hormones other than endogenous opioids also have an effect on antibody response, and other mechanisms may be involved in the differential response to primary and secondary antibody responses following exercise. (30)

Although the focus of our study was to use moderate exercise training to enhance immune response to vaccination, moderate exercise training is also believed to reduce infections. In one randomized controlled study of 36 young women (mean age=35 years), exercise subjects (45 minutes of walking, 5 days a week) experienced 50% less (one-half) days with upper respiratory tract infection symptoms during the 15-week period compared with a sedentary control group. (31)

Conclusions

Conditioning exercise is an important therapeutic intervention used by physical therapists in the management of patients with many clinical problems. The effects of exercise on the cardiopulmonary and musculoskeletal systems have been well characterized. (32) However, the effects of exercise on the immune system are only beginning to be explored. Moderate exercise training may enhance immune function, whereas intense training suppresses it. (33) Because patients with compromised immune systems (eg, aged patients, those with cancer or human immunodeficiency virus infection, and those who have had organ transplants or surgery) frequently receive physical therapy interventions, an understanding of the effect of exercise on immune function and the physiological mechanisms underlying these effects is important. Potentiation of immune responses by moderate exercise could improve the quality of life of different individuals and may expand the role of physical therapy in an important area of health care.

The findings of our study support the hypothesis that a shorter exercise program of 2 weeks may be sufficient to enhance secondary antibody response. Confirming these findings in humans, using a moderate-intensity exercise program over a short duration (thereby improving adherence), may offer a viable strategy to enhance antibody response to vaccinations.

Table 1.
Experimental Design to Evaluate the Effects of an 8-Week Exercise
Program Versus a 2-Week Exercise Program on the Secondary Antibody
Response

Sedentary Group                    8-Week Exercise Group

Primary immunization               Primary immunization
Rest for 3 weeks                   Rest for 3 weeks
Start sedentary protocol and       Start exercise protocol and
  continue for 8 weeks               continue for 8 weeks
Secondary immunization             Secondary immunization
Continue sedentary protocol for 9  Continue exercise protocol for 9
  days                               days
Rest for 1 day                     Rest for 1 day
Bleed and assess secondary         Bleed and assess secondary
  antibody response (day 10)         antibody response (day 10)
Continue sedentary protocol for    Continue exercise protocol for
  another 9 days                     another 9 days
Rest for 1 day                     Rest for 1 day
Bleed and assess secondary         Bleed and assess secondary
  antibody response (day 20)         antibody response (day 20)

Sedentary Group                    2-Week Exercise Group

Primary immunization               Primary immunization
Rest for 3 weeks                   Rest for 3 weeks
Start sedentary protocol and       Start exercise protocol and
  continue for 8 weeks               continue for 2 weeks
Secondary immunization             Secondary immunization
Continue sedentary protocol for 9  Continue exercise protocol for 9
  days                               days
Rest for 1 day                     Rest for 1 day
Bleed and assess secondary         Bleed and assess secondary
  antibody response (day 10)         antibody response (day 10)
Continue sedentary protocol for    Continue exercise protocol for
  another 9 days                     another 9 days
Rest for 1 day                     Rest for 1 day
Bleed and assess secondary         Bleed and assess secondary
  antibody response (day 20)         antibody response (day 20)

Table 2.
Experimental Design to Evaluate the Effects of 3 Weeks of Exercise
Prior to the Primary Immunization on Primary and Secondary
Antibody Responses

Sedentary Group                     Exercise Preprimary Immunization
                                    Group

Start sedentary protocol and        Start exercise protocol and
  continue for 3 weeks                continue for 3 weeks
Primary immunization                Primary immunization
Continue sedentary protocol for 2   Continue exercise protocol for 2
  weeks                               weeks
Bleed and assess primary antibody   Bleed and assess primary antibody
  response and give secondary         response and give secondary
  immunization                        immunization
Continue sedentary protocol for 9   Continue exercise protocol for 9
  days                                days
Rest for 1 day                      Rest for 1 day
Bleed and assess secondary          Bleed and assess secondary
  antibody response (day 10)          antibody response (day 10)
Continue sedentary protocol for 9   Continue exercise protocol for 9
  days                                days
Rest for 1 day                      Rest for 1 day
Bleed and assess secondary          Bleed and assess secondary
  antibody response (day 20)          antibody response (day 20)

Table 3.
Number of Subjects and Mean and Standard Deviation Values of Mouse
Serum Anti-Human Serum Albumin Antibody Levels (in Micrograms per
Milliliter)

                                Sedentary

Mouse Group/Measurement         n    [bar]X  SD

8-week exercise group            9
  Secondary response on day 10        182.5  101.6
  Secondary response on day 20        425.9  342.2

2-week exercise group            9
  Secondary response on day 10        182.5  101.6
  Secondary response on day 20        425.9  342.2

Exercise preimmunization        10
  Primary response                     21.8   22.9 (a, b)
  Secondary response on day 10        183.4  171.6 (a)
  Secondary response on day 20        227.1  193.1 (b)

                                Exercise

Mouse Group/Measurement         n   [bar]X   SD

8-week exercise group            7
  Secondary response on day 10        549.4    376.5 *
  Secondary response on day 20        905.3    538.8 *

2-week exercise group            8
  Secondary response on day 10        890.3  1,149.6 *
  Secondary response on day 20      1,047.1  1,098.2

Exercise preimmunization         8
  Primary response                     20.9     13.6 (c)
  Secondary response on day 10        239.9    145.4 (c)
  Secondary response on day 20        436.6    204.3 *, (c)

* P < .05 compared with corresponding sedentary group.

(a,b,c) Values sharing lower-case letters are different (P < .05).

Table 4.
Results of 2-Way Repeated-Measures Analysis of Variance Comparing
Mouse Serum Anti-Human Serum Albumin Antibody Levels Among Exercise
Condition and Time of Measurement

Source of Variance    df  SS         MS         F      P

Exercise               1  104181.19  104181.19   2.55   .1296
Time of measurement    2  871575.18  435787.59  36.16  <.0001 (a)
Exercise x time of
  measurement          2  105141.59   52570.80   4.36   .0211 (a)
Error                 16  652929.74   40808.11
Error (time)          32  385608.71   12050.27

(a) P [less than or equal to] .05.

* The Jackson Laboratory, 600 Main St, Bar Harbor, ME 04609.

([dagger]) Molecular Devices Corp, 1311 Orleans Dr, Sunnyvale, CA 94089-1136.

([double dagger]) Road Master Corp, 4501 Old Troup Hwy, Tyler, TX 75707.

([section]) Rohm & Haas Co, Independence Mall W, Philadelphia, PA 19105.

([parallel]) Jackson Immunoresearch, 872 W Baltimore Pike, West Grove, PA 19390.

(#) Merck & Co Inc, West Point, PA 19486.

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ZF Kapasi, PT, PhD, is Assistant Professor, Division of Physical Therapy, Department of Rehabilitation Medicine, Emory University School of Medicine, 1441 Clifton Rd NE, Atlanta, GA 30322 (USA) ([e-mail]). Address all correspondence to Dr Kapasi.

PA Catlin, PT, EdD, is Professor and Director, Division of Physical Therapy, Department of Rehabilitation Medicine, Emory University School of Medicine.

MA Adams, PT, MPT, is Staff Physical Therapist, HEALTHSOUTH Rehabilitation Hospital of Central Kentucky, Elizabethtown, Ky.

EG Glass, PT, MPT, is Contract Physical Therapist, 627 Coventry Rd, Decatur, Ga.

BW McDonald, PT, MPT, is Staff Physical Therapist, Bannock Regional Medical Center, Pocatello, Idaho.

AC Nancarrow, PT, MPT, is Staff Physical Therapist, Thomas Jefferson University Hospital, Philadelphia, Pa.

Ms Adams, Ms Glass, Mr McDonald, and Ms Nancarrow were graduate students, Department of Rehabilitation Medicine, Division of Physical Therapy, Emory University, during this study, which was undertaken in partial fulfillment of the requirements for their Master of Physical Therapy degree.

Dr Kapasi provided concept/idea and research design, and Dr Catlin, Ms Adams, Ms Glass, Mr McDonald, and Ms Nancarrow provided research design. Dr Kapasi, Ms Adams, Ms Glass, Mr McDonald, and Ms Nancarrow provided data collection. All authors provided writing, data analysis, project management, and consultation (including review of manuscript before submission). Dr Kapasi and Dr Catlin provided subjects, fund procurement, facilities/equipment, and institutional liaisons. Ms Adams, Ms Glass, Mr McDonald, and Ms Nancarrow provided clerical support.

This study was approved by the Institutional Animal Care and Use Committee of Emory University School of Medicine.

This research was presented at the 5th International Society of Exercise and Immunology (ISEI) Symposium; May 29-31, 2001; Baltimore, Md.

This article was submitted September 23, 2002, and was accepted February 10, 2003.