Abdominal breathing increases salivary IgA secretion.
Today, the average life expectancy is over 80 yrs of age in many countries. Although a major cause of death is malignancy, pneumonia is also a major cause of deaths in the elderly. Pneumonia ranks as the fourth leading cause of death in Japan in people 60 yrs of age or older, and the percentage of elderly deaths due to pneumonia is increasing. In order to prevent the development of pneumonia, it is necessary to improve mucosal immunity in the upper respiratory tract.
Regular exercise has received considerable attention for its positive influence on health preservation in the elderly and those with disabilities. While many studies have investigated the role of treadmill exercise on health and well-being, these studies have primarily focused on the professional athlete (10,17). Relatively speaking, only a few reports have focused on the effects of exercise in the general population and chronically ill older adults (14,27).
Secretory immunoglobulin A (SIgA) is the dominant immunoglobulin isotype found on mucosal surfaces where it acts as a first line of defense against microbial invasion. It is composed of two molecules of IgA, joining (J) chain and a polymeric immunoglobulin receptor (pIgR) (16). IgA and J chain are synthesized by plasma cells in the submucosa and secreted as dimeric (d) IgA. dIgA bind to pIgR on the basement membrane of mucosal epithelial cells. The dIgA-pIgR complex is transported to the apical membrane and released to mucosal surface as SIgA. SIgA has been shown to inhibit the attachment of viruses and bacteria, to neutralize toxins, and to show antibody-dependent cytotoxicity (12,30,34). Recent investigations (7,8) suggest that the SIgA concentrations vary throughout the day due to a range of variables including dietary factors, daily mood, and exercise.
Cortisol is a steroid hormone. It is an important member of the glucocorticoid family. Cortisol is secreted from the adrenal cortex via the hypothalamic-pituitary-adrenal axis in response to stressors (including physical exertion). Increased cortisol levels are associated with anxiety, depression, and intensive physical exercise (23). Cortisol is involved catabolic processes as it reduces protein synthesis, increases protein degradation and inhibits inflammatory processes and immunity. Measuring cortisol concentration in saliva provides an important reference for blood cortisol levels. Cortisol monitoring during sports detects the stress response to physical exertion in exercise or training (24,32,33).
The present study investigated the effects of two different simple exercises, treadmill and abdominal breathing, on the concentration of SIgA and cortisol in saliva. Two generations of subjects were examined (40-yr olds vs. 60-yr olds) in order to compare age differences. We hypothesized that the data would support the development of future prevention program for older individuals.
The study protocol received ethical approval in accordance with the committee of Nihon University School of Dentistry. Fifty-four healthy adult subjects in their 40s (16 male and 16 female) or 60s (11 male and 11 female) volunteered to participate in this study (Table 1). The subjects consisted of two groups: those who exercised on treadmill (TM group, 16 subjects in their 40s and 10 subjects in their 60s) and those who performed abdominal breathing supervised by an instructor (AB group, 16 subjects in their 40s and 12 subjects in their 60s). The TM group exercised for 30 min at 6 km x [h.sup.-1] while the AB group performed exercises for 30 min.
Assay Procedures: Saliva Collection
All subjects rinsed their mouths with 50 ml of water 20 min before starting the exercises. During the exercises, the subjects were prohibited from taking any food or drink. The saliva was collected from each subject into 50 ml plastic test tubes before and after exercise. All samples were centrifuged at 14,000 rpm for 5 min, and supernatant was maintained at -20[degrees]C until examination.
The concentration of total protein in each saliva sample was measured using the DC protein assay kit (BIO RAD). Each saliva sample was diluted 5 times with PBS 0.05% Tween20 (PBS-T). Five ml of saliva and standard solution was mixed with 25 [micro]l of reagent A and 200 [micro]l of reagent B in a micro-titer plate. The reaction mixture was maintained at room temperature (RT) for 15 min. The amount of absorption of each mixture was measured with a 595 nm plate reader (BIO RAD). Bovine serum albumin was used as standard protein.
The SIgA concentration in the saliva was measured with a EIA-SlgA test kit (Medical & Biological Laboratories). Each saliva sample was diluted 40 times with a reaction buffer and 10 [micro]l of the diluted sample and standard solution was added to 400 [micro]l of reaction buffer. Styrene beads coated with antihuman SC antibody were incubated with each sample and standard solution for 1 hr at RT. After being adequately washed with PBS, the beads were reacted with HRP conjugated anti-human IgA antibody. The beads were rewashed with PBS and, then, in an incubated o-phenylene diamine solution containing [H.sub.2][O.sub.2] (OPD) for 20 min to colorization. [H.sub.2]S[O.sub.4] solution was added to the reaction mixture to stop the reaction. The amount of absorption of each reaction mixture was detected with a spectrophotometer (Beckman).
The FSC concentration in each saliva sample was measured using ELISA. Fifty ml of anti-free human SC antibody (Nordic) diluted 1000 times with 1% BSA-PBS were applied to each well of a micro-titer plate. The wells were washed three times with PBS-T and, then, 200 [micro]l of 1% BSA-PBA were applied for 1 hr to block nonspecific binding. Then, the solution was discarded. Fifty [micro]l of the samples or standard solution were applied to each well and incubated for 1 hr at RT. After washing was completed, 50 [micro]l of Hrp-anti human SC antibody (DAKO) were added to the wells and incubated for 1 hr at RT. One hundred [micro]l of OPD solution were added to each well for colorization of HRP-conjugated antibody (Hrp) and incubated for 20 min. Twenty-five [micro]l of 2M [H.sub.2]S[O.sub.4] were added to stop the reaction. The amount of absorption of each reaction mixture was measured on the plate reader. The FSC used as standard solution was purified from human colostrums previously prepared in our laboratory (4).
The concentration of cortisol in each saliva sample was measured using the SALIVARY CORTISOL ENZYME IMMUNOASSAY KIT (Salimetrics). Twenty-five ml of samples and standard was applied to each well of the microtiter plate. Fifty-five [micro]l of Hrp conjugated cortisol were applied to each well and incubated for 1 hr at RT. After washing was completed, 200 [micro]l of OPD solution were added to each well for colorization and incubated for 25 min. Twenty-five [micro]l of 2M [H.sub.2]S[O.sub.4] were added to stop the reaction. The amount of absorption of each reaction mixture was measured on the plate reader.
The values are expressed as the mean [+ or -] SD for all the subjects. All statistical comparisons of data obtained before and after exercise were performed using the Student's paired t-test. A alpha level of P=0.05 was considered to be statistically significant.
Concentration of Total Protein
The total protein concentration of each sample is shown in Figure 1. The average concentration of total protein before exercise in the AB group of the subjects in their 40s was 2.5 [+ or -] 0.5 mg/ml that of the subjects in their 60s was 2.5 [+ or -] 1.2 mg/ml. In the TM group, the average concentration of total protein before exercise in the subjects in their 40s was 2.9 [+ or -] 0.7 mg/ml and that of the subjects in their 60s was 2.5 [+ or -] 3.1 mg/ml. The concentration of total protein in saliva measured after exercise was not statistically different (P>0.05) from before exercise.
[FIGURE 1 OMITTED]
Concentration of SIgA
The concentration of SIgA in saliva showed some differences with respect to age in the AB group (Figure 2). In the AB group, the average concentration of SIgA before exercise in the subjects in their 40s was 234 [+ or -] 45 mg/ml and that of the subjects in their 60s was 367 [+ or -] 76 mg/ml. In the TM group, the average concentration of SIgA before exercise in the subjects in their 40s was 365 [+ or -] 66 mg/ml and that of the subjects in their 60s was 463 [+ or -] 115 mg/ml. The concentrations of SIgA in the TM group were higher than those in the AB group. In the AB group, the average concentration of SIgA measured after exercise in the subjects in their 40s was 384 [+ or -] 52 mg/ml and that of the subjects in their 60s was 595 [+ or -] 172 mg/ml. The concentration of SIgA significantly increased after exercise in the AB group. However, no differences were observed in SIgA concentration measured before and after exercise in TM group.
[FIGURE 2 OMITTED]
Concentration of FSC
In the AB group, the average FSC concentration in saliva measured before exercise in the subjects in their 40's was 2.9 [+ or -] 0.4 mg/ml and that in the subjects in their 60's was 2.0 [+ or -] 0.3 mg/ml (Figure 3). In the TM group, the average FSC concentration in saliva measured before exercise in the subjects in their 40's was 5.6 [+ or -] 1.2 mg/ml and that in the subjects in their 60's was 6.0 [+ or -] 1.8mg/ml. The FSC concentration in the subjects in their 60's in the AB group were significantly increased. The concentrations of FSC in saliva in the TM group were higher than those in the AB group. However, no significant differences were found in the ratio of concentrations measured before and after exercise.
[FIGURE 3 OMITTED]
Concentration of Cortisol
The concentrations of cortisol in saliva measured after exercise were decreased in all subjects (Figure 4). In the AB group, the average cortisol concentration measured before exercise in the subjects in their 40s was 0.121 [+ or -] 0.02 [micro]g/dl and that in the subjects in their 60s was 0.104 [+ or -] 0.01 mg/dl. In the TM group, the average cortisol concentration measured before exercise in the subjects in their 40s was 0.118 [+ or -] 0.01 [micro]g/dl and that the subjects in their 60s was 0.122 [+ or -] 0.01 [micro]g/dl. In the AB group, the average cortisol concentration measured after exercise in the subjects in their 40's was 0.100 [+ or -] 0.01 [micro]g/dl and the the subjects in their 60s was 0.080 [+ or -] 0.01 mg/dl. In the TM group, the average cortisol concentration measured after exercise in their 40s was 0.082 [+ or -] 0.02 [micro]g/dl and that in the subjects in their 60s was 0.086 [+ or -] 0.0 [micro]g/dl.
[FIGURE 4 OMITTED]
Saliva encourages soft tissue repair by reducing clotting time and accelerating wound contraction. The protective function of saliva results from the salivary role in the maintenance of ecological balance in the oral cavity. This is carried out by debridement/lavage, aggregation and the reduction of adherence by both immunological and non-immunological means, and direct antibacterial activity. Saliva also possesses anti-fungal and anti-viral properties. Saliva effectively maintains pH in the oral cavity, thus contributing to the regulation of plaque pH. It also helps to neutralize reflux acids in the esophagus (11). SIgA is the most abundant antimicrobial protein in mucus secretions, including saliva in the oral cavity. Salivary IgA functions as part of innate immunity. It is considered the best indicator of mucosal immunity since it act as the first line of defense by neutralizing and preventing viral pathogens from entering the body via mucosal surfaces (28).
In the present study, we demonstrated that the SIgA secretion is significantly increased following abdominal breathing (AB) supervised by an instructor in subjects in their 40s and 60s. On the other hand, 30 min of treadmill exercise did not significantly alter the SIgA level in saliva. This is an interesting finding since many studies have indicated that physical activities and susceptibility to upper respiratory tract infections are associated with decreased SIgA secretion in saliva in a "J-shaped" relationship (20). Decreases in the SIgA levels have been shown to be associated with increased incidences of upper respiratory tract infections in elite athletes and college football players (3,5,6,9,21,22). This discrepancy may indicate subtle differences in the subject characteristics and the quality and type of their exercise programs. Our subjects exercised at recreational levels while others studies have focused on elite athletes.
Polymeric Ig receptor (pIgR) present on mucosal epithelial cells can bind to dimeric IgA and pentameric IgM. The pIgR-dimeric IgA complex is transported from the basal membrane to the apical membrane of epithelial cells and released on the mucosal surface as SIgA. The extra cellular domain of pIgR cleaved from the cell membrane is combined with pIgs. However, 60% of the total pIgR in mucosal secretions is present as pIgR without dimeric IgA or IgM as the so-called free secretory component (FSC) (1). There exist few reports regarding FSC concentrations in secretions such as saliva because of technical difficulties associated with measuring methods. We developed a specific antibody against polyclonal and monoclonal anti-human SC. In addition, the functions of FSC have not been made clear. The inhibition of adhesions of E coli and Candida to epithelial cell lines has recently been reported (2,31). Additionally, the FSC inhibits the chemotactic activity of IL-8 by using the carbohydrate of FSC. Interestingly, since FSC is thought to posses anti-inflammatory and anti-infective properties (13,19), it is important to point out that FSC increased in the 60-yr-old subjects in the AB group (while the levels of FSC measured in the TM group were higher than those measured in the AB group).
The cortisol present in saliva originates from passive diffusion of cortisol from the blood. Thus, the concentration of cortisol found in saliva closely reflects the levels of unbound cortisol in blood (32,33). Monitoring the salivary cortisol level is good method of evaluating health or disease under stressful condition. Both the noninvasiveness and ease of sampling and storage of saliva for testing are considered to be important. The salivary cortisol level is an inexpensive, yet valid and reliable measure of the level of bioactive cortisol in the body. It represents responses to short, high intensity bouts of exercise, and hormonal changes to exercise show greater sensitivity in saliva than corresponding blood measurements (29). That is why salivary cortisol concentration quickly increases following sporting events such as rugby matches, competitive kickboxing matches, wrestling competitions and Bruce treadmill test (15,18,25,26).
The findings in the present study indicate that the cortisol level is decreased after exercise both abdominal breathing and treadmill exercises. Additionally. no difference in cortisol levels was observed between subjects in their 40s and those in their 60s. The results suggest that the abdominal breathing and treadmill exercise performed in this study were not significantly stressful for the subjects. That is, the intensity of the recreational exercises used in the present were not stressful, but rather had a relaxing effect on the subjects. It is noteworthy that the level of SIgA increased with mild exercise such as the abdominal breathing. It may be possible to apply this finding to elite athletes who engage in heavy physical exercise in order to prevent decrease in SIgA secretion in saliva. Hence, these results should be useful in preventing upper respiratory infection in the elderly and those with any disabilities.
This study was supported in partly by the Sato Fund, Nihon University School of Dentistry (20102011), and by Uemura Fund Nihon University School of Dentistry.
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Hatsumi Inagawa [1,2], Takashi Iwase , Koichi Nagai , Shuichi Watanabe , Kazuo Komiyama 
 Department of Pathology, Nihon University School of Dentistry, Tokyo, Japan;  Department of Physiology, Saitama Medical University, Saitama, Japan
Address for correspondence: Dr. Kazuo Komiyama, Department of Pathology, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo,101-8310, Japan. Email:email@example.com
Table 1. Number of Subjects and Average of Age in Each Group. 40-yr olds 60-yr olds AB group 16 (44.5 [+ or -] 2.6) 12(66.6 [+ or -] 1.6) TM group 16 (46.5 [+ or -] 2.4) 10(64.6 [+ or -] 2.7)
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|Title Annotation:||immunoglobulin A|
|Author:||Inagawa, Hatsumi; Iwase, Takashi; Nagai, Koichi; Watanabe, Shuichi; Komiyama, Kazuo|
|Publication:||Journal of Exercise Physiology Online|
|Date:||Oct 1, 2012|
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