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2 Research and Development Department, House Wellness Foods, Itami 664-0011, Japan; 3 Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and 4 PS Clinic, Medical LTA, Hakata-ku, Fukuoka 812-0025, Japan
* To whom correspondence should be addressed. E-mail: hirose_yoshitaka{at}house-wf.co.jp.
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ABSTRACT |
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 TOP ABSTRACT IntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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and IL-4–producing cluster of differentiation (CD)4+ T cells (Th1:Th2 ratio), and the serum IgG4:IgG ratio were measured every 4 wk or at wk 0 and wk 12. Health-related QOL was assessed using a self-rating questionnaire with 26 items. Among the measured biomarkers, the percent change in Con A-induced proliferation and the Th1:Th2 ratio in the HK-LP group was greater than those in the control group (P = 0.036 and P = 0.002, respectively). The degree of improvement in QOL was higher in the HK-LP group than in the control group at wk 8 (P = 0.049) and tended to be higher at wk 12 (P = 0.092). These results suggest that a daily intake of HK-LP augments acquired immunity, especially Th1-related immune functions in healthy subjects, thereby improving the health-related QOL.
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Introduction |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Various lines of evidence in human studies have revealed that foods and their components can improve immune function in a wide range of human subjects whose immune function is poorer than expected. Systematically analyzing such evidence, Kaminogawa et al. (9) have concluded that the improvement of immune function by food ingredients can be evaluated by measuring the general biomarkers for innate immunity [phagocytic activity and natural killer (NK) activity] and acquired immunity (delayed-type hypersensitivity, antigen-specific antibody production, proliferative response of T cells, and T cell number). Moreover, improvement of these biomarkers has been shown to correlate with reduction of the incidence of pathogenic infection (9).
Poor immune function likely results not only in high susceptibility to infections but also deterioration of the health-related quality of life (QOL). In fact, among patients with chronic fatigue syndrome, individuals with lower T cell proliferation have been reported to show poor health-related QOL scores (10). In addition, we have previously reported that nigerooligosaccharide, which is an immunopotentiating agent, augments T-cell mitogen response in association with improvement of the health-related QOL in the elderly (11). Therefore, enhancement of immune function may lead to improvement of the health-related QOL.
The objective of this study was to examine whether intake of HK-LP affects innate and acquired immune functions and health-related QOL in healthy subjects.
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Subjects and Methods |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Preparation of HK-LP. LP20 (House Wellness Foods), which contains 20% HK-LP and 80% dextrin, was used in this study. HK-LP was prepared on the basis of the method previously described (5).
Experimental design. Sixty healthy subjects were enrolled in a randomized, double-blind, placebo-controlled, parallel study. The subjects were randomly assigned by age (middle-aged or elderly) and sex into 2 groups using a sequential series of numbered, sealed envelopes, each containing 1 food ingredient assigned in a computer-randomized fashion. After the assignment, the subjects consumed a hard gelatin capsule containing 50 mg of LP20 (1 capsule/d) or a matching placebo capsule in which dextrin was substituted for LP20 for 12 wk. Throughout the study, the general health of subjects was assessed every 4 wk via direct interviews conducted by the health care provider. Subjects were asked to confirm their compliance with the dietary regimens. Blood samples were collected every 4 wk. Biomarkers for immune function were measured by a clinical laboratory testing company, SRL, every 4 wk. The IgG4:IgG ratio was measured at 0 and 12 wk. Safety assessments such as anthropometric measurements, biochemical examinations of blood, hematological assessments, and urine tests were done at 0 and 12 wk. The study was conducted at PS Clinic (Fukuoka, Japan) from May to August 2005.
Biomarkers for innate immunity. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-Conray centrifugation technique (Ficoll-Conray, density = 1.077; centrifuged at 400 x g for 30 min). The NK activity of PBMC was assessed by a standard chromium release method using K562 tumor cells at an effector:target ratio of 20:1 for 3.5 h (12). The neutrophil phagocytosis function was analyzed by flow cytometry (FACSCalibur; Becton Dickinson) after uptake of fluorescein isothiocyanate (FITC)-labeled latex beads (Polyscience) by whole blood. The results are expressed as the percentage of FITC-positive cells, which correspond to cells phagocytosing 1 or more beads, vs. total neutrophils. Cell surface expression of cluster of differentiation (CD)64 on monocytes was analyzed by FACSCalibur after whole blood samples were stained with FITC-conjugated CD64-specific mAb (Becton Dickinson). The results are expressed as the mean fluorescence intensity of CD64-positive monocytes.
Biomarkers for acquired immune functions.
PBMC (5 x 108 cells/L) were cultured with or without suboptimal dose (2 mg/L) of concanavalin A (Con A) for 72 h at 37°C and pulse labeled with 37 kBq of [3H]-thymidine during the last 8 h. DNA synthesis was assessed by measuring thymidine uptake. Percentages of INF- and IL-4-producing CD4+ T cells (Th1:Th2 ratio) were determined by single-cell measurement of intracellular cytokines using flow cytometry as described (13). Serum IgG was measured quantitatively by the latex-agglutination method using N-assay TIA IgG-SH (Nittobo) and IgG4 was measured by nephelometry using IgG subclass BS-NIA IgG4 (The Binding Site).
Health-related QOL. Health-related QOL was assessed every 4 wk using a self-rating questionnaire with 26 items, as shown in Supplemental Table1 (11). Each question was scored from 1 to 3; a higher score indicated a better QOL. Increase in the total QOL score compared with that of the baseline was defined as improved QOL, and unchanged and decreased total QOL scores were defined as unchanged and deteriorated QOL, respectively. QOL changes were scored as follows: improved QOL, 1; unchanged QOL, 0; and deteriorated QOL, –1.
Statistical analysis. We compared baseline values between groups using the unpaired t test for age and immune functions or the Mann-Whitney U test for total QOL score. We calculated Spearman correlation coefficients between age and immune functions. Because the methods for assessing immune function are based on a highly complicated process including cell culture, we thought that intra-assay variation would increase over time. Therefore, we compared the measurements between groups at the same time point but not within a single individual over time. Percent changes from the baseline in immune functions other than the IgG4:IgG ratio were analyzed by 2-way ANOVA, followed by comparison between groups at each time point using the unpaired t test. We compared QOL change scores between groups by the Mann-Whitney U test. All analyses were performed in all subjects as well as in subjects by age (middle-aged, 40–64 y, and elderly, >64 y) and sex (male and female) using Dr. SPSS software version 8.0J (SPSS Japan). Differences of P < 0.05 were considered significant.
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Results |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Effects of dietary intervention on health-related QOL. The QOL change score, the degree of improvement in QOL, was higher at 8 wk (P = 0.049) and tended to be higher at 12 wk (P = 0.092) in the HK-LP group than in the control group (Table 2).
Safety assessments. Three adverse events in the control group and 6 in the HK-LP group were recorded during the study. The adverse events other than mentioned above were knee arthralgia in the control group and Meniere's syndrome, dizziness, and acute urticaria in the HK-LP group. All adverse events were judged to be unrelated to the dietary intervention. Among the safety assessments that we performed, the percent change in lymphocyte count was greater in the HK-LP group than in the control groups (P < 0.05), but the changes were within the range of reference values.
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Discussion |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Because there are many reports of improvement of immune function by food-derived components in healthy individuals, systematic analysis of these reports enables us to advocate reliable biomarkers for assessing the immunomodulating effects of food-derived substances. By doing this, Kaminogawa et al. (9) have advocated general biomarkers for assessing immune function. To examine the effects of HK-LP intake on immune function in healthy individuals, we measured such general biomarkers, including phagocytic activity, NK activity and the proliferative response of T cells. Among these biomarkers, the proliferative response of T cells was considerably augmented by HK-LP. The acquired immunity such as the proliferative response of T cells reportedly decreases as a result of aging and of stress (14–18). In accordance with these observations, Con A-induced proliferation was decreased in association with aging in this study. The poorer acquired immunity was reported to correlate with the risk of various infectious diseases (14–18). Therefore, the fact that intake of HK-LP augmented the proliferative response of T cells during the dietary period suggests that HK-LP is beneficial for the prevention of infectious diseases.
Several lines of evidence show that dietary consumption of probiotic lactic acid bacteria can enhance the activity of NK cells in healthy adult subjects (19,20). Although the intake of HK-LP did not affect NK activity in all subjects (Table 2), it did affect NK activity in men and the middle-aged subjects. We do not know why other subjects did not respond to HK-LP, but the reason may be related to the strong influence of the menstrual cycle on NK activity in females (21) and to age-related changes in the responsiveness to external stimuli in the elderly. HK-LP did not enhance the neutrophil phagocytosis function, although lactic acid bacteria have been reported to enhance phagocytic activity (19,20). We assessed the neutrophil phagocytosis function by a conventional clinical method for assessing neutrophil dysfunction, in which functional neutrophils that could phagocytose 1 or more beads exceeded 90% of the total neutrophils in most healthy subjects. Thus, the method is not appropriate for evaluating the effect of immunomodulators on phagocytic activity in healthy subjects. To clarify the effects of HK-LP on innate immunity in healthy subjects, further examinations are needed.
We examined the effects of the intake of HK-LP on Th1-related immune function by determining the Th1:Th2 ratio of CD4+ T cells. Intake of HK-LP considerably augmented the Th1:Th2 ratio in healthy subjects, as was observed previously in a study in which a number of lactic acid bacteria skewed T cells toward Th1 polarization in vitro (7). This result suggests that HK-LP may decrease risk for the development of infectious and allergic disease associated with a low Th1:Th2 ratio. We also measured the expression of CD64 on monocytes and the serum IgG4:IgG ratio as a candidate for a biomarker for Th1-related immune function on the basis of observation that in vivo treatment with INF- in healthy human subjects led to an increased expression of CD64 (FcRI) on monocytes (22) and that higher Th1:Th2 ratios enhanced IgG4 antibody production from PBMC in subjects who responded to bee venom (23). However, intake of HK-LP did not affect either the expression of CD64 on monocytes or the serum IgG4:IgG ratio in all subjects (Table 2). Therefore, it seems that these biomarkers are not appropriate for assessing Th1-related immune function in healthy subjects.
In the measurements of either Con A-induced proliferation or the Th1:Th2 ratio, a time point effect was observed (Table 2). Because the methods for measurement of these biomarkers are based on a highly complicated process including cell culture, measurement noise must be considered. Intra-assay variation is thought to be lower in measurements at the same time point than in those at different time points. In addition, measurement reliability can be enhanced by measuring a control specimen. Therefore, we considered that the comparison between groups at the same time point was reliable but that those over time were not. However, we could not exclude the possibility that Con A-induced proliferation and Th1:Th2 ratio varied seasonally.
QOL has been defined by the WHO as "the state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity." Schipper suggested that QOL has 4 components: physical and occupational functions, social interaction, psychological state, and somatic sensation (24). To investigate the effects of HK-LP on health, we measured health-related QOL using a self-rating questionnaire aimed at both physical and psychological states. The degrees of improvement in the health-related QOL were higher from wk 8 in the HK-LP group following the augmentation of the immune function such as Con A-induced proliferation and the Th1:Th2 ratio, which were seen from wk 4. These results suggest that improvement of the health-related QOL by HK-LP is accomplished by the enhancement of immune function in healthy subjects.
In conclusion, we demonstrated that a daily intake of HK-LP enhanced acquired immunity, especially Th1-related immune functions in healthy subjects, in addition to inducing subsequent improvements of the health-related QOL. Our results suggest that intake of HK-LP may be useful for prevention and treatment of infectious and allergic diseases induced by a weak Th1-type immune response.
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FOOTNOTES |
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5 Abbreviations used: CD, cluster of differentiation; Con A, concanavalin A; FITC, fluorescein isothiocyanate; HK-LP, heat-killed Lactobacillus plantarum L-137; NK, natural killer; PBMC, peripheral blood mononuclear cell; QOL, quality of life; Th, T helper.
Manuscript received 21 June 2006. Initial review completed 16 July 2006. Revision accepted 24 September 2006.
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LITERATURE CITED |
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