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Keywords:

  • Probiotic;
  • Adjuvant effect;
  • Immunomodulation;
  • Lactobacillus GG;
  • Lactococcus lactis

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Thirty healthy volunteers were randomised into three different treatment groups and consumed Lactobacillus GG, Lactococcus lactis or placebo (ethyl cellulose) for 7 days. On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection. All subjects responded well to the vaccine, but no significant differences were observed in numbers of IgA-, IgG- and IgM-secreting cells among the different groups. There was a trend towards a greater increase in specific IgA among the subjects receiving the vaccine in combination with Lactobacillus GG. Those receiving L. lactis with their vaccine evinced significantly higher CR3 receptor expression on neutrophils than those receiving either the placebo or Lactobacillus GG. These results indicate that probiotics may influence differently the immune response to oral S. typhi vaccine and that the immunomodulatory effect of probiotics is strain-dependent.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

More than 400 species of bacteria are estimated to reside in the human gastrointestinal tract and these endogenous bacteria comprise a complex human intestinal microflora [1]. Even more species may be present but not culturable by the traditional plate count method [2]. The intestinal microflora is known to contribute to the development and function of the mucosal barrier and colonisation resistance in the human intestine. Mechanisms may include competition for nutrients and adhesion receptors, production of antimicrobial substances and stimulation of gut-associated lymphoid tissue. The resulting enhancement of the gut mucosal barrier may prevent the invasion of pathogens and assist in handling antigens [3]. The maintenance of a healthy human intestinal microflora is beneficial for health and well-being [4].

Probiotic microbes offer one means of modulating the human intestinal microflora [4–6]. Probiotics are defined as microbial cell preparations or components of microbial cells which have a beneficial effect on the health and well-being of the host [7]. Although viable probiotics are generally recommended, health-promoting effects of inactive probiotics have also been observed [8]. Most current probiotics are lactic acid bacteria, especially the Lactobacillus and Bifidobacterium strains, which often possess adhesive properties and the ability to colonise, at least transiently, the human gastrointestinal tract [6]. Data from animal studies have indicated that probiotic lactic acid bacteria can significantly influence the immune responses of host animals in promoting the production of the secretory IgA, enhancing phagocytosis, altering the balance of Th1 and Th2 and the cytokine production profile [9]. However, the immunomodulatory properties of probiotic lactic acid bacteria in human have not as yet been well characterised.

Lactobacillus GG (ATCC 53103) is a widely used and extensively studied probiotic strain [10]. It can adhere to human intestinal cells and mucus, and transiently colonise the human gastrointestinal tract [11–14]. Lactobacillus GG has been shown to prevent antibiotic-associated diarrhoea and traveller's diarrhoea and to promote recovery from rotavirus diarrhoea. It has alleviated symptoms of atopic dermatitis and modulated immune responses in milk-hypersensitive subjects [15–21]. The mechanisms behind these documented clinical effects are related to the enhancement of intestinal immune functions [5,9]. Lactococcus lactis is one of the dominant micro-organisms present in the traditional Scandinavian fermented milk product ‘Viili’. L. lactis has also been reported to have immune-enhancing properties [22]. Schiffrin and his coworkers likewise report immunomodulation of human blood cells following fermented milk administration [23]. It has been shown that specific fermented milks may also enhance the immune response to oral vaccine [24].

To study the immunomodulatory effect of these probiotics, Lactobacillus GG and L. lactis were given to human subjects orally immunised with Salmonella typhi Ty21a vaccine.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

2.1Volunteer participants

A total of 30 healthy adult volunteers (females=15, males=15) aged from 20 to 50 were recruited from among the students and staff members of the University of Turku and the National Public Health Institute, Finland. Criteria for eligibility included that the subjects were free from infections caused by Salmonella and Escherichia coli, and had not received antibiotic treatment within the previous 2 months, or vaccination with S. typhi during a 5-year period preceding the study. Informed consent was obtained from volunteers and the Ethical Committee of University Turku had approved the study protocol.

2.2Study design

Volunteers were divided into three different groups and given orally lyophilised Lactobacillus GG (Valio Ltd., Helsinki, Finland) 4.0×1010 colony forming units (cfu) day−1, L. lactis (Valio Ltd., Helsinki, Finland) 3.4×1010 cfu day−1, or placebo (ethyl cellulose) for 7 days. On days 1, 3 and 5 of the administration, all volunteers consumed an attenuated S. typhi Ty21a oral Vivotif™ vaccine capsule (Swiss Serum and Vaccine Institute, Bern, Switzerland) as indicated by the manufacturer. Blood samples were collected 1 day before and 7 days after intake of the vaccine. During the study, all subjects were asked to avoid consumption of fermented dairy products and other probiotic products.

2.3ELISPOT assay

The total numbers of immunoglobulin–secreting cells (ISC) and the cells secreting antibodies specifically (sASC) against S. typhi Ty21a were enumerated by ELISPOT assay as previously described [18]. In short, mononuclear cells, mainly lymphocytes, were obtained by FicollPaque (Pharmacia AB, Uppsala, Sweden) centrifugation of heparinised blood samples. Isolated cells were washed three times in Hank's buffered salt solution (HBSS) (Flow Laboratories, Irvine, UK), suspended in culture medium and adjusted to a concentration of 2×106 cells ml−1. These cells were incubated in antigen- or antibody-coated, flat-bottomed 96-well microtiter plates (Immunoplate R I, a/s Nunc, Roskilde, Denmark) to allow the antibodies they secreted to react with the antigen near them. The antibodies from each cell were visualised by application of enzyme-labelled antiserum followed by a substrate-agarose overlap and observing the coloured spots. For determination of ISC and sASC, the plates were coated with anti-Ig to human IgA, IgM or IgG, or with a whole cell preparation of formalin-killed Salmonella strain SL 2404 sharing with the vaccine strain the 0-9,12 Ag. An immune response was assumed if the number of sASC cells exceeded 0.5 sASC per 106 cells.

Rabbit IgG to human IgA (α-chain) and IgM (μ-chain) were obtained from Dakopatts NS, Roskilde, Denmark. Goat anti-human IgG (γ-chain-specific) F(ab′)2 fragment of antibody, goat anti-human IgG (γ-chain-specific), IgA (α-chain-specific) and IgM (μ-chain-specific) alkaline phosphatase conjugate came from Sigma Chemical Co., Ltd., St. Louis, MO, USA.

2.4Receptor expression analysis

HBSS without Ca2+ and Mg 2+ ions (pH 7.4) was made and supplemented with 0.1% gelatin. Isotypic controls (IgG1-FITC, IgG1-PE and IgG2a-PE), anti-CR1(CD35-FITC), anti-FcγRI(CD64-FITC), anti-FcγRII(CD32-PE), anti-FcγRIII(CD16-FITC) were purchased from Immunotech (A Coulter Company, Marseille, France). Anti-CD3(CD11b-PE) was obtained from Biodesign International (Kennebunk, ME, USA).

The expression of complement receptors (CR1, CR3), receptors for IgG (FcγRI, FcγRII, FcγRIII) on neutrophils and monocytes was assayed by the methods described in [25]. Briefly, peripheral EDTA-anti-coagulated (1.5 mg EDTA ml−1 blood) blood samples were collected by venopuncture. The leukocytes were isolated by lysing the erythrocytes with ammonium chloride (1.5 ml blood, 8.5 ml of 0.83% ammonium chloride) at room temperature for 15 min. After lysis, the leukocytes were centrifuged (4000×g for 10 min) and resuspended in 500 μl ice-cold HBSS. Leukocytes (3×105) were incubated with monoclonal antibodies for 30 min at 4°C in a volume of 90 UL. The control sample was incubated with isotype-matched monoclonal antibodies directed to irrelevant antigens. After incubation, cells were washed with cold HBSS. Flow cytometric analysis was made with a Counter EPICS XL (Miami, FL, USA) flow cytometer with an argon ion laser.

2.5Statistical analysis

ISC and sASC numbers were given as geometric means with 95% confidence interval (CI). Receptor expression was presented as change % and S.D. Kruskal–Wallis test and two-tailed independent Student's t test were used for comparison of differences.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

3.1Immune response in ISC and sASC anti-S. typhi Ty21a

The number of ISC and sASC anti-S. typhi Ty21a cells in the subjects prior to and after administration of the attenuated S. typhi Ty21a oral vaccine with their test supplement were examined by ELISPOT assay (Tables 1 and 2). Prior to oral immunisation, the cells secreting antibodies against S. typhi Ty21a in all subjects were negative (sASC per 106 cells <0.5), and the differences in ISC counts among the study groups were not significant. The subjects responded well to S. typhi Ty21a oral vaccine and ISC and sASC were increased in most of the individuals after oral immunisation. No significant difference was found among the volunteer groups. A tendency was observed for more volunteers given Lactobacillus GG to evince a high number of IgA sASC anti-S. typhi Ty21a, with a maximum of 350 per 106 cells, compared to the L. lactis and placebo control groups with a maximum of 83 and 109.4 per 106 cells (Fig. 1).

Table 1.  Anti-S. typhi Ty21a sASC per 106 cells in subjects before and after oral immunisation with S. typhi Ty21 a oral vaccine
  1. aSpecific anti-S. typhi Ty21a sASC per 106 cells is expressed as geometric means with 95% CI.

  2. bSample time: 0: day prior to study; 8: day after 7 days of lactic acid bacteria and vaccine administration.

  3. cStudy groups: LGG: Lactobacillus GG group; L: L. lactis group; P: placebo (ethyl cellulose) group.

  4. dThe exponential transformed values were analysed. In the analysis of covariance, the four groups formed the grouping factor, and the corresponding baseline (I) value was included as a covariate.

 Sample time (day)LGGc (n=10)L (n=10)P (n=9)P-valued
IgA0b0.12 (0.10–0.10)a0.10 (0.10–0.10)0.21 (0.10–0.60) 
 821.0 (17.5–115.6)18.2 (6.90–79.4)28.3 (16.9–43.8)0.81
IgG00.18 (0.10–0.60)0.38 (0.10–1.88)0.26 (0.10–0.60) 
 81.65 (0.10–19.4)3.46 (0.60–26.0)2.73 (1.25–28.3)0.66
IgM00.17 (0.10–0.60)0.20 (0.10–0.10)0.12 (0.10–0.10) 
 87.28 (1.90–41.9)9.62 (2.50–95.0)22.1 (13.0–45.6)0.67
Table 2.  Number of ISC per 106 cells in subjects before and after oral immunisation with S. typhi Ty21a oral vaccine
  1. aISC per 106 cells expressed as geometric means with 95% CI.

  2. bSample time: 0: day prior to study; 8: day after 7 days of lactic acid bacteria and vaccine administration.

  3. cStudy groups: LGG: Lactobacillus GG group; L: L. lactis group; P: placebo (ethyl cellulose) group.

  4. dThe exponential transformed values were analysed. In the analysis of covariance, the four groups formed the grouping factor, and the corresponding baseline (I) value was included as a covariate.

 Sample time (day)LGGc (n=10)L (n=10)P (n=9)P-valued
IgA0b1251 (1040–2440)a1232 (840–1800)2028 (1240–2600) 
 81353 (1000–1920)1475 (880–2560)2252 (1640–3480)0.41
IgG01934 (1800–2480)1999 (1480–3040)2167 (1440–2750) 
 82075 (1600–2440)1952 (1720–2480)2429 (1920–3360)0.89
IgM0295 (160–520)480 (280–480)324 (200–853) 
 8503 (320–1000)642 (240–1200)375 (160–920)0.85
image

Figure 1. Number of IgA sASC against S. typhi Ty21a in each individual subject after oral vaccine intake.

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3.2Immune responses in FcγR receptor and complement receptor expression

The receptor expression results are presented in Table 3. They indicate altered receptor expression during the administration of oral vaccine and fermented milk. There were no significant differences between the groups in baseline values of receptor expressions. However, the volunteers receiving L. lactis had significantly higher CR3 receptor expression values than those receiving either the placebo or Lactobacillus GG after intake of the S. typhi Ty21a vaccine.

Table 3.  Changes in FcγR and CR receptor expression of neutrophils and monocytes in subjects after oral immunisation
  1. aResults expressed as change % and S.D.

  2. bStudy groups: LGG: Lactobacillus GG group; L: L. lactis group; P: placebo (ethyl cellulose) group.

ReceptorLGGb (n=10)L (n=10)P (n=9)ANOVA P-valueKruskal–Wallis P-value
CR1
neutrophils97.5±24.8a120.0±29.996.0±31.10.320.22
monocytes96.4±23.8136.4±59.1118.3±34.10.290.42
CR3
neutrophils97.8±16.5149.2±41.9112.6±30.30.020.03
monocytes101.6±28.8145.9±51.5137.8±41.30.190.27
FcγRI
neutrophils166.0±115.3147.1±129.6107.9±53.50.280.41
monocytes108.1±41.9111.0±28.6103.8±42.10.980.83
FcγRII
neutrophils95.0±11.4100.8±7.898.0±12.30.400.40
monocytes101.3±45.9101.1±58.7152.0±83.00.290.39
FcγRIII
neutrophils95.9±9.197.9±16.9101.7±16.10.830.80
monocytes110.7±54.3118.2±44.394.4±41.80.710.62

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Among the mechanisms where by probiotics influence human immunity we may envisage a strengthening of the gut mucosal barrier and an influence on intestinal immunity [18,26,27], this possibly also including the adjuvant effects. It has been reported that fermented milk containing L. johnsonii La1 and bifidobacteria can increase the specific IgA titer to S. typhi Ty21a in human volunteers [24]. This would indicate that specific lactic acid bacteria possessing the ability to persist in the intestinal tract can act as adjuvants to the humoral immune response. In clinical and animal studies, probiotics such as Lactobacillus GG have been seen to increase the numbers of sASC against β-lactalbumin and casein in patients with Crohn's disease and milk protein in suckling rats, and IgA sASC and serum IgA titers to rotavirus in rotavirus patients [18,26,27]. These results suggested that Lactobacillus GG might possess adjuvant potential, a conception which led to the hypothesis of adjuvant effects. In the present study, the adjuvant activity of Lactobacillus GG was evaluated using S. typhi Ty21a oral vaccine as test antigen. Although no significant differences were observed among the study groups, Lactobacillus GG stimulated IgA sASC responses against S. typhi Ty21a in a greater number of subjects than placebo and L. lactis. The potential of Lactobacillus GG to stimulate a human specific response as an adjuvant agent requires further assessment.

Lactobacillus GG is a strain which possesses the ability to adhere to human intestinal cells and mucus [11–14]. In a similar manner, the L. lactis strain has been shown to be adhesive [23]. Lactobacillus GG has been reported to increase antigen transfer across Peyer's patches and enhanced antigen-specific immune defence in the suckling rat [27]. In clinical studies, Lactobacillus GG has alleviated intestinal inflammation and promoted the endogenous barrier mechanism in atopic dermatitis and food allergy [20]. Bovine casein hydrolysed with Lactobacillus GG-derived enzymes has been found to suppress lymphocyte proliferation in healthy adults and to down regulate anti-CD3 antibody-induced IL-4 production in atopic children [28,29]. Both active and inactive forms of Lactobacillus GG have significantly shortened the duration of rotavirus-induced diarrhoea. However, only active Lactobacillus GG significantly increases the level of specific IgA-secreting cells to rotavirus [19]. Such findings must lead to the conclusion that the immunomodulatory effect of Lactobacillus GG observed in the present study may be attributed to the enhancement of intestinal barrier function and the immunomodulation of lumen antigens caused by this bacterium. Since it has been shown that milk as a carrier protects Lactobacillus GG and results in higher fecal recovery, it may be important to conduct a study using Lactobacillus GG-fermented milk as a carrier and use the same dose. Similarly, Link-Amster and colleagues observed immune enhancement when lactic acid bacteria were administered in fermented milk in conjunction with the vaccine [24]. With these observations in mind, the present authors have also the opinion that the adjuvant effects of Lactobacillus GG may be related to the viability of this bacterium and its colonisation in the human intestine, although components of the cell wall such as peptidoglycan are generally thought to determine the adjuvant effects of micro-organisms [30]. Further investigation should therefore be undertaken while the colonisation of Lactobacillus GG in the human intestine is enhanced with fermented milk and extended administration.

Instead of promoting specific IgA immune responses, L. lactis significantly increased CR3 receptor expression on the neutrophils. CR3 is a multifunctional receptor, and the number of CR3 on phagocyte leukocytes varies considerably depending on the activation state of the cells [25]. CR3 receptor expression has been used to monitor changes in phagocytosis and inflammation caused by antigens and probiotic exposure [21]. In the present study, the increase in CR3 expression on neutrophils induced by L. lactis can be regarded as an alteration of phagocytosis, indicating that this bacterium can influence the non-specific immune response although it did not enhance specific immune responses. The L. lactis used in the present study is an isolate from the traditional Scandinavian fermented milk known as ‘Viili’. Viili has long been consumed by Scandinavian people and is generally believed to exert health-promoting effects. The results obtained in the present study suggest that the health benefits of this soured milk may be partly attributed to the immunomodulatory effects of the lactic acid bacteria such as L. lactis present in it. L. lactis is phylogenetically different from Lactobacillus GG, isolated from the human intestinal microflora. This bacterium cannot colonise the human intestine, as does Lactobacillus GG. The different performances of Lactobacillus GG and L. lactis in stimulating the human immune response may be attributed to differences in the mechanisms by which these respective bacteria influence immunity. The results here suggest that the immunomodulatory effect of probiotics is a strain-dependent characteristic and species speciality in the host. Probiotic strains should thus be evaluated on their own merits; extrapolation from other species or strains is not appropriate. The use of fermented milks as protective carriers for probiotics should also be further assessed.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References
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