an> 1010
CFU mL–1 and that of OVA was 10 μg per rat on 3 occa-
sions (Table 1).
Immunological assay (IMA). Blood samples were
individually collected from immunized rats by a tail
bleed on days 0, 7, 14 and 21 for the analysis of systemic
OVA-specific antibodies (10e10 bateria, on 3 occasions).
Fresh fecal pellets were individually collected lyophi-
lized from the same rat groups on days 0, 7, 14 and 21.
The samples were treated as previous described [9] (Ma
et al., 2011). The supernatants were analyzed for
OVA-specific sIgA levels to study the secretory mucosal
immune response. Both the serum was analyzed with
goat anti-rat IgG and the fecal specific antibody titers
were analyzed with goat anti-rat IgA by ELISA, respec-
tively. OVA (1.0 μg·mL–1) was diluted in a coating
buffer (50 mM sodium carbonate, 30 mM sodium bicar-
bonate, pH 9.6) and allowed to adsorb to 96-well plates
Table 1. The SD rats and the vaccination groups.
Groups (rats) Vaccinations agents Dose ((C.F.U. mL–1) + μg OVA)
A (n = 12) rBI-LTB + OVA 1.0 1010 + 10 μg
B (n= 12) wBI + OVA 1.0 1010 + 10 μg
C (n = 12) OVA 10 μg
D (n = 10) PBS /
(1.0 μg each well) overnight at 4˚C. The plates were
blocked with 5% skim milk for 1h at room temperature
and a twofold serial dilution of serum was added. After
incubation, the plates were washed three times with PBS
with 0.05% Tween 20 (PBS-Tween), and then 1.0
mg· mL –1 of goat anti-rat IgG mAb or anti-rat sIgA mAb
conjugated to HRP (1.0 μg·mL–1, BOSTER, China) was
added and incubated again.
The OD450 value was measured on a Molecular De-
vices SpectroMax Plus spectrophotometer. Endpoint titers
were determined as the dilution of each sample showing
a 2.1-fold higher level of absorbance at 450 nm than that
of the negative control samples. Average OD450 values
for the animals were calculated.
Tissue samples preparation. Following collection of
blood and fecal samples, two of vaccinated rats were
randomly anaesthetised and sacrificed from each group
and the jejunum samples were isolated from each rat on
days 7, 14 and 21, respectively. Following wash with
PBS, the jejunum samples were fixed with 4% polyoxy-
methylene for 24 h. Then the fixed jejunums were rinsed
with 95% ethyl and successively dehydrated 10 m with
ethyl from concentration 90%, 95% and 100 %, respec-
tively. After treating with dimethylbenzene, the samples
were embedded 4 h with paraffins at 56˚C and cut into
slices (<5 μm). Then the slices were treated with acetone
containing 1% APES (3-aminopropyl triethoxysilane)
and dried at 37˚C.
Immunohistochemical staining sections (IHCS). 1)
The prepared slice samples were twice treated 40 m with
dimethylbenzene to dewax and then successively dehy-
drated with ethyl from concentration 100%, 95%, 90%,
80% to 70 % for 30 s, respectively; 2) Following rinse 5
m with PBS three times, each slice was incubated 30 m
with 50 μL peroxidase blocking solution (198 mL metha-
nol + 2 mL 30 % hydrogen peroxide) at 25˚C; 3) After
rinsing 5 m with distilled water, the slices were put into
0.01 M citric acid solution (pH 6.0) and heat 15 m at
95˚C. Then, the slices were naturally cooled to 25˚C and
rinsed with PBS three times; 4) Incubated with OVA (1.0
μg·mL–1) at 37˚C for 3 h and then rinsed 5 m with PBS
three times; 5) Following blocked with 5% skim milk,
each slice was incubated at 37˚C for 30 m and then
sucked up the serum with filter paper; 6) Incubated with
OVA antibody (goat anti-OVA/HRP) at 37˚C for 3 h and
then rinsed 5 m with PBS three times; 7) Incubated 3 m
with 100 μL DBA solution and rinsed with tap water; 8)
Following counterstain 3 m with hematine, then rinsed
with tap water and overnight at 50˚C; 9) Coverslip with
neutral balsam.
ICH analysis (ICHA). All slices were surveyed and
took pictures with Leica DM2000 microscope. Each slice
randomly selected ten visual fields to statistical analysis
Copyright © 2012 SciRes. IJCM
Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances
the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat
389
by Qwin image manipulation tools. The yellowish-brown
value stands for the positive cell area.
Statistical analysis. The data was statistically evalu-
ated by the SPSS 19.0 statistical software package (SPSS
Inc., Chicago, IL) and a value of p < 0.05 was considered
significant.
3. Results
IMA. Serum and mucosal OVA-specific antibody levels
were measured by ELISA. High levels of serum OVA-
specic IgG were observed on day 14 after the secondary
booster vaccination, with an endpoint titer of 206 and
reached a novel increased level in following booster dose
on day 21 with the titer of 566 in groups A (n = 12).
However, in group B (n = 12), the OVA-specic IgG
titer just reached to 95 on 14 d and to 183 on 21 d. In the
other way, we did not find the OVA-specic IgG in
group C (n = 12) and D (n = 10). Statistically, there was
a significant difference in the IgG titer between the
groups A and B for the adjuvant function of LTB (p <
0.05) (Figure 1). The level of variation in the responses
was 20.48 in group A and 20.18 in group B between in-
dividual animals on day 21.
Similar to the serum IgG titer variation, the fecal sIgA
titer in group A was greater than that in group B. High
levels of OVA-specic sIgA was observed on day 14
after the secondary booster vaccination, with an endpoint
titer of 7.57 and reached a novel increased level in fol-
lowing booster dose on day 21 with the titer of 54.67 in
groups A (n = 12). However, in group B (n = 12), the
OVA-specic sIgA titer just reached to 3.02 on 14 d and
to 9.70 on 21 d. However, we did not find the OVA-
specic sIgA in group C (n = 12) and D (n = 10). The
Group A
Group B
Group C
Group D
7 14 21
day (d)
700
600
500
400
300
200
100
0
Sorum l
g
G tite
r
Figure 1. The serum IgG titer in different groups post-im-
munization. The group A rats were immunized orally with
rBI-LTB + OVA, group B with wBI + OVA, group C with
OVA, and group D were treated with PBS (10e10 bateria,
on 3 occasions). The group A shown significant difference,
compared with group B (p < 0.05). However, there was no
statistically significant difference IgG titer between the
group C and group D for no detectable spec i fic IgG.
results suggest that rBI-LTB expressed in BI had muco-
sal adjuvant activity in group A. Statistically, there was a
significant difference in the sIgA titer between the
groups A and B for the adjuvant function of LTB (p <
0.05) (Figure 2). The level of variation in the responses
was 10.88 in group A and 0.61 in group B between indi-
vidual animals on day 21.
ICHA. Examined under a microscope, the OVA-spe-
cific sIgA producing cells were stained in yellow-
ish-brown and distributed in jejunal mucosa, submucosa,
external and internal of intestinal crypt, intestinal lamina
propria and serous membrane (Figures 3 (a)-(h)). How-
ever, the colored positive cells in serous membrane were
obviously fewer than that in other type cells. So the re-
sults suggest that intestinal mucosa and submucosa was
the main field of sIgA secretion.
Analysis the specific sIgA producing cells on 7 d
post-vaccination statistically, we found that the positive
cells were increased appreciably group A than that in
group B, but there was no significant difference each
other. However, the positive cells were increased sig-
nificantly from secondary and third booster in group A
than that in group B (p < 0.05). As was expected, the
positive cells did not find in group C and D (Figure 4).
4. Discussion
Appropriate mucosal adjuvant is essential for oral immu-
nization to elicit immune response. Besides of the
ADP-ribosylating enterotoxins (CT and LTB), the other
two bacterial products, synthetic oligodeoxy-nucleotides
containing unmethylated CpG dinucleotides (CpG ODN),
and mono-phosphoryl lipid A (MPL), were used As
mucosal adjuvants. Both MPL and CpG act through
Group A
Group B
Group C
Group D
7 14 21
day (d)
60
50
40
30
20
10
0
Fecal sIgA tite
r
Figure 2. The fecal sIgA titer in different immunization
groups in SD rat. The group A rats were immunized orally
with rBI-LTB + OVA, group B with wBI + OVA, group C
with OVA, and group D were treated with PBS (10e10
bateria, on 3 occasions). The group A shown significant
difference, compared with group B (p < 0.05). However,
there was no statistically significant difference sIgA titer
between the group C and group D for no detectable specific
sIgA.
Copyright © 2012 SciRes. IJCM
Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances
the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat
Copyright © 2012 SciRes. IJCM
390
(a) (b)
(c) (d)
(e) (f)
(g) (h)
Figure 3. Immunohistochemic al evaluation of OVA-specific producing cells in jenunal mucosa. (a), (b) Jenunal mucosal sec-
tions on 14 d and 21 d in group A (magnification 200 and 400, respectively). (c), (d) Sections on 14 d and 21 d in group B
(magnification 200 and 400, respectively). (e), (f) Sections on 14 d and 21 d in group C (magnification 200 and 400, respec-
tively). (g), (h) sections on 14 d and 21 d in group D (magnification 400 and 200, respectively). The OVA-specific sIgA pro-
ducing cells were stained with goat anti-OVA/HRP in yellowish-brown and distributed in jejunal mucosa, submucosa (arrows
indicating mucosal locations of sIgA producing cells).
Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances
the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat
391
7 14 21
day (d)
0.06
0.05
0.04
0.03
0.02
0.01
0
sIgA secretion cells (gray value)
Group
A
Group B
Group C
Group
D
Figure 4. The specific sIgA secretion cells measured with
yellow-brownish value. The positive cells, stained with goat
anti-OVA/HRP, were increased significantly from sec ondar y
and third booster in group A than that in group B ( p < 0.05).
However, the positive cells did not find in group C and D.
MyD88-dependent and -independent pathways and the
adjuvant activities of CpG and MPL are due to several
different effects they have on innate and adaptive im-
mune responses [5].
LTB is a nontoxic molecule with potent biological
properties and is a powerful mucosal and parenteral ad-
juvant that induces a strong humoral and mucosal im-
mune response against co-administered antigen or cou-
pled antigens [8,9,15,17-19]. The adjuvant mechanism of
LTB appears to be related with its capacity to: 1) en-
hance antigen presentation via MHC class I and MHC II;
2) activate selective differentiation of lymphocytes; 3)
influence DCs maturation and activation; 4) induce B7-2
expression on APCs for subsequent co-stimulatory sig-
naling to CD4+ T cells and; 5) increase the expression of
activation markers on B lymphocytes (MHC class II, B7,
CD40, CD25 and ICAM-1) [4,20].
Oral vaccination is based on antigen delivery to the
gastrointestinal tract, the largest mucosal surface and the
central site of IgA secretion [21]. Since the intestinal
mucosa is the natural site of BI colonization, rLTB pro-
ducing in BI easily crosses the epithelial layer to the area
rich in cells of the mucosal immune system. BI-LTB
appears to be one of the best forms of LTB adjuvant de-
livery system [9]. Because of the same reason, the sev-
eral strains LAB has been genetically modified as a
promising oral recombinant vaccine delivery system for
inducing efficient mucosal immunity as well as system-
atic immunity [9,22-26].
Bifidobacterial cell wall preparation (whole pepti-
doglycan, WPG) has been documented with adjuvant
activity and the activity of WPG is related to their ability
to induce a reduction in regulatory T cells (Tregs) activ-
ity [27,28]. A previous document was demonstrated that
LAB might be promising adjuvants in vaccines due to
their capability to reduce functional activity of Tregs,
thereby speeding up vaccine-induced immune responses
[29]. Another document suggests that WPG of bifido-
bacterium induces IL-12 secretion in DCs from bone
marrow and rhIL-12, as adjuvant, has been shown to
augment both cellular and humoral immunity [30-33]. In
this study, we find that wild type BI performed weak
adjuvant activity to OVA oral administration, which
suggests that WPG of BI might be promising adjuvant
activity in OVA.
There are many more experiments have been done in
mice where there is more immunological reagents for
these types of investigation. In this study, we use of the
rat model is novel for oral vaccination. One reason for
choosing rat is that a report used of rat for investigation
of the role of intestinal bifidobacteria on immune system
development [34]. They found that intestinal bifidobacte-
ria plays an important role of development of both the
gut and systemic immunity in early life. Neonatal SD rats
supplemented daily with bifidobacteria could promote
the maturation of DCs and its expression of IL-12 locally
in the gut, favour the development of Th1 response by
increasing the local and systemic expression of IFNγ and
ensure the intestinal Treg response by promoting the lo-
cal expression of IL-10 [34].
There is no purified LT-B plus OVA as a comparable
control to compare the organism expressing LT-B in this
study. One reason is that the nontoxic CT plus OVA or
LT-B plus OVA confirmed that the enterotoxin B subunit
acts as a mucosal adjuvant intranasally immunized
[35,36]. Oral vaccination is not an economically afford-
able way for purified LT-B or other enterotoxin B sub-
unit because it needs high dose enterotoxin B subunit.
However, BI expressing LT-B can easily get over it.
5. Acknowledgements
This study was funded by the National Natural Science
Foundation of China (No. 30972585).
REFERENCES
[1] J. Sanchez and J. Holmgren, “Cholera Toxin—A Foe & a
Friend,” Indian Journal of Medical Research, Vol. 133,
No. 2, 2011, pp. 153-163.
[2] T. Yamamoto and T. Yokota, “Plasmids of Enterotoxi-
genic Escherichia coli H10407: Evidence for Two Heat-
Stable Enterotoxin Genes and a Conjugal Transfer Sys-
tem,” Journal of Bacteriology, Vol. 153, No. 3, 1983, pp.
1352-1360.
[3] T. Yamamoto, T. Tamura and T. Yokota, “Primary Struc-
ture of Heat-Labile Enterotoxin Produced by Escherichia
coli Pathogenic for Humans,” Journal of Biological
Chemistry, Vol. 259, No. 8, 1984, pp. 5037-5044.
[4] V. P. da Hora, F. R. Conceição, O. A. Dellagostin and D.
L. Doolan, “Non-Toxic Derivatives of LT as Potent Ad-
juvants,” Vaccine, Vol. 29, No. 8, 2011, pp. 1538-1544.
Copyright © 2012 SciRes. IJCM
Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances
the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat
392
doi:10.1016/j.vaccine.2010.11.091
[5] L. C. Freytag and J. D. Clements, “Mucosal Adjuvants,”
Vaccine, Vol. 23, No. 15, 2005, pp. 1804-1813.
doi:10.1016/j.vaccine.2004.11.010
[6] N. A. Williams, “Immune Modulation by the Cholera-
Like Enterotoxin B-Subunits: From Adjuvant to Immu-
notherapeutic,” Journal of Medical Microbiology, Vol.
290, No. 4-5, 2000, pp. 447-453.
doi:10.1016/S1438-4221(00)80062-4
[7] T. O. Nashar, Z. E. Betteridge and R. N. Mitchell, “Evi-
dence for a Role of Ganglioside GM1 in Antigen Presen-
tation: Binding Enhances Presentation of Escherichia coli
Enterotoxin B Subunit (EtxB) to CD4(+) T Cells,” Inter-
national Immunology, Vol. 13, No. 4, 2001, pp. 541-551.
doi:10.1093/intimm/13.4.541
[8] E. Fingerut, B. Gutter, M. Goldway, D. Eliahoo and J.
Pitcovski. “B Subunit of E. coli Enterotoxin as Adjuvant
and Carrier in Oral and Skin Vaccination,” Veterinary
Immunology and Immunopathology, Vol. 112, No. 3-4,
2006, pp. 253-263. doi:10.1016/j.vetimm.2006.03.005
[9] Y. P. Ma, Y. L. Luo, X. P. Huang, F. Z. Song and G. L.
Liu, “Construction of Bifidobacterium infantis as a Live
Oral Vaccine that Expresses Antigens of the Major Fim-
brial Subunit (CfaB) and the B Subunit of Heat-Labile
Enterotoxin (LTB) from Enterotoxigenic Escherichia
coli,” Microbiology, Vol. 158, No. 2, 2012, pp. 498-504.
[10] C. M. M. Hayward, P. O’Gaora, D. B. Young, G. E. Grif-
fin, J. Thole, T. R. Hirst, L. R. R. Castello-Branco and D.
J. M. Lewis. “Construction and Murine Immunogenicity
of Recombinant Bacille Calmette Guerin Vaccine Ex-
pressing the B Subunit of Escherichia coli Heat Labile
Enterotoxin,” Vaccine, Vol. 17, No. 9-10, 1999, pp. 1272-
1281. doi:10.1016/S0264-410X(98)00350-8
[11] N. Goto, J. I. Maeyama, Y. Yasuda, M. Isaka, K. Matano,
S. Kozuka, T. Taniguchi, Y. Miura, K. Okhuma and K.
Tochikubo, “Safety Evaluation of Recombinant Cholera
Toxin B Subunit Produced by Bacillus brevis as a Muco-
sal Adjuvant,” Vaccine, Vol. 18, No. 20, 2000, pp.
2164-2171. doi:10.1016/S0264-410X(99)00337-0
[12] M. Isaka, Y. Yasuda, S. Kozuka, T. Taniguchi, K. Ma-
tano, J. Maeyama, T. Komiya, K. Ohkuma, N. Goto and
K. Tochikubo, “Induction of Systemic and Mucosal An-
tibody Responses in Mice Immunized Intranasally with
Aluminium-Non-Adsorbed Diphtheria Toxoid Together
with Recombinant Cholera Toxin B Subunit as an Adju-
vant,” Vaccine, Vol. 18, No. 7-8, 1999, pp. 743-751.
doi:10.1016/S0264-410X(99)00258-3
[13] P. Slos, P. Dutot, J. Reymund, P. Kleinpeter, D. Prozzi,
M. P. Kieny, J. Delcour, A. Mercenier and P. Hols, “Pro-
duction of Cholera Toxin B Subunit in Lactobacillus,”
FEMS Microbiology Letters, Vol. 169, No. 1, 1998, pp.
29-36. doi:10.1111/j.1574-6968.1998.tb13295.x
[14] S. Liljeqvist, P. Samuelson, M. Hansson, T. N. Nguyen,
H. Binz and S. Stahl, “Surface Display of the Cholera
Toxin B Subunit on Staphylococcus xylosus and Staphy-
lococcus carnosus,” Applied and Environmental Micro-
biology, Vol. 63, No. 7, 1997, pp. 2481-2488.
[15] E. Fingerut, B. Gutter, R. Meir, D. Eliahoo and J. Pit-
covski, “Vaccine and Adjuvant Activity of Recombinant
Subunit B of E. coli Enterotoxin Produced in Yeast,”
Vaccine, Vol. 23, No. 38, 2005, pp. 4685-4696.
doi:10.1016/j.vaccine.2005.03.050
[16] F. Guarner and J. R. Malagelada, “Gut Flora in Health
and Disease,” The Lancet, Vol. 361, No. 9356, 2003, pp.
512-519. doi:10.1016/S0140-6736(03)12489-0
[17] A. da Silva Ramos Rocha, F. R. Conceição, A. A. Grass-
mann, V. L. Lagranha and O. A. Dellagostin. “B Sub-
unit of Escherichia coli Heat-Labile Enterotoxin as Ad-
juvant of Humoral Immune Response in Recombinant
BCG Vaccination,” Canadian Journal of Microbiology,
Vol. 54, No. 8, 2008, pp. 677-686. doi:10.1139/W08-056
[18] F. R. Conceicao, A. N. Moreira and O. A. Dellagostin, “A
Recombinant Chimera Composed of R1 Repeat Region of
Mycoplasma hyopneumoniae P97 Adhesin with Escheri-
chia coli Heat-Labile Enterotoxin B Subunit Elicits Im-
mune Response in Mice,” Vaccine, Vol. 24, No. 29-30,
2006, pp. 5734-5743. doi:10.1016/j.vaccine.2006.04.036
[19] R. Weltzin, B. Guy, W. D. Thomas Jr., P. J. Giannasca
and T. P. Monath, “Parenteral Adjuvant Activities of Es-
cherichia coli Heat-Labile Toxin and its B Subunit for
Immunization of Mice against Gastric Helicobacter pylori
Infection,” Infection and Immunity, Vol. 68, No. 5, 2000,
pp. 2775-2782. doi:10.1128/IAI.68.5.2775-2782.2000
[20] T. O. Nashar, T. R. Hirst and N. A. Williams, “Modu-
lation of B-cell Activation by the B Subunit of Es-
cherichia coli Enterotoxin: Receptor Interaction Up-Re-
gulates MHC Class II, B7, CD40, CD25 and ICAM-1,”
Immunology, Vol. 91, No. 4, 1997, pp. 572-578.
doi:10.1046/j.1365-2567.1997.00291.x
[21] J. R. McGhee, J. Mestecky, M. T. Dertzbaugh, J. H. El-
dridge, M. Hirasawa and H. Kiyono, “The Mucosal Im-
mune System: From Fundamental Concepts to Vaccine
Development,” Vaccine, Vol. 10, No. 2, 1992, pp. 75-88.
doi:10.1016/0264-410X(92)90021-B
[22] J. K. Liu, X. L. Hou, C. H. Wei, L. Y. Yu, X. J. He, G. H.
Wang, J. S. Lee and C. J. Kim, “Induction of Immune
Responses in Mice after Oral Immunization with Recom-
binant Lactobacillus casei Strains Expressing Entero-
toxigenic Escherichia coli F41 Fimbrial Protein,” Applied
and Environmental Microbiology, Vol. 75, No. 13, 2009,
pp. 4491-4497. doi:10.1128/AEM.02672-08
[23] M. Medina, E. Vintiñi, J. Villena, R. Raya and S. Alvarez,
Lactococcus lactis as an Adjuvant and Delivery Vehicle
of Antigens against Pneumococcal Respiratory Infections,”
Bioengineered Bugs, Vol. 1, No. 5, 2010, pp. 313-325.
doi:10.4161/bbug.1.5.12086
[24] C. H. Wei, J. K. Liu, X. L. Hou, L. Y. Yu, J. S. Lee and C.
J. Kim, “Immunogenicity and Protective Efficacy of
Orally or Intranasally Administered Recombinant Lacto-
bacillus casei Expressing ETEC K99,” Vaccine, Vol. 28,
No. 24, 2010, pp. 4113-4118.
doi:10.1016/j.vaccine.2009.05.088
[25] J. Wells, “Mucosal Vaccination and Therapy with Ge-
netically Modified Lactic Acid Bacteria,” Annual Review
of Food Science and Technology, Vol. 2, 2011, pp. 423-
445. doi:10.1146/annurev-food-022510-133640
Copyright © 2012 SciRes. IJCM
Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances
the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat
Copyright © 2012 SciRes. IJCM
393
[26] S. Yamamoto, J. Wada, T. Katayama, T. Jikimoto, M.
Nakamura, S. Kinoshita, K. M. Lee, M. Kawabata and T.
Shirakawa, “Genetically Modified Bifidobacterium Dis-
playing Salmonella-Antigen Protects Mice from Lethal
Challenge of Salmonella Typhimurium in a Murine Ty-
phoid Fever Model,” Vaccine, Vol. 28, No. 41, 2010, pp.
6684-6691. doi:10.1016/j.vaccine.2010.08.007
[27] K. Sekine, E. Watanabe-Sekine, T. Toida, T. Kasashima,
T. Kataoka and Y. Hashimoto, “Adjuvant Activity of the
Cell Wall of Bifidobacterium infantis for in Vivo Immune
Responses in Mice,” Immunopharmacol Immunotoxicol,
Vol. 16, No. 4, 1994, pp. 589-609.
doi:10.3109/08923979409019741
[28] E. G. Schmidt, M. H. Claesson, S. S. Jensen, P. Ravn and
N. N. Kristensen, “Antigen-Presenting Cells Exposed to
Lactobacillus acidophilus NCFM, Bifidobacterium bifidum
BI-98, and BI-504 Reduce Regulatory T Cell Activity,”
Inflammatory Bowel Diseases, Vol. 16, No. 3, 2010, pp.
390-400.
[29] D. Paineau, D. Carcano, G. Leyer, S. Darquy, M. A. Al-
yanakian, G. Simoneau, J. F. Bergmann, D. Brassart, F.
Bornet and A. C. Ouwehand, “Effects of Seven Potential
Probiotic Strains on Specific Immune Responses in
Healthy Adults: A Double-Blind, Randomized, Con-
trolled Trial,” FEMS Immunology and Medical Microbi-
ology, Vol. 53, No. 1, 2008, pp.107-113.
doi:10.1111/j.1574-695X.2008.00413.x
[30] L. L. Zhao and Q. Cheng, “The Influence of Whole Pep-
tidoglycan of Bifidobacterium on Interleukin-12 Produced
by Dendritic Cells Derived from Bone Marrow of Mice in
Vitro,” Chinese Journal of Microecology, Vol. 21, No. 10,
2009, pp. 896-901.
[31] O. Hamid, J. C. Solomon, R. Scotland, M. Garcia, S. Sian,
W. Ye, S. L. Groshen and J. S. Weber, “Alum with Inter-
leukin-12 Augments Immunity to a Melanoma Peptide
Vaccine: Correlation with Time to Relapse in Patients
with Resected High-Risk Disease,” Clinical Cancer Re-
search, Vol. 13, No. 1, 2007, pp. 215-222.
doi:10.1158/1078-0432.CCR-06-1450
[32] M. A. Jacobson, E. Sinclair, B. Bredt, L. Agrillo, D.
Black, C. L. Epling, A. Carvidi, T. Ho, R. Bains, V.
Girling and S. P. Adler, “Safety and Immunogenicity of
Towne Cytomegalovirus Vaccine with or without Adju-
vant Recombinant Interleukin-12,” Vaccine, Vol. 24, No.
25, 2006, pp. 5311-5319.
doi:10.1016/j.vaccine.2006.04.017
[33] A. K. Wright, I. Christopoulou, S. El Batrawy, J. Limer
and S. B. Gordon, “rhIL-12 as Adjuvant Augments Lung
Cell Cytokine Responses to Pneumococcal Whole Cell
Antigen,” Immunobiology, Vol. 216, No. 10, 2011, pp.
1143-1147.
[34] P. Dong, Y. Yang and W. P. Wang, “The Role of Intesti-
nal Bifidobacteria on Immune System Development in
Young Rats,” Early Human Development, Vol. 86, No. 1,
2010, pp. 51-58. doi:10.1016/j.earlhumdev.2010.01.002
[35] P. N. Boyaka, M. Ohmura, K. Fujihashi, T. Koga, M.
Yamamoto, M. N. Kweon, Y. Takeda, R. J. Jackson, H.
Kiyono, Y. Yuki and J. R. McGhee, “Chimeras of Labile
Toxin One and Cholera Toxin Retain Mucosal Adjuvan-
ticity and Direct Th Cell Subsets via Their B Subunit,”
Journal of Immunology, Vol. 170, No. 1, 2003, pp. 454-
462.
[36] S. Yamamoto, H. Kiyono, M. Yamamoto, K. Imaoka, K.
Fujihashi, F. W. Van Ginkel, M. Noda, Y. Takeda and J.
R. McGhee, “A Nontoxic Mutant of Cholera Toxin Elic-
its Th2-Type Responses for Enhanced Mucosal Immu-
nity,” Proceedings of the National Academy of Sciences
USA, Vol. 94, No. 10, 1997, pp. 5267-5272.
doi:10.1073/pnas.94.10.5267