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Consumption of Bifidobacterium animalis subsp. lactis BB-12 reduces TNF-α
secretion from LPS-stimulated PBMCs, but does not alter serum TNF-α levels
1
Meng ,
1
Fleming ,
1
Lee ,
2
Ba ,
2
Furumoto ,
*
0.8
0.4
Gut transit time
≥ 24h
A
Randomization
Period 2
A
Period 3
A
Period 4
A
B
B
B
B
C
C
C
C
D
D
D
D
4 weeks
4 weeks
4 weeks
Sample collection
All
subjects
freeliving
1.0
0.5
P
A
C
ST
PO
Figure 1. The effect of treatment on TNF-α secretion from BB-12 or LPS stimulated PBMCs.
PBMCs were stimulated with 10μg/ml BB-12 or LPS, and culture supernatants were collected
post 4h stimulation. (A) TNF-α secretion from BB-12 stimulated PBMCs and (B) TNF-α secreted
from LPS stimulated PBMCs were assessed by ELISA. Data are presented as LSMEANS±SEM
(N=30). Repeated measure ANOVA demonstrated significant differences in TNF-α secretion
from BB-12 stimulated PBMCs (p=0.0032) and TNF-α (p=0.0328) secretion from LPS stimulated
PBMCs among baseline and treatments. Bonferroni’s post-hoc test found lower TNF-α
(p=0.0025) secretion from LPS stimulated PBMCs following YS-Post treatment compared to
baseline.
(4 weeks)
500
0
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
Baseline Serum TNF- (pg/ml)
r= 0.1676
p= 0.4929
2000
1500
1000
500
0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
YS-Post Serum TNF- (pg/ml)
Figure 3. The correlation between plasma TNF-α and TNF-α secreted from LPS-stimulated
PBMCs. Serum TNF-α data was not normally distributed, so it was transformed using the
logarithm function, which yielded a normally distributed data set. No significant correlation was
found using the Pearson Correlation Coefficient test at baseline (A) or after treatment with YSPost (B).
Summary
• Subjects who consumed a yogurt smoothie with BB-12 added post
fermentation had significantly lower TNF-α from LPS stimulated
PBMCs.
• Plasma TNF-α was not correlated with TNF-α from LPS stimulated
PBMCs at any time point.
• No treatment effect was observed on plasma levels of TNF-α.
Conclusions
3
2 week compliance break
change to next treatment
Immune Outcomes:
• TNF-α secretion from PBMCs in response to LPS stimulation.
• Serum TNF-α quantification.
• Evaluation of treatment effects on TNF-α.
• Comparison of these two assay methods and tissue compartments in
regards to TNF-α quantification.
1000
0.0
Plasma TNF-α Before and After YS-Post Treatment
Treatment groups include:
• A: yogurt smoothie without added BB-12 (YS);
• B: yogurt smoothie with BB-12 added post fermentation (YS-Post);
• C: yogurt smoothie with BB-12 added pre-fermentation (YS-Pre);
• D: a tablet containing BB-12 (TAB).
1500
P
*
YS-Post LPS-stimulated TNF-  (pg/ml)
1.5
TNF- (pg/ml)
Period 1
2000
C
p= 0.0328
Study Design
Screening
r= -0.3161
p= 0.1627
Treatments
Treatments
Randomized, 4-period cross-over free-living design
2500
A
ST
PO
PR
E
YS
B
L
0.0
Correlation Between In Vitro and In Vivo TNF-α
Baseline LPS-stimulated TNF-  (pg/ml)
TNF- (ng/ml)
p= 0.0032
PR
E
1. To determine if BB-12 will alter innate and/or adaptive immune
responses of human subjects.
2. To determine the appropriate tissue compartment and assay to
quantify changes in inflammatory mediators in this model.
1.2
B
L
Aims
1
Rogers
Results
TNF-α Secretion from BB-12 or LPS stimulated PBMCs
TNF- (ng/ml)
Increasing evidence suggests that probiotic bacteria can modulate inflammatory
responses. However the immune response varies by species and strain of
organism, as well as delivery matrix of the probiotic. Furthermore the tissue
compartment and assay method by which inflammatory mediators are quantified
in clinical trials is a debated issue. The goal of the current study was to evaluate
the effect of one probiotic species, Bifidobacterium animalis subsp. lactis BB-12
at a dose of log 10 ± 0.5 CFUs/day on both plasma- and in vitro cultured
peripheral blood mononuclear cells (PBMC)-derived inflammatory mediators in a
partially blinded, 4-period crossover, free-living study. Healthy adults (n=30)
aged 18-40 years were recruited, and received 4 treatments in a random order:
1) yogurt smoothie alone; smoothie with organism added 2) before or 3) after
fermentation, or 4) organism given in capsule form. At baseline and after each
treatment, plasma was collected and PBMCs were isolated and stimulated in
vitro with LPS as an inflammatory stimulus. Inflammatory cytokines were
quantified in each compartment at baseline and after each treatment.
Participants who consumed yogurt smoothies with BB-12 added postfermentation had a significant reduction in TNF-α secretion from LPS-stimulated
PBMCs compared to baseline (p=0.039). However, there was no treatment
effect observed in plasma TNF-α levels. Additionally, plasma TNF-α levels were
not correlated with TNF-α produced by cultured PBMCs in the presence of
stimulation. These results demonstrate that quantification of inflammatory
mediators in the plasma of healthy adults may not be adequate to detect the
effect of probiotic consumption on inflammatory responses. The assessment of a
nutritional intervention on in vitro cultured PBMCs in response to LPS
stimulation may be a more sensitive assay method to detect probiotic-induced
changes in inflammatory responses. These findings demonstrate that selection
of the tissue compartment and the assay method to evaluate inflammatory
response is a critical variable in clinical trial design.
4 weeks
1
Kris-Etherton ,
Results
Abstract
18-40 years old
BMI:22-40 kg/m2
2
Roberts ,
Huicui
Jennifer A.
Yujin
Zhaoyong
Emily J.
Robert F.
Penny M.
Connie J.
1Department of Nutritional Sciences, 2Department of Food Science, Pennsylvania State University, University Park, PA, 16802.
YS
Hannah
1
VanEvery ,
p= 0.2509
1
Probiotic consumption may alter inflammatory mediators, however,
these effects may not be robust enough to detect in plasma. The
assessment of probiotic consumption on in vitro cultured PBMCs in
response to LPS stimulation may be a more sensitive and accurate
assay method to detect probiotic-induced changes in inflammatory
responses.
0
These findings demonstrate that selection of the tissue
compartment and the assay method to evaluate inflammatory
response is a critical variable in clinical trial design.
2
Baseline
YS-Post
Acknowledgements
Figure 2. The effect of treatment on serum TNF-α. Serum was isolated from peripheral blood
samples drawn at baseline and after treatment with YS-Post. TNF-α was assed by Quest
Diagnostics using a high-sensitivity ELISA assay. Serum TNF-α data was not normally
distributed, so it was transformed using the logarithm function, which yielded a normally
distributed data set. A paired t-test demonstrated no significant differences in serum TNF-α
among baseline and post YS-Post treatment.
Funding provided by the Dairy Research Institute (CJR) and the CTSI TL1
Training Program (HLV).
Contact information:
Connie Rogers
Email: [email protected]
Tel: 814-867-3716
Hannah VanEvery Email: [email protected]
Tel: 515-231-5640