Powerpoint presentatie Corporate NL€¦ · Selection of test persons (inclusion and exclusion...
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DiAgRaMs Folkert Faber, Maaike Smelt, Nynke Leistra, Doede Binnema
Transcript of Powerpoint presentatie Corporate NL€¦ · Selection of test persons (inclusion and exclusion...
DiAgRaMsFolkert Faber, Maaike Smelt, Nynke Leistra, Doede Binnema
Programme
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- Introduction project Diagrams
- Logistics organisation TEMPO2.0 Hanzehogeschool
- Bicycle ergometer tests Hanzehogeschool
- Some conclusions and remarks
Aim of the Diagrams project
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2013-2017
Standardization of the bicycle ergometer test as stress model to assess nutritional effects on intestinal function and immune
responsiveness in healthy young men
Aim of the Diagrams project
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Standardization of the bicycle ergometer test as stress
model to assess nutritional effects on intestinal function
and immune responsiveness in healthy young men
Endurance tests (stress tests) used to induce intestinal permeability
Not properly validated stress tests available (inter-lab variability)
Not clear how exercise-intensity influences the biomarkers
Intestinal permeability linked to health
What are reliable biomarkers for the intestinal permeability?
Can nutrition influence (protect) intestinal permeability?
Aim of the Diagrams project
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Intestinal epithelial barrier:
role in maintaining health
Microbial flora
Immune system
Permeability linked to health effects
(e.g. Chronic inflammation)
Intestinal permeability (integrity)
Physical exercise can influence
intestinal permeabilityKonig. Clin Transl Gastroenterol. 2016 Oct; 7(10): e196.
Aim of the Diagrams project
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Intestinal permeability (integrity) Biomarkers
Gastro-intestinal: e.g. citrullin, IFABP
Immunological: e.g. leukocytes (granulocyte, lymphocyte,
monocyte subpopulations), IL-8, IL-10)
General: e.g. insulin, cortisol, electrolytes, et cetera
Intervention: Multi-sugar: Lactulose, Rhamnose solutions
Amino acids: Glutamine/Alanine solutions
Blood, saliva and urine
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Phase 3: Interventions studies (possibly with less healthy population)
General Outline Diagrams project
Phase 1: “GRINTA”
Bicycle ergometer tests (5 bicycle ergometer test; 15 persons; Wageningen)
Analysis of various biomarkers at different time points
Statistical selection of relevant biomarkers at relevant time points
Phase 2: “TEMPO2.0”
Interlab-variability: Validation of bicycle ergometer test on selected biomarkers.
Setup (as indicated in METC)
4 exercise protocols (including rest)
7 test persons in Utrecht (Hogeschool Utrecht)
7 test persons in Groningen (Hanzehogeschool)
General Outline Diagrams project
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monocentre: Wageningen
autumn 2014
15 volunteers
Rest condition
4 exercise protocols of 1h with different intensities and/or hydration status
• P1 = rest protocol
• P2 = 1h 70% Wmax
• P3 = 1h 70% Wmax, dehydrated cond.
• P4 = 1h 50% Wmax
• P5 = 1h 55/85% Wmax
• > 80 parameters
GRINTA!
multicentre: Utrecht and Groningen
Spring 2017
14 volunteers
Rest condition
3 exercise protocols differentiating in duration and intensity
• P1 = rest protocol
• P2 = 1h 70% Wmax
• P4 = 1h 50% Wmax
• P6 = 30 min 70% Wmax
± 35 parameters
TEMPO2.0
TEMPO2.0 @ Hanze UAS
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Logistical organisation of TEMPO2.0 at the Hanze
Approval of the METC (some delay due to changes in the protocol)
Organizing the bicycle ergometer tests:
Selection of test persons (inclusion and exclusion criteria)
o Organize meeting to inform test persons ZP11
o Schedule for medical screening UMCG
o Schedule form maximal capacity tests (VO2max; Wmax) Sport facility labs
Planning of bicycle ergometer tests:
o Fitting in the educational program of ILST
o Fitting in the availability of ergometers (Sport studies)
TEMPO2.0 @ Hanze UAS
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Physical exercise
Health status
Food intake
Healthy males
18-35 years
Cycling experience
BMI 18,5-25 kg/m²
Valid VO2max-test
Suitable veins
Immune-/GI-related
disease
Use of NSAIDS
Use of hard drugs
Use of pro-/prebiotica
Smoking
Participation in other
studies
Blood donor
TEMPO2.0 @ Hanze UAS
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Hanze: If possible, the research at the Hanze is linked to education
Institute of Life Science & Technology
Doede Binnema (KCBBE)
Maaike Smelt (lecturer/researcher)
Folkert Faber (lecturer/researcher)
Nynke Leistra (process manager)
Various instructors supervising students
Students:
- Arnau Sangenis (internship period on Flow Cytometry and IFAB-Elisa)
- Various 3rd year students Minor Medical Biology:
BML-Research
BML-Medical Diagnostics
ILST: Zernikeplein 11: Van Doorenveste
TEMPO2.0 @ Hanze UAS
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School of Sport Studies
Sporting facilities (bicycle ergometers)
SportsFieldLab Groningen: “Inspanningslab”
General Practice Centre (StudentArts)
Sports doctor (Zernikeplein and UMCG)
Qualified nurse (blood sampling)
Volunteers/test subjects/students
Zernikeplein 17: Willem Alexander SportcentrumTEMPO2.0 connecting Schools of Hanze
Challenging for ILST:
First time research-participation in METC study
Combining TEMPO2.0 with educational curriculum
Tuning of activities between all participants (scheduling)
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Scheduling of bicycle ergometer tests during minor Medical Biology (Feb.-June 2017)
Logistical organisation TEMPO2.0
Logistical organisation TEMPO2.0
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Scheduling
Information: 10 test persons were selected based on profiles (March 29)
Screening:
Sign up for ECG (April 14)
(Medical consults Hans Zwerver/Bram Bessem)
Max. tests (Wmax and VO2max) (April 18 or 19; Ruby Otter)
SportFieldsLab Sport Studies (ZP17)
Test weeks:
4 weeks (“Wednesday group” and “Thursday group”)
week 19: May 10 and11 (12)
week 20: May 17 and 18 (19)
week 22: May 31, June 1 (2)
week 23: June 7 and 8 (9)
Unfortunately, 3 persons withdrew participation 7 test subjects
Logistical organisation TEMPO2.0
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Test protocols
A. 60 min rest
B. 60 min. 70% Wmax
C. 30 min. 70% Wmax
D. 60 min. 50% Wmax
Group 1 Group 2
Nummer 10-mei 17-mei 31-mei 7-jun 6-jul Nummer 11-mei 18-mei 1-jun 8-jun 6-jul
31 A D B C 35 A D B C
32 B A X D C 36 X B C D A
33 C B D A 37 C B D A
38 D C A B
Date Date
subject age weight length BMI VO2max Wmax Wmax/kgyears kg cm kg/m2 ml/min·kg Watt Watt/kg
31 32,0 88,6 188 25,1 49 406 4,6
32 26,7 80,1 186 23,2 58 468 5,8
33 22,9 82,0 188 23,2 60 531 6,5
35 22,3 72,2 178 22,8 58 388 5,4
36 19,7 61,4 174 20,3 61 348 5,7
37 26,8 69,5 181 21,2 57 368 5,3
38 18,8 69,9 171 23,9 66 442 6,3
average 24,2 74,8 180,9 22,8 58,4 421,6 5,7
std dev 4,29 8,53 6,33 1,49 4,75 58,80 0,59
Gro
up
1G
rou
p 2
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Organisation of endurance tests
Overview protocol for test persons
Before the test day:
- Keep a diary (exercise, intake food, health remarks)
- Evening:
- Approx. 18:00 use meal (handed out)
- until 23:00 few snacks (handed out)
During the test day:
- 8:00 – 16:00 Endurance tests and sampling for biomarkers (next slide)
After test day
- 8:00: come to lab in fastened state
- Blood and saliva sampling (24h)
- Use breakfast at Hanze
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Organisation of endurance tests
Fastened state
Collect urine
Weigh test person
Apply blood cannula, fill in questionnaire
Basis line blood and saliva
Small breakfast (2 crackers + peanut butter + tea)
Every hour: 200 ml of water
Start bicycling T=0
Take lactulose/rhamnose
Sample blood T=30
Sample blood and saliva T=60
Stop bicycling T=60
Glutamine/alanine solution
Continue sampling
Lunch
Check diary, hand out meals, instructions
Next day: return for blood and saliva sample
Phase II TEMPO 2.0; sampling
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T=0 (min) T=60 T=120 T=360min
Endurance protocol (A, B, D)
Protocol (C)
T=30 T=90 T=180 T=240 T=300 T=24hrs
T=30 T=90 T=330minT=0 T=60 T=150 T=210 T=270 T=24hrs
• T=0: Lactulose & Rhamnose drink to measure trans- and paracellular intestinal permeability
• T=30/60: Glutamine-Alanine dipeptide solution to measure small intestinal enterocyte function
• Subjects were euhydrated and received standardized meals (breakfast
after the endurance test)
Scheduling sampling
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Preparation of large amount of sampling vials (with labels)
Logistical organisation TEMPO2.0
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StudentArts(General Practice Center) SportsFieldLab
ILST labs
processing
samples
De Plint(rest/work facility)
Sporting facilities(bicycle ergometers)
Bicycle ergometer tests
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Preliminary results
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citrullin
Wageningen GroningenUtrecht
glutamine bolus
Citrulline
1. Post-prandial increase citrullin levels in all protocols with peak after 1h
2. Glutamine bolus in 0.5h protocol at T30, post-prandial peak 0.5h earlier
3. 1h 70% and 1h 50% protocols: Utrecht-Wageningen comparable levels (1.5x), Groningen
lower levels (1.25x)
4. 1h 70%: post-exercise and post-prandial levels in Groningen are comparable
5. 0.5h 70% protocol: Utrecht-Groningen comparable levels
Normalized
concentrations
Citrullin is linked to glutamine conversion in entrocytes and thus to entrocyte activity
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Preliminary results iFABP
iFABP
1. In all centres 1h 50% Wmax follows levels in rest condition levels
2. Levels 1h 70% are comparable in all centres
3. Utrecht: comparable levels 1h and 0.5h 70% Wmax
4. Groningen: 0.5h 70% shows lower levels than 1h 70% Wmax
Wageningen Utrecht Groningen
Heavy exercise appears to induce short term enterocyte damage
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Analysis of leukocyte populations
Procedure:• Collection in Na-Heparin tubes for Flow Cytometry • Lysis of RBCs in small WB sample & counting of total leukocyte numbers• Staining, using 3 different antibody cocktails to discriminate between leukocyte subsets• Lysis of RBCs and analysis by flow cytometry (Accuri C6, FlowJo software)
• Staining was optimized by 3rd year BML-MD students during theme 9 & 10 2016_2017• Staining was performed by 3rd year BML-research students during theme 12 2016_2017
Cocktail M1 M2 M3 M4
Granulocytes CD15 CD16 CD62L CD193
Monocytes CD14 CD16 CD62L HLA-DR
Lymphocytes CD3 CD19 CD56 CD16
Leukocytes are part of the immune system, which is very complex
Leukocytes can be labelled with specific tags to identify subpopulations
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Leukocytes and exercise
• Total leukocyte numbers increase after (heavy) exercise
• In response to intestinal epithelial stress and injury?
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Gating strategy granulocytes
Of “Live cells” Of “Granulocytes” Of “CD15+CD16+” Of “CD16-CD15dim”
FSC FSC CD16-PerCP
CD
15
-FIT
C
SSC
SSC
CD16-PerCP
CD
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L-A
PC
CD15-FITC
CD
19
3-P
E
GranulocytesNeutrophils
Basophils
Rest
Regular neutrophils
Suppressorneutrophils
Eosinophils
Cocktail M1 M2 M3 M4
Granulocytes CD15 CD16 CD62L CD193
Monocytes CD14 CD16 CD62L HLA-DR
Lymphocytes CD3 CD19 CD56 CD16
• Spectral overlap: compensation samples (beads)
• All gates were set based on Fluorescence Minus One
(FMO) samples
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0
1
2
3
0 60
18
0
36
0
0 60
18
0
36
0
0 60
18
0
36
00 60
18
0
36
0
60' rest 30' 70%60' 70% 60' 50%
*
***
****
***
**
***
CD
15
+C
D1
6- C
D1
93
+ (%
of
gra
nu
loc
yte
s)
Eosinophils
0
50
100
150
0 60
18
0
36
0
0 60
18
0
36
0
0 60
18
0
36
00 60
18
0
36
0
60' rest 30' 70%60' 70% 60' 50%
**
* *
CD
15
+C
D1
6+ (
% o
f g
ran
ulo
cy
tes
)
0.00
0.05
0.10
0.15
0.20
0.25
0 60
18
0
36
0
0 60
18
0
36
0
0 60
18
0
36
00 60
18
0
36
0
60' rest 30' 70%60' 70% 60' 50%
CD
15
- CD
16
+ (
% o
f g
ran
ulo
cy
tes
)
Neutrophils
0
5.0×1009
1.0×1010
1.5×1010
0 60
18
0
36
0 0 60
18
0
36
0
0 60
18
0
36
00 60
18
0
36
0
****
*
**
*
CD
15
+C
D1
6+ (#
/L)
60' rest 30' 70%60' 70% 60' 50%
0
2.0×1007
4.0×1007
6.0×1007
8.0×1007
0 60
18
0
36
0 0 60
18
0
36
0
0 60
18
0
36
00 60
18
0
36
0
***
***
CD
15
+C
D1
6- C
D1
93
+ (#
/L)
60' rest 30' 70%60' 70% 60' 50%
0
2.0×1006
4.0×1006
6.0×1006
8.0×1006
1.0×1007
0 60
18
0
36
0 0 60
18
0
36
0
0 60
18
0
36
00 60
18
0
36
0
CD
15
- CD
16
+ (
#/L
)
60' rest 30' 70%60' 70% 60' 50%
Granulocytes following exerciseBasophils
• Within the granulocytes, exercise appears to induce a redistribution of subsets favoring neutrophils over eosinophils• However, also during the rest-protocol, these changes are observed (less prominent)• Following exercise, blood neutrophil numbers increase, whereas eosinophil numbers decrease• The distribution of suppressor and regular neutrophils is not affected by any exercise protocol
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Preliminary conclusions and remarks
• Large amount of biomarkers still needs to be analyzed
• Statistical analysis of obtained data still in progress, but….
• Exercise appears to induce short term intestinal stress and immunologic changes:
• Altered levels are found in all volunteers
• Kinetic effects of all biomarkers (0-24 hr)
• Relationship between exercise intensity and effect on different parameters
• Results show similar responses: GRINTA! (Wageningen), TEMP2.0 (Utrecht/Groningen)
• Standardization of bicycle ergometer tests is possible!
Remarks:
• Intestinal stress trigger for immune activation/recruitment?
• Do these changes reflect the changes in individuals with compromised health?
• Short term exercise (30 min; 70% Wmax) seems effective for primary biomarkers
protocol for elderly people, or with compromised health (e.g. obesities, diabetes)?
Phase 3: Intervention study (Winclove; FrieslandCampina)?
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REMARKS
We managed implement a human clinical study in collaboration with Sport studies and UMCG
- First-time successful completion of human study at ILST (METC)
- Cooperation between institutes stimulated (between schools and between UAS)
- Enthusiastic and motivated students and teachers
- Increased knowledge in institutes
- Possibilities for scientific publications
(PhD Shirley Kartaram)
- Valorisation of knowledge in other projects
- PR activities
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Acknowledgements
Research team @ HANZE:Maaike SmeltNynke LeistraCynthia KoxDoede Binnema
All study subjectsAll participating MD and BMR students, especially Arianne Ebbinge for doing additional work!
Research team @ HU, UU, WUR, Avans:Shirley KartaramRaymond PietersRenger WitkampJohan Garssen Klaske van NorrenEric Schoen Harriet WittinkTim BlatterLaura M’RabetMarc Teunis
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333-12-2018
Phase II TEMPO 2.0; Analytes in urine
Procedure:• Urine is collected and stored at -
20C• Fluid intake and urine output are
measured
Biomarkers:• Lactulose/Rhamnose ratio
Results:• Not yet analyzed
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Citrulline as a marker for enterocyte activity
L-Glutamine-L-Alanine dipeptide
353-12-2018
iPlasma iFABP as a marker for intestinal damage
• iFABP is a enterocyte-specificintracellular protein involvedin fatty acid/lipid metabolism
• Plasma iFABP is a sign of enterocyte damage/stress
