Indole-3-PROpionic Acid Clinical Trials - a Pilot Study Part 2

FoxP3+CD25+CD127- regulatory T cells expressed as a percentage of single, live CD3+CD4+CD8- lymphocytes. Analysed in freshly isolated peripheral blood mononuclear cells using a Symphony A3 flowcytometer.

Brain-derived neurotrophic factor measured in platelet-free plasma samples using ELISA or mesoscale.

Th1/Th2 ratio calculated from T cell profiling of freshly isolated peripheral blood mononuclear cells (PBMCs) using a Symphony A3 flowcytometer.

Th1 cells are defined as the CXCR3+CCR4-CCR6-CCR10- non-Treg population and expressed as a percentage of single, live CD3+CD4+CD8-CD45RA- lymphocytes.

Th2 cells are defined as the CXCR3-CCR4+CCR6-CCR10- non-Treg population and expressed as a percentage of single, live CD3+CD4+CD8-CD45RA- lymphocytes.

Panel antigens: FVS780, CD3, CD4, CD8, CD45RA, CCR7, CXCR3, CCR6, CCR4, CCR10, CD25, CD127, FoxP3.

Th17/mTreg ratio calculated from T cell profiling of freshly isolated peripheral blood mononuclear cells (PBMCs) using a Symphony A3 flowcytometer.

Th17 cells are defined as the CXCR3-CCR4+CCR6+CCR10- non-Treg population and expressed as a percentage of single, live CD3+CD4+CD8-CD45RA- lymphocytes.

mTreg (memory Tregs) are defined as the FoxP3+CD25+CD127-CD45RA- population and expressed as a percentage of single, live CD3+CD4+CD8-CD45RA- lymphocytes

Panel antigens: FVS780, CD3, CD4, CD8, CD45RA, CCR7, CXCR3, CCR6, CCR4, CCR10, CD25, CD127, FoxP3.

Th17.1 cells in freshly isolated peripheral blood mononuclear cells (PBMCs) characterized using a Symphony A3 flowcytometer.

Th17.1 cells are defined as the CXCR3+CCR4-CCR6+CCR10- non-Treg population and expressed as a percentage of single, live CD3+CD4+CD8-CD45RA- lymphocytes

Panel antigens: FVS780, CD3, CD4, CD8, CD45RA, CCR7, CXCR3, CCR6, CCR4, CCR10, CD25, CD127, FoxP3.

C-reactive protein (CRP) measured in plasma (mg/L) as a biomarker of infection and systemic inflammation. Lower-limit of quantification: 0,4 mg/L. Values below 0,4 mg/L are imputed as 0,2 mg/L.

Plasma triglycerides (mmol/l).

Non-HDL cholesterol calculated as total cholesterol minus HDL cholesterol (mmol/l)

Proinsulin C-peptide (pmol/l) measured in plasma.

Plasma glucose (mmol/l). Participants abstain from eating and drinking after 22.00 the day before. Only water is allowed. Fasting blood samples are taken between 8.00-10.00 in the morning.

F2-isoprostanes with a specific focus on 8-iso-prostaglandin F2α measured in blood or morningurine samples as a marker of lipid oxidation.

8-oxo-dG (8-Oxo-2'-deoxyguanosine) measured in blood or morningurine samples as a marker of DNA-related stress damage.

Targetted and untargetted liquid-chromatography mass-spectrometry-based metabolomics of serum samples. Targetted analyses aim to quantify indole-3-propionic acid and its metabolites (biomarker of compliance as well as absorptive and metabolic capacity), other bacterial- and host metabolites of tryptophan as well as short-chain fatty acids.

Plasma cholesterol (mmol/l).

Plasma very low-density lipoproteins (mmol/l).

Plasma low-density lipoprotein (LDL) (mmol/l).

Plasma high-density lipoprotein (HDL) (mmol/l).

Malondialdehyde measured in blood or morningurine samples as a marker of oxidative stress.

Protein carbonyls measured in blood or morningurine samples as a marker of protein oxidation.

HbA1c (IFCC, mmol/mol) measured in whole blood. Estimated average glucose values (mmol/l) are also calculated automatically from HbA1c by our laboratory.

Targetted and untargetted T cell profiling of freshly isolated peripheral blood mononuclear cells (PBMCs) using a Symphony A3 flowcytometer. Panel antigens: FVS780, CD3, CD4, CD8, CD45RA, CCR7, CXCR3, CCR6, CCR4, CCR10, CD25, CD127, FoxP3.

Target populations:

Naïve: CD45RA+CCR7+, Central memory: CD45RA-CCR7+, Effector memory: CD45RA-CCR7-, TEMRA: CD45RA+CCR7-, Th1: CXCR3+CCR4-CCR6-CCR10-, Th2: CXCR3-CCR4+CCR6-CCR10-, Th17: CXCR3-CCR4+CCR6+CCR10-, Th17.1: CXCR3+CCR4-CCR6+CCR10-, Th22: CXCR3-CCR4+CCR6+CCR10+, as well as expression of chemokine receptors on cytotoxic T cells and regulatory T cells.

An untargetted approach may be employed to allow for unbiased identification of changes in novel, yet uncharacterized populations.

Characterization of the gut microbiome using molecular biology methods such as 16s rRNA sequencing.

Targetted and untargetted liquid-chromatography mass-spectrometry-based metabolomics. Targetted analyses aim to quantify indole-3-propionic acid, other bacterial- and host metabolites of tryptophan as well as short-chain fatty acids.

Endotoxin, capsular polysaccharides or other bacterial polysaccharides measured in blood samples as biomarker of bacterial translocation across the intestinal epithelium.

Measurement of pre-haptoglobin 2 in blood samples as a biomarker of intestinal permeability.

Measurements of intestinal fatty acid binding protein in blood samples as a biomarker of enterocyte damage.

Measurement of citrulline in blood samples as a biomarker of intestinal function.

Calprotectin measured in fecal samples as a biomarker of intestinal inflammation.

Neopterin measured in morningurine samples as a biomarker of systemic inflammation.

Soluble urokinase plasminogen activator receptor (suPAR) measured in blood samples.

Flow cytometric analyses of microvesicles/ microparticles in platelet-free plasma as biomarker of systemic inflammation.

Flow cytometric analyses of endothelial progenitor cells measured in platelet-free plasma as biomarker of systemic inflammation

Self-reported (questionnaire) gastrointestinal symptoms of bloating, pain, rumbling, flatulence, constipation, hard stools and diarrhea evaluated using a visual analogue scale as well as a question on the frequency of defecation.

Measurement of transit time through the digestive system using a so-called maize test. Participants consume 100 grams of sweet maize in the morning between 5.30-10.00 while still in a fasting state. The exact date and time of consumption is registered and so is the exact date and time when maize is observed for the first time in feces. Transit time is expressed as the difference between the ingestion and fecal excretion timepoints in hours.

Classification of stool consistency using the Bristol Stool Chart. Numerical scale ranging from 1 (separate hard lumps) to 7 (liquid consistency with no solid pieces) with one-unit increments.

pH of collected fecal samples

Characterization of IgA, IgG and IgM-coating of bacteria from fecal samples using bacterial flow cytometry.

Plasma immunoglobulin G (g/l)

Plasma immunoglobulin A (g/l)

Total leucocytes as well as basophils, eosinophils, lymphocytes, monocytes, neutrophils as well as the joint group of metamyelo-, myelo- and promyelocytes. All counted in whole blood samples (10^9/l).

Hemoglobin measured in whole blood (mmol/l).

Plasma alanine transaminase (ALT, U/l) as a biomarker of liver function.

Alkaline phosphatase (U/l) as a biomarker of liver function.

Aspartate aminotransferase also known as aspartate transaminase measured in plasma (U/l) as a biomarker of liver damage.

Bilirubins measured in plasma (µmol/L) as a biomarker of liver function.

Coagulation factors II + VII + X (INR: International Normalized Ratio) measured in plasma as a biomarker of liver function.

Lactate dehydrogenase measured in plasma (U/l).

Plasma creatinine (µmol/L) as a biomarker of kidney function.

Estimated glomerular filtration rate (eGFR/ 1,73m² (ml/min)) as a biomarker of kidney function.

Albumin measured in plasma (g/l).

Plasma 25-OH-vitamin D (D3+D2) (nmol/L)

Thyroid-stimulating hormone (TSH) measured in plasma (mU/L)

Blood pressure (mm Hg) defined as the lowest value obtained across three measurements.