Association of Constipation Inflammatory and Microbial Translocation Markers

Study Design and Population

Patients and methods 2.1. Patient selection Two hundred hemodialysis patients are recruited. Functional constipation will be diagnosed according to the Rome IV criteria. Organic lesions of the abdominal cavity and the pelvic floor are excluded in all patients by endoscopic (sigmoidoscopy, colonoscopy, gastroduodenoscopy), radiologic and ultrasonographic evaluation.

This study will be carried out in accordance with the Declaration of Helsinki as revised in 2008. All patients will give written informed consent to participate in the study. All patients in the renal division of Tungs Taichung Metroharbour Hospital were prospectively and consecutively included. The inclusion criteria are

• Hemodialysis for more than 3 months
• Age older than 20 years old

The exclusion criteria are a history of chronic inflammatory disease, the presence of current infection, collagen vascular disease or immuno-modulatory or immuno-suppressive treatment.

Measurements of inflammatory cytokines, etotaxin-1, and amyloid A

The plasma levels of MCP-1, IL-6, CRP, IL-17A and calprotectin are tested by commercially available human ELISA kit respectively, according to the manufacturer's instruction. Human serum eotaxin1 levels are determined using a commercial ELISA kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer's protocol. The commercially available ELISA procedure is used to measure serum amyloid A concentration in serum,

Endotoxin and Endotoxin Ig M as well as IgG core antibody

Endotoxin is measured in serum by Limulus Amebocyte Lysate QCL-1000 from Lonza (catalogue # 50-647U)(EU/ml). Serum or plasma samples are diluted at 1∶5 ratio with LAL reagent water. Endotoxin IgG core antibodies are measured in plasma using an ELISA kit from Cell Sciences Inc (catalogue # HK504). Units are expressed as GMU/ml which are IgG standard median units based on medians of ranges of 1000 healthy adults by the manufacturer.

Determination of CD14 and CD16 Mononuclear Phenotype

Peripheral blood will be collected by venipuncture using ethylenedi- aminetetraacetic acid (EDTA) as an anticoagulant. For cytometric analysis, monoclonal antibodies against CD14 (fluorescein isothiocyanate (FITC) conjugated; clone RMO52; Beckman Coulter, Miami, FL, USA), CD16 (phycoerythrin (PE) conjugated; clone 3G8; Beckman Coulter, Miami, FL, USA), CD45 (phycoerythrin cyanin-5 (PC5); clone J33; Beckman Coulter, Miami, FL, USA), and CD56 (clone IM2073; Beckman Coulter, Miami, FL, USA) are used. Briefly, 100 l of the whole blood is stained with saturating amounts of the above mentioned monoclonal antibodies and corresponding isotype controls. After incubation for 15 min at room temperature in the dark according to the manufacturer's recommendations, OptiLyse C (Beckman Coulter, Miami, FL) is added to lyse RBC and the samples are fixed. Fixed cells are analyzed by flow cytometry within 6 hours.

Determination of leukocyte and monocyte subset distribution is performed using a FC500-Cytometer (Beckman Coulter), and CXP analysis software (version 2.2) is used (Schroers et al., 2005). Monocytes are identified as CD45 positive and CD56 negative cells exhibiting a specific forward and sideward scatter profile. Monocytes are then gated in an SSC/CD dot plot, identifying monocytes as CD86 cells with monocyte scatter properties. Subsets of CD14 monocytes with and without CD16 are defined according to the surface expression pattern of the lipopolysaccharide receptor CD14 and the CD16 (Fcgamma receptor III). One million cells are analyzed from each sample, and the percentage of CD16 positive mononuclear cells (CD14+/CD16+ and CD14++/CD16+) and the number of cells out of the total monocytes are compared using fluorescent microbeads (Flow-Count, Beckman Coulter). The CD86 antibody (clone HA5.2B7; Beckman Coulter, Miami, FL, USA) is used in this study.

sCD14, LBP, zonulin and I-FABP assays

LBP and sCD14 markers are used to monitor the microbial translocation while Zonulin and I-FABP are measured to explore intestinal permeability: LBP plasma levels are measured using Enzyme Immunoassay for Quantification of free human LBP ELISA kit, according to the manufacturer's recommendations. Dilution factor is 1:800. Plasma level of sCD14 is measured using Human sCD14 kit, according to the manufacturer's specifications. Dilution factor is 1:200. Zonulin serum levels are measured using Human zonulin ELISA kit. I-FABP plasma levels are measured by ELISA kit according to the manufacturer's recommendations.

Inclusion criteria

• Hemodialysis for more than 3 months and Age older than 20 years old

Exclusion criteria

• he exclusion criteria are a history of chronic inflammatory disease, the presence of current infection, collagen vascular disease or immuno-modulatory or immuno-suppressive treatment.