Association of Constipation Inflammatory and Microbial Translocation Markers
Condition: Constipation; Inflammation
MeSH terms: Inflammation; Constipation
Study type: Observational

Summary: 
      Growing evidences showed that patients with chronic constipation accompanied with intestinal
      dysbiosis. Gut dysbiosis is a harbinger of chronic inflammation, yet the underlying basis is
      unclear. Plasma level of microbial translocation is a marker of mucosal permeability.
      Increased mucosal permeability ignites elevated microbial translocation and is a source of
      systemic immune activation in CKD patients. The passage of microbial components from the
      gastrointestinal tract into the systemic circulation may be an important contributor to the
      chronic inflammatory process and subsequent atherosclerosis development. We plan to determine
      the association constipation with biomarkers of inflammation such monocyte activation and
      associated cytokines as well as markers of microbial translocation including endotoxin and
      its antibodies, intestinal barrier proteins of 200 hemodialysis patients.
    

Detailed description: 
      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

        1. Hemodialysis for more than 3 months

        2. 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.
    

Observational model: Case-Crossover
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        hemodialysis (HD) patients
      
Biospecimen description: 
      plasma
    
Keywords: constipation; microbial translocation; inflammatory markers