Exosomes and microvesicles in intestinal luminal fluids and stool and use of same for the assessment of inflammatory bowel disease
The present disclosure relates to methods of collecting intestinal luminal fluid (ILF) exosomes and microvesicles (EMV) and isolating corresponding mRNA in order to diagnose and treat inflammatory bowel diseases (IBD). In particular, certain embodiments relate to the method of capturing EMV from ILF applied to a filter device that is capable of capturing EMV. Nucleic acids such as mRNA can be isolated from the EMV using an oligo(dT)-coated plate designed to accommodate the filter device and then used for further molecular analysis. Quantification of the collected nucleic acids may then be used in the diagnosis and/or treatment of IBD.
1. A method of treating an inflammatory bowel disease (IBD) in a human subject, the method comprising:
(A) having a vesicle-containing sample obtained from said human subject sent to a laboratory for the laboratory to perform an assay comprising the following steps (1)-(3):
(1) capturing at least a portion of vesicles from said sample on or in a vesicle-capture material, thereby generating a vesicle sample;
(2) quantifying an expression level of at least two genes in said vesicle sample, wherein said at least two genes is selected from the group consisting of epithelial cell adhesion molecule (EPCAM), mucin 2 (MUC2), trefoil factor 1 (TFF1), defensin alpha 3 (DEFA3), lymphocyte common antigen (CD45), transforming growth factor beta 1 (TGFB1), and S100 calcium binding protein A9 (S100A9);
(3) determining that said subject has said IBD due to said expression level of each of said at least two genes being significantly different from the expression level of the respective gene in a vesicle sample of a healthy human control subject not suffering from said IBD, thereby diagnosing said subject as having IBD; and
(B) administering an effective amount of an IBD medication to the human subject having said IBD, wherein said IBD medication is selected from the group consisting of an aminosalicylate, a corticosteroid, an immunosuppressor, and a TNF-alpha inhibitor, thereby treating said IBD in said human subject.
2. The method of claim 1 , wherein said vesicle-containing sample is selected from the group consisting of a blood sample, a urine sample, a saliva sample, and an intestinal fluid sample.
3. The method of claim 1 , wherein capturing comprises passing said vesicle-containing sample through a filter comprising glass fiber.
4. The method of claim 1 , further comprising lysing said vesicle sample on or in said vesicle-capture material.
5. The method of claim 1 , wherein said inflammatory bowel disease is selected from the group consisting of ulcerative colitis and Crohn's disease.
6. The method of claim 1 , wherein said vesicle-containing sample is a stool sample.
7. The method of claim 6 , further comprising diluting the stool sample in liquid.
8. The method of claim 1 , wherein quantifying an expression level of at least two genes in said vesicle sample comprises quantifying said expression level of three genes.
9. The method of claim 1 , wherein the vesicle-containing sample comprises a fluid sample and capturing further comprises:
receiving said fluid sample; and
passing said fluid sample through a vesicle capture material adapted to capture a captured vesicle population within said vesicle capture material.
10. The method of claim 9 , wherein quantifying comprises:
positioning said vesicle capture material and said captured vesicle population adjacent to a substrate comprising immobilized oligo(dT);
applying lysis buffer to said vesicle capture material, thereby lysing said captured vesicle population;
hybridizing at least two mRNA from said captured vesicle population to said substrate;
synthesizing directly on said substrate at least two cDNA from said at least two mRNA; and
quantifying by PCR analysis of said at least two cDNA an expression level of at least two marker mRNA in said captured vesicle population.
11. The method of claim 9 , wherein receiving comprises receiving said fluid sample from a medical professional.
12. The method of claim 9 , wherein said fluid sample comprises a stool sample from a human being.
13. The method of claim 9 , wherein said fluid sample comprises an intraluminal fluid sample from a human being.
14. The method of claim 13 , wherein said intraluminal fluid is selected from the group consisting of a blood, a urine, a saliva, and an intestinal fluid.
15. The method of claim 14 , wherein said vesicle capture material comprises glass fiber.
16. The method of claim 15 , wherein passing comprises loading said fluid sample into a device comprising a loading reservoir removably attached to a tip, wherein said vesicle capture material is housed within said tip.
17. An assay for quantifying an expression level of one or more mRNA in vesicles of an intraluminal fluid sample, the assay comprising:
capturing said vesicles from said intraluminal fluid on a vesicle capture material by passing said intraluminal fluid through said vesicle capture material;
lysing said vesicles on said vesicle capture material, thereby generating a lysate;
passing said lysate from said vesicle capture material to a substrate comprising immobilized oligo(dT);
hybridizing said one or more mRNA from said lysate to said substrate;
synthesizing directly on said substrate cDNA from said one or more mRNA hybridized to said substrate; and
using said cDNA for real-time quantitative polymerase chain reaction, thereby quantifying the expression level of said one or more mRNA in said vesicles of said intraluminal fluid sample, wherein each of said one or more mRNA corresponds to a gene selected from the group consisting of epithelial cell adhesion molecule (EPCAM), mucin 2 (MUC2), trefoil factor 1 (TFF1), transforming growth factor beta 1 (TGFB1), and S100 calcium binding protein A9 (S100A9).