Use and identification of biomarkers for gastrointestinal diseases

a biomarker and gastrointestinal disease technology, applied in the field of identification of biomarkers for gastrointestinal diseases, can solve the problems of no universally accepted molecular markers that identify gastrointestinal stem cells, poor prognosis for patients with advanced crc, and no specific biomarker to aid

Inactive Publication Date: 2010-04-15
ALFAGENE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0065]FIG. 11 shows RT-PCR amplicons derived from normal cells and gastrointestinal diseased cells. Lane 1: positive control; Lane 2: normal CRC cell line; Lane 3, diseased CRC cell line; Lane 4: non-active ulcerative colitis cell line; Lane 5: active ulcerative colitis cell line; Lane 6: non-active Crohn's disease cel

Problems solved by technology

There are no universally accepted molecular markers that identify gastrointestinal stem cells.
Many treatments focus on symptom relief, and are not curative.
The prognosis for patients with advanced CRC generally is poor.
Currently, no specific biomarker exists to aid in early diagnosis and/or management of CRC.
However, none of these genes or molecules have proved to be specific.
This partially may be due to the fact that studies that compare differential gene expression between tumor and normal colorectal epithelium have been limited by the lack of paired normal and tumor-derived colorectal epithelial cell lines from the same individual.
However, significant difficulties for identifying these biomarkers exist.
Additionally, many individuals do not avail themselves of colonoscopy (the best means of early detection) due to its costly and invasive nature.
Further, the vast majority of CRC biomarkers that have been discovered do not have a function in vivo, are not very effective in early detection of CRC, recognize only a small subset of cancers,

Method used

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  • Use and identification of biomarkers for gastrointestinal diseases
  • Use and identification of biomarkers for gastrointestinal diseases
  • Use and identification of biomarkers for gastrointestinal diseases

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation and Culture of HIPEC and HITEC Lines

[0207]1.1. Isolation of Human Gastrointestinal Segment-Specific Stem-Cell-Like Progenitor Cells and Establishment of Epithelial Cell Lines in Long Term Culture

[0208]Human intestinal primary epithelial cell lines (HIPEC) and human intestinal tumor-acquired epithelial cell lines (HITEC) are derived from gastrointestinal epithelial stem cell-like progenitor cells obtained from normal colon epithelium and from cancerous colon epithelium. Normal intestinal epithelial cells (IECs) are obtained from patients with colon cancer using surgical specimens excised at least 10 cm away from the tumor. Cancer tissue IECs are excised from the same patient. Both surgical specimens are made available from the Departments of Surgery and Pathology at the Robert Wood Johnson University Hospital.

[0209]1.1(a) Tissue Isolation

[0210]Surgical specimens of tissue are transported from an operating room to the laboratory within 30 minutes of surgery. The specimens ar...

example 2

Proof of Epithelial Origin of HIPEC Lines

[0222]2.1(a) Cytokeratin 18 Staining

[0223]Cells of epithelial origin are known specifically to contain cytoskeletal keratin. The intermediate filament system of intestinal epithelial cells in vivo contains cytokeratin 18 and 8. In order to confirm the epithelial origin of the HIPEC cells in culture, these cells are stained with an antibody (anti-cytokeratin 18) against one of the two intestinal epithelial filament proteins.

[0224]Gastrointestinal stem-cell-like progenitor cells, as isolated in Example 1 (see above), are grown on cover slips (Fisher Scientific). After permeabilization and fixation, cells are stained with anti-CK-18 antibody. The cover slips are washed three times in PBS / BSA and subsequently fixed in cold methanol at −20° C. for 10 minutes. The coverslips then are incubated in 2% FCS / PBS for 5 minutes. A FITC conjugated antibody against anti-human cytokeratin (Sigma) is used to detect the presence of cytokeratin and is incubated...

example 3

Characterization of HIPEC Lines

[0229]3.1. Morphology

[0230]The morphology of the HIPEC cell lines is examined using light microscopy. The epithelial origin of the cells is confirmed by staining with anti-cytokeratin 18. Electron microscopy is used to evaluate the ultrastructural characteristics of the cells. The cells were found to have a structural organization typical of intestinal epithelial cells as well as microvilli found at the apical membrane and a basement membrane formed at the base of the monolayer.

[0231]3.2. HIPEC Lines are Non-Transformed

[0232]Soft agar was prepared as follows: a) 1% agarose solution (5 ml) was added into a 10 cm2 petri dish until the plate was completely covered; b) the agarose was pipetted off leaving a thin film of agarose on the bottom of the petri dish; c) after drying the plate for 20 minutes with the lid on, 10 ml of cell suspension (0.5×106) was added on the top of the agarose film; d) medium was replaced every 4-5 days. Suspensions (2.0 ml) of n...

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Abstract

The described invention relates to the identification of biomarkers for gastrointestinal diseases and provides methods utilizing the biomarkers for in drug discovery, monitoring of treatment efficacy, and diagnostics. The invention further provides methods for identifying a therapeutic target to treat ulcerative colitis, colorectal cancer, and Crohn's disease.

Description

CROSS REFERENCES[0001]This application claims the benefit of priority to U.S. provisional application 61 / 195,646, filed 9 Oct. 2008, which is incorporated in its entirety herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to the identification of biomarkers for gastrointestinal diseases and further provides methods utilizing such biomarkers in drug discovery, the monitoring of treatment efficacy, and diagnostics.BACKGROUND OF THE INVENTION[0003]Components of the Human Gastrointestinal Tract[0004]The gastrointestinal tract is a continuous tube that extends from the mouth to the anus. On a gross level, the gastrointestinal tract is composed of the following organs: the mouth, most of the pharynx, the esophagus, the stomach, the small intestine (duodenum, jejunum and ileum), and the large intestine (cecum including the appendix, colon and rectum) (Tortora, G. J., et al., Principles of Anatomy and Physiology, Wiley & Sons, Inc. Hoboken, N.J. 2006). Each segmen...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/25C40B30/00
CPCC12Q1/6886C12Q2600/136C12Q2600/158G01N33/5044G01N33/57446G01N2500/10G01N2800/065G01N33/57419C12N5/0679C12N5/068C12Q1/68G01N33/5073G01N33/53
Inventor PANJA, ASIT
Owner ALFAGENE BIOSCI
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