New road map through the land of IBD.
- SourceAvailable from: Giovanni Parmigiani[Show abstract] [Hide abstract]
ABSTRACT: Ulcerative colitis (UC) and Crohn's disease (CD) are inflammatory bowel diseases (IBD) with variable, overlapping clinical features and complex pathophysiologies. To identify pathogenic processes underlying these disease subtypes, we used single endoscopic pinch biopsies to elucidate patterns of gene expression in active and inactive areas of UC and CD and compared these to infectious colitis and healthy control samples. Unsupervised classification of a total of 36 samples yielded promising separation between the affected IBD, unaffected IBD, non-IBD colitis, and normal control samples, suggesting each sample type had a distinctive gene expression pattern. Genes differentially expressed in the CD samples compared to in the controls were related to IFNgamma-inducible TH1 processes (IFITM1, IFITM3, STAT1, and STAT3) and antigen presentation (TAP1, PSME2, PSMB8). The most noticeable change in the UC samples was reduced expression of genes regulating biosynthesis, metabolism, and electrolyte transport (HNF4G, KLF5, AQP8, ATP2B1, and SLC16A). Twenty-five percent of genes down-regulated in the UC samples were also down-regulated in the infectious colitis samples. Unaffected biopsy samples of IBD patients also registered differences expression of genes compared to in the normal controls. Of these differentially expressed genes, only 2 were up-regulated, PSKH1, a regulator of mRNA processing, and PPID, a suppressor of apoptosis. The study shows that the gene expression patterns of IBD, CD in particular, are quite different from those of infectious colitis, highlighting distinctive expression of genes and pathways in UC and CD.Inflammatory Bowel Diseases 08/2007; 13(7):807-21. · 5.12 Impact Factor
- Gastroenterology 11/2007; 133(4):1327-39. · 12.82 Impact Factor
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ABSTRACT: The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn's disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N = 6), UC (N = 6), and colonic cancer (N = 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor alpha that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N = 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI alpha expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation.Journal of Proteome Research 04/2007; 6(3):1114-25. · 5.06 Impact Factor
New Road Map Through the Land of IBD
immunofluorescence microscopy to investigate the mecha-
nisms underlying the inherent intestinal tissue injury seen in
both ulcerative colitis (UC) and Crohn’s disease (CD). In this
study the authors recruited patients with active inflammatory
bowel disease (IBD), as defined by a CD activity index ?150
and colitis activity index ?9 for CD and UC, respectively.
Endoscopic samples were taken from macroscopically in-
flamed areas, confirmed by histological examination, and
compared with the normal mucosa from control subjects.
Frozen tissue sections from these biopsies were then serially
treated with 32 different fluorescent-labeled antibodies and
imaged. The resulting data for all antibodies were integrated
to generate a matrix of combinatorial molecular phenotypes
(CMP), consisting of the presence or absence for each of the
32 epitopes distributed in a topographic manner over the
entire section. As such, a 2D representation of protein asso-
ciations was created on the histological map of the intestinal
mucosa. Wilcoxon rank-sum and Student’s t-test were used to
identify CMPs that differed in a statistically significant man-
ner between the biological classes. Hub analysis was used to
identify markers that colocalized with a particular marker of
The authors report finding 1337 CMP motifs that dif-
fered between CD and controls, 2930 that differed between
UC and controls, and 2599 that differed between CD and UC.
As it is difficult to determine which motifs are pathogenic and
which are bystanders, the authors then asked focused ques-
tions about CD3?T cells to determine if specific combina-
tions of protein colocalizations might differentiate between
the disease cohorts. Through this analysis the authors deter-
mined that naive T cells are increased and resistant to apop-
totic signals in the inflamed mucosa in CD. However, the
regulation of apoptosis was distinct in various T-cell sub-
populations. Furthermore, CD4?CD25?T regulatory cells
were found to be elevated only in UC, whereas CD4?CD7?
memory T cells were increased in both CD and UC. Using
erndt et al recently reported a provocative new approach
that uses Multi-Epitope-Ligand-Cartographie (MELC)
hub analysis, the authors were able to identify colocalization
patterns for NF-?b and PARP that distinguished UC from
CD. In summary, the authors have presented a usage for the
MELC technology to interrogate protein networks in a topo-
graphical context to better understand IBD pathogenesis.
With the recent explosion in high-throughput “-omic” technol-
ogies, there is an ever-increasing and confusing morass of bio-
logical data that is accompanied by not insignificant false-posi-
tive and -negative rates. However, through the integration of
orthogonal “-omic” approaches we can better understand the
relationships between individual genes/proteins to differentiate
between relevant disease pathophysiology and epiphenomenon.1
Recent genome-wide association studies for IBD susceptibility
loci have identified several genes, most notably NOD2 and
IL-23R.2,3Transcriptome studies of endoscopic pinch biopsies
have demonstrated that genes involved in Th1 processes and
antigen presentation are upregulated, whereas in UC genes that
regulate biosynthesis, metabolism, and electrolyte transport are
downregulated.4Tissue proteome studies have demonstrated
increased proteins associated with signal transduction, stress
responses, and energy metabolism.5However, it is only through
the integration of the growing number of high-throughput data-
sets that we might extract additional relevant biological infor-
mation. This approach is illustrated in our recent colorectal
cancer biomarker discovery studies, which integrated plasma
proteome data and tissue transcriptomic data to prioritize candi-
date biomarkers for orthogonal antibody validation (Hung et al,
Traditional “shotgun” proteomics approaches are per-
formed on tissue lysates to determine global protein differ-
ences between 2 biological states.8Unfortunately, proteins
require proper colocalization within a framework that is or-
ganized in time and space for their functionality. Whereas
this information is not captured by traditional proteomics
approaches, the MELC technology is able to provide such
valuable toponomic data. Nonetheless, MELC does suffer
from being an “unsupervised supervised approach.” This
method is unsupervised in that the CMPs can be clustered
without knowledge of the disease classes. However, it is
supervised in that it is dependent on carefully characterized
antibodies against previously known epitopes. As such, the
MELC technology can only uncover novel colocalizations
Copyright © 2008 Crohn’s & Colitis Foundation of America, Inc.
Published online 14 February 2008 in Wiley InterScience (www.
Inflamm Bowel Dis ● Volume 14, Number 6, June 2008
and interactions between known proteins. Nonetheless, these
2 approaches may be coupled in a complementary fashion.
Shotgun proteomics may be used to identify novel proteins of
interest, whereas MELC can place these proteins in the con-
text of a protein interaction network. Taken together, the
MELC technology can now add an extra dimension of infor-
mation to traditional proteomics approaches.
As highlighted previously, analysis of enormous high-
throughput datasets has yielded limited biological significance.
However, the integration of orthogonal “-omic” datasets can
yield additional biologically significant information. Indeed,
such an analysis might identify further biological importance
among the thousands of CMP motifs that were identified with
only 32 antibodies. As such, the MELC technology now adds
the toponome to our growing arsenal against IBD.
Kenneth E. Hung, MD, PhD
Instructor of Medicine
Harvard Medical School
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