INHIBITORS OF IGFBP3 BINDING TO TMEM219 FOR TREATMENT OF INTESTINAL DISEASES
20200316168 ยท 2020-10-08
Inventors
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K38/177
HUMAN NECESSITIES
A61P1/14
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to an inhibitor of IGFBP3 binding to TMEM219 and uses thereof for the treatment and/or prevention of an intestinal disease in a subject, in particular the treatment and/or prevention of diabetic enteropathy or inflammatory bowel disease.
Claims
1. An inhibitor of IGFBP3, wherein the inhibitor is an extracellular fragment of TMEM219, and wherein the inhibitor inhibits or blocks the interaction of IGFBP3 with its receptor TMEM219.
2.-4. (canceled)
5. The inhibitor of claim 1, wherein the fragment of TMEM219 is a fragment comprising an extracellular domain of TMEM219 capable of binding IGFBP3.
6. The inhibitor of claim 5, wherein the inhibitor is ecto-TMEM219 (SEQ ID NO: 3).
7.-9. (canceled)
10. A pharmaceutical composition comprising the inhibitor of claim 1 and pharmaceutically acceptable carriers.
11.-18. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0125] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION OF THE INVENTION
Example 1
[0145] Material and Methods
[0146] 60 individuals with long-standing T1D (T1D+ESRD) registered on the waiting list for simultaneous pancreas-kidney transplantation (SPK) were enrolled in the study and compared with 20 healthy subjects matched for age and gender (CTRL). Assessment of gastrointestinal symptoms, intestinal motility and intestinal mucosa pathology defined DE. CoSCs were identified on colonic purified crypts based on the expression of CoSC specific markers (flow-cytometry, RT-PCR, Western Blot, transcriptome profiling). CoSCs self-renewal properties were assessed by evaluating the % of in vitro developed mini-guts and by characterizing their expression of cell lineages markers in different conditions (
Patients and Study Design
[0147] 60 individuals with T1D+ESRD registered on the waiting list for simultaneous pancreas-kidney transplantation (SPK) matched for (age 41 to 43 years old), gender, and duration of T1D (29.41.8 years) were enrolled in the study. 20 healthy subjects matched for age and gender (CTRL), with normal renal function and normal glycometabolic parameters, were studied as well. T1D+ESRD subjects were all on intensive insulin treatment at the time of enrollment in the study, while the CTRL group was not being administered any medication. All T1D+ESRD subjects were on the same treatment as antiplatelet therapy (ASA) and anti-hypertension (angiotensin-converting-enzyme inhibitors), while 40 out of 60 received statins when enrolled in the study. Subjects with clear signs of inflammatory bowel diseases as well as celiac disease were not enrolled.
[0148] T1D+ESRD individuals were followed up for 8 years (mean follow-up: 8.61.1 years) after receiving either SPK (n=30) or K+T1D (n=30) transplantation according to the macroscopic surgical evaluation at the time of transplantation. Individuals taking an oral anticoagulant agent were not included. SPK individuals were all insulin-independent for the entire follow-up period, whereas K+T1D individuals were on intensive subcutaneous insulin therapy. All subjects provided informed consent before study enrollment. Studies not included in the routine clinical follow-up were covered by an appropriate Institutional Review Board approval (Enteropatia-trapianto/01 Secchi/Fiorina).
Transplantation and Immunosuppression
[0149] Organs for transplantation were obtained from deceased donors through the North Italia Transplant organ procurement consortium (NITp, Milan). After induction with ATG (thymoglobulin, IMTIX, SANGSTAT), immunosuppression was maintained using cyclosporine (through levels between 100-250 ng/ml) or FK506 (through levels between 10-15 ng/ml), mycophenolate mofetil (500-2000 mg/day), and methylprednisolone (10 mg/day). Steroids were withdrawn within 3-6 months after transplantation. All patients included in the T1D+ESRD and SPK groups were on anti-platelet therapy (80% ASA and 20% ticlopidine) to prevent graft or fistula thrombosis. Metabolic status, renal function and blood pressure were examined during enrolment and after transplantation every 2 years thereafter. The estimate glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) formula (Levey et al., 1999).
The Gastrointestinal Symptom Rating Scale (GSRS)
[0150] Gastrointestinal symptoms were evaluated by GSRS questionnaire in healthy subjects, in longstanding T1D individuals (T1D+ESRD) and in SPK and K+T1D groups at 2, 4 and 8 years after transplantation. The Gastrointestinal Symptom Rating Scale (GSRS) is a questionnaire consisting of 15 items with a seven-graded Likert scale defined by descriptive anchors (Svedlund et al., 1988). The questionnaire was originally constructed as an interview-based rating scale designed to evaluate a wide range of gastrointestinal symptoms and was later modified to become a self-administered questionnaire. The higher the scores, the more severe the symptoms: the scale ranges from a minimum value of 1 to a maximum value of 7. If an individual's participation in the study is discontinued, the value at the last available observation will be carried forward in the analysis. The items can be grouped into five dimensions previously identified on the basis of a factor analysis: abdominal pain syndrome (three items), reflux syndrome (two items), indigestion syndrome (four items), diarrhea syndrome (three items) and constipation syndrome (three items).
Anorectal Manometry
[0151] Data on anorectal manometry were already available in healthy subjects, and were compared with those obtained by performing anorectal manometry in long-standing T1D individuals (T1D+ESRD) using a custom-designed, open-tip, 14-Fr diameter, PVC probe with seven lumens 15 and a 4-cm latex balloon tied at the end of the probe (Bioengineering Laboratories Plc., Milan, Italy) (Carrington et al., 2014; Remes-Troche et al., 2010). The sphincter length was measured after a 10-minute run-in period, anal pressure was recorded for 15 minutes in resting conditions. Subjects were then instructed to squeeze the anus as tightly as possible and for as long as possible-for at least 20 seconds. Inventors' study evaluated the following items: Resting Tone, Contraction Tone, Reflex Response, and Urgency Response.
Pathology, Immunohistochemistry and Electron Microscopy
[0152] Colorectal endoscopy procedure was performed in healthy subjects, in long-standing T1D individuals (T1D+ESRD) at baseline and in SPK and K+T1D groups at 2, 4, and 8 years after transplantation using a Welch Allyn optic sigmoid scope. Intestinal mucosal samples were fixed in buffered formalin (formaldehyde 4% w/v and acetate buffer 0.05 M) and routinely processed in paraffin wax. 3 m-thick sections of each enrolled case were stained with Hematoxylin & Eosin (H&E) for morphological evaluations. For immunohistochemistry, 3 m-thick sections were mounted on poly-L-lysine coated slides, deparaffinized and hydrated through graded alcohols to water. After antigen retrieval, performed by dipping sections in 0.01 M citrate buffer, pH 6 for 10 minutes in a microwave oven at 650W as well as endogenous peroxidase activity inhibition, performed by dipping sections in 3% hydrogen peroxide for 10 minutes, incubation with primary antibodies was performed at 4 C. for 18-20 hours, followed by the avidin-biotin complex procedure (Hsu et al., 1981). Immunoreactions were developed usmg 0.03% 3,3 diaminobenzidine tetrahydrochloride, and then sections were counterstained with Harris' hematoxylin. The following antibodies were used: Ki67 (monoclonal, clone MIB M1B1, 1:100 dilution, Dako, Carpinteria, Calif., USA), aldehyde dehydrogenase (monoclonal, clone 44/ALDH, 1:1000 dilution, Transduction Laboratories, Franklin Lakes, N.J., USA), EphB2 (monoclonal, clone 48CT12.6.4, 1:200 dilution, Lifespan Biosciences, Seattle, Wash., USA), LGR5 (monoclonal, clone 2A2, 1:100 dilution, Origene Technologies, Rockville, Md., USA), hTERT (monoclonal, clone Y182, 1:500 dilution, Millipore, Billerica, Mass., USA), glicentin (polyclonal, 1:1250 dilution, Milab, Malmo, Sweden), pancreatic polypeptide (polyclonal, 1:500 dilution, Peninsula, Belmont, Calif., USA), PYY (polyclonal, 1:1000 dilution, Biogenesis, Bournemouth, UK), serotonin (monoclonal, clone YC5, 1:50 dilution, Biogenesis), somatostatin (polyclonal, 1:550 dilution, Dako), IGF-I (polyclonal, 1:500, Abcam) and IGF-IR (polyclonal, 1:100, Cell Signaling Technologies), (Fiorina et al., 2003). For ultrastructural studies, samples were fixed for 2 hours at 4 C. in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3. They were post-fixed in 1% osmium 15 tetroxide for 1 hour at room temperature, then dehydrated and embedded in Epon-Araldite. Ultrathin sections were cut with a diamond knife and mounted on 200-mesh nickel grids, previously coated with a Formvar film. Ultrathin sections were stained with aqueous uranyl acetate and Reynold's lead citrate solutions and subsequently examined with a Philips Morgagni 268D electron microscope. Cases were grouped according to the number of neuroendocrine vesicles (n>3 and n<3) for statistical analysis. For crypt isolation, tissue was collected in a sample containing a mixture of antibiotics and processed as described in the next paragraph. The immunostaining intensity for EphB2 was graded as 1 (negative EphB2 gradient to few cells positive per crypt per field) to 5 (strong EphB2 gradient in all longitudinal crypts). An anti-IGFBP3 primary antibody (polyclonal, 1:50 dilution, Sigma Aldrich) was immunohistochemically tested in liver biopsies 25 from patients with type 1 diabetes. Liver biopsies without pathological findings were used as controls. All of these tissue samples came from the files stored at the Unit of Pathology of the Department of Biomedical, Biotechnological, and Translational Sciences, University of Parma, Parma, Italy. The immunostaining intensity was graded as 1 (mild), 2 (moderate), and 3 (strong), while its diffusion as 1 (focal), 2 (zonal), and 3 (diffuse).
Immunoflurescence
[0153] Immunofluorescence samples obtained from liver biopsies were observed using a confocal system (LSM 510 Meta scan head integrated with the Axiovert 200 M inverted microscope; Carl Zeiss, Jean, Germany) with 63 oil objective. Images were acquired in multitrack mode, using consecutive and independent optical pathways. The following primary antibodies were used: rabbit IGFBP3 (1:10, Sigma) mouse Hep Par-1 (1:20, monoclonal, Dako), mouse CD163 (1:10, cloneMRQ26, CellMarque).
[0154] Mini-guts co-cultured with/without IGFBP3, with/without long-standing T1D serum+high glucose (35 mM Glucose) and those obtained from crypts of T1D+ESRD individuals, were stained with Vimentin, Citocheratin 20, Aldheide Dehydrogenase and Synaoptifisin for immunofluorescence analysis to assess expression of cell lineages markers (
In Situ Hybridization
[0155] Paraffin sections of human colon mucosa were de-paraffinized and re-hydrated according to standard procedures. After treatment of sections using 0.2M HCl for 15 minutes at room temperature, sections were washed 3 times in PBS and incubated for 15 min at 37 C. in proteinase K (30 g/ml in PBS). 0.2% glycine in PBS was added for 1 minute in order to neutralize Proteinase K activity, and samples were washed twice in PBS. After post-fixation in 4% PFA for 10 min at room temperature and 3 washes in PBS, histone acetylation was achieved by incubating samples two times for 5 min in an aqueous solution containing 1.5% triethanolamine, 0.15% HCl, and 0.6% acetic anhydride. Samples were then washed and pre-hybridized for 1 hour at 68 C. in hybridization solution (50% formamide, 5SSC, pH4.5, 2% Blocking Reagent (Roche), 0.05% CHAPS (Sigma), 5 mM EDTA, 50 g/ml Heparin (Sigma) and 50 g/ml yeast RNA. For TMEM219, the digoxigenin-labelled probe was diluted 750 ng/ml in hybridization solution and incubated for 24 hrs at 65 C. Post-hybridization washes were performed 320 min in 50% Formamide/2SSC at 65 C. Sections were rinsed in TBS-T buffer (0.1M TrisHC1 pH7.5, 0.15M NaCl, 0.1% Tween20) and blocked for 30 min at room temperature in Blocking Solution (0.5% Blocking Reagent, 10% sheep serum in TBS-T). Sheep anti-DIG antibody (Fab fragment, Roche) was diluted 1/2000 in Blocking Solution and incubated overnight at 4 C. After this, samples were washed in TBS-T and then in NTM buffer (0.1M Tris pH9.5, 0.1M NaCl, 0.05M MgCl2) and developed in NBT/BCIP solution (Roche) for 24 hrs.
CoSC Characterization
Crypt Purification
[0156] Muscle layer and sub-mucosa were carefully removed from human fresh rectal biopsy specimens, and mucosa was incubated with a mixture of antibiotics (Normocin, [Invivogen, San Diego, Calif. 92121, USA], Gentamycin [Invitrogen, Carlsbad, Calif., USA] and Fungizone [Invitrogen]) for 15 minutes at room temperature (RT). Next, tissue was cut into small pieces and incubated with 10 mM Dithiotreitol (DTT) (Sigma, St. Louis, Mo. 63103, USA) in PBS 2-3 times for 5 minutes at RT. Samples were then transferred to 8 mM EDTA in PBS and slowly rotated for 60-75 minutes at 4 C. Supernatant was replaced by fresh PBS, and vigorous shaking of the sample yielded supernatants enriched in colonic crypts. Fetal bovine serum (FBS, Sigma) was added to a final concentration of 5%, and fractions were centrifuged at 40g for 2 minutes in order to remove single cells. This washing procedure was repeated 3 times with Advanced DMEM/F12 (ADF, Gibco) medium supplemented with 2 mM GlutaMax (Invitrogen), 10 mM HEPES (Sigma), and 5% FBS (Sigma).
[0157] 200-300 isolated human colonic crypt units were mixed with 50 l matrigel and plated on pre-warmed 24-well culture dishes as already described. After solidification (15-20 minutes at 37 C.), crypts were overlaid with 600 l complete crypt culture medium [Wnt3a-conditioned medium and Advanced DMEM/F12 (Life Technologies, Grand Island, N.Y.) 50:50, supplemented with Glutamax, 10 mM HEPES, N-2 [lx], B-27 without retinoic acid [lx], 10 mM Nicotinamide, 1 mM N-Acetyl-L-cysteine, 50 ng/ml human EGF (Life Technologies, Grand Island, N.Y.), 1 g/ml RSPO1 (Sino Biological, Beijing, China), 100 ng/ml human Noggin (Peprotech, Rocky Hill, N.J., USA), 1 g/ml Gastrin (Sigma-Aldrich, St. Louis, Mo.), 500 nM LY2157299 (Axon MedChem, Groningen, The Netherlands), 10 M SB202190 (Sigma) and 0.01 M PGE2 (Sigma)]. Medium was replaced every other day. Rock inhibitor Y-27632 (10 M, Sigma) was added to the cultures for the first 2-3 days. Purified crypts were directly cultured for 8 days. Cell Lineages markers for enterocytes and enteroendocrine cells were assessed in the mini-guts and in the EphB2.sup.+ and EphB2.sup. sorted single cells with RT-PCR by testing: CHGA, KRT20 and EPCAM (Life Technologies, Grand Island, N.Y.). Colony forming efficiency (%) was evaluated on freshly isolated crypts in order to exclude that the bioptic procedure and the isolation processing could have compromised their efficiency in forming mini-guts in in vitro culture. DAPI staining was performed to confirm number of nuclei in freshly isolated crypts from CTRL and T1D+ESRD subjects. Developed mini-guts with at least 1 crypt domain were also counted and percentage was calculated in order to add a more quantitative criteria to measure developed mini-guts (
Glucose levels (T1D+ESRD vs. CTRL, 17847.5 vs 905.5 mg/dl, p0.0001);
Insulin levels (T1D+ESRD vs. CTRL, 12.94.6 vs 5.81.6 IU/ml, p=0.009).
Flow Cytometry
[0158] The expression of the CoSC markers EphB2 (APC anti-human EphB2 antibody, R&D, Minneapolis, Minn.) and LGR5 (PE anti-human LGR5, Origene, Rockville, Md.) was determined by flow cytometry by excluding CD45- and -positive cells (V450 anti-human CD45 and CD11b, BD Biosciences, San Jose, Calif.). Propidium iodide (PI) was added (10 g/ml) to exclude dead cells. EphB2+ cells were also sorted by flow cytometry to obtain a single cell suspension for culturing purposes. Intracellular detection of human-tert (hTERT) was performed by permeabilizing cells and staining with primary anti-human hTERT antibody (GeneTex, Irvine, Calif.) followed by DAPI anti-goat secondary antibody (Life Technologies). With regard to the analysis, cells were all first gated as PI.sup. before the assessment of other surface or intracellular markers. Samples were run on a BD LSR-Fortessa and analyzed by FSC Express 3.0 (DeNovo Software, Los Angeles, Calif., USA).
In Vitro Mini-Gut Generation Study
[0159] Crypts were isolated from healthy subject rectal biopsy samples and cultured as previously described to generate mini-guts. To create hyperglycemic conditions, the culturing medium was modified by adding glucose at different concentrations (35 mM: high glucose; 5 mM: normal glucose). To mimic uremic conditions, human uremic serum obtained from long-standing T1D individuals with ESRD was added to crypts, which were cultured as reported in the crypt culturing methods section. After 8 days, crypts were collected, and the morphology, mini-gut growth, expression of intestinal signature markers (EphB2, LGR5, h-TERT), IGF-IR and TMEM219 (Life Technologies), and Caspase 9 (Life Technologies) were examined using RT-PCR. A pan-caspase inhibitor (caspase inhibitor Z-VAD-FMK, 20 mM, Promega, Madison, Wis.), a Caspase 8 selective inhibitor (Z-IETD-FMK, BD Pharmingen), a Caspase 9 selective inhibitor (Z-LEHD-FMK, BD Pharmingen), a caspase3 inhibitor Z-DEVD-FMK (BD Pharmingen) were used in vitro in mini-guts to confirm the antiapoptotic effect of IGFBP3.
[0160] To culture isolated crypts with crypts culturing medium containing healthy subjects human serum, namely CTRL serum, in place of regular FBS, L-Wnt3 cells were grown in 10% CTRL serum to generate conditioned medium that was further added 50:50 to Advanced DMEM/F 12 medium in order to obtain the crypts culture medium as previously described (see Crypt purification).
[0161] To assess the properties of sorted EphB2.sup.+ cells in generating mini-guts, 2000 sorted cells were mixed with 50 l matrigel and plated on pre-warmed 24-well culture dishes. After solidification of the matrigel (10-15 min at 37 C.), cells were overlaid with single cell growth medium (=complete crypt culture medium+10 M Rock inhibitor Y-27623). Medium was replaced with fresh single cell growth medium every other day. Rock inhibitor was included in the culture medium for seven to nine days.
Immunoblotting
[0162] Total proteins of intestinal bioptic samples were extracted in Laemmli buffer (Tris-HCl 62.5 mmol/1, pH 6.8, 20% glycerol, 2% SDS, 5% -mercaptoethanol) and their concentration was measured (Lowry et al., 1951). 35 g of total protein was electrophoresed on 7% SDS-PAGE gels and blotted onto nitrocellulose (Schleicher & Schuell, Dassel, Germany). Blots were then stained with Ponceau S. Membranes were blocked for 1 h in TBS (Tris [10 mmol/1], NaCl [150 mmol/1]), 0.1% Tween-20, 5% non-fat dry milk, pH 7.4 at 25 C., incubated for 12 h with 200 mg/ml of a polyclonal anti-goat EphB2 antibody or polyclonal anti-goat LGR5 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) or monoclonal IGF-IR (Santa Cruz Biotechnology) and polyclonal TMEM219 (R&D, Minneapolis, Minn.) diluted 1:200 or with a monoclonal mouse anti-(3-actin antibody (Santa Cruz Biotechnology) diluted 1:1000 in TB S-5% milk at 4 C., washed four times with TBS-0.1% Tween-20, then incubated with a peroxidase-labeled rabbit anti-goat IgG secondary antibody (or rabbit anti mouse for (3-actin) diluted 1:1000 (Santa Cruz Biotechnology) in TBS-5% milk, and finally washed with TBS-0.1% Tween-20. The resulting bands were visualized using enhanced chemiluminescence (SuperSignal; Pierce, Rockford, Ill., USA).
Live Imaging of Intestinal Crypt Growth
[0163] Live imaging of mini-guts, obtained by purification and culture of intestinal crypts of CTRL, T1D+ESRD and SPK individuals, was performed on a Zeiss Axiovert S 100 equipped with environmental control (from Oko-Lab, Italy) with a chamber in which a humidified premixed gas consisting of 5% CO.sub.2 and 95% air was infused, and the whole setup was set at 37 C. Images were acquired at 20-minute intervals for 72 hours. Images were acquired and processed using Time Lapse (Oko-Lab, Italy) and, if necessary, image editing was performed using Adobe Photoshop Elements 7.0.
Morphology Imaging Analysis
[0164] The images of mini-guts were taken at day 0, 5 and 8 days by inverted microscopy Leica DH/RB and acquired with Axio Vision AC Release 4.3. Pictures reported in figures represent mini-guts at day 5, 10 magnification.
Transcriptome Profiling
[0165] Total RNA was isolated from purified intestinal crypt suspension using the RNeasy Mini Kit (Qiagen, Valencia, Calif.) with on-column DNase I digestion. Next, 3 g total RNA from each sample was reverse-transcribed using the RT2 First Strand kit (C-03; SABiosciences, Frederick, Md.). The inventors used the Human Stem Cell RT2 Profiler PCR Arrays (PAHS-405Z), the human Stem Cell Signaling PCR Array (PAHS-047Z) and a custom array with the following genes: AXIN2, OLFM4, BMI1, RNF43, CDCA7, SLC12A2, CDK6, SOX9, DKC1, ZNRF3, ETS2, EPHB2, FAM84A, LGR5, GPX2, ACTB (SABiosciences). The Profiler PCR Arrays measure quantitatively the expression of a panel of genes using SYBR Green-based real-time PCR (Kosinski et al., 2007). To assess the transcriptome profiling of apoptotic markers and oxidative stress markers the Human Apoptosis PCR Arrays (PAHS-012Z, SABiosciences) and the Human Oxidative Stress PCR Arrays (PAHS-065Z, SABiosciences) were used.
qRT-PCR Analysis
[0166] RNA from purified intestinal crypts was extracted using Trizol Reagent (Invitrogen), and qRT-PCR analysis was performed using TaqMan assays (Life Technologies, Grand Island, N.Y.) according to the manufacturer's instructions. The normalized expression values were determined using the AACt method. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) data were normalized for the expression of ACTB, and AACt values were calculated. Statistical analysis compared gene expression across all cell populations for each patient via one-way ANOVA followed by Bonferroni post-test for multiple comparisons between the population of interest and all other populations. Statistical analysis was performed also by using the software available RT.sup.2 profiler PCR Array Data Analysis (Qiagen). For two groups comparison Student t test was employed. Analysis was performed in triplicates after isolation of fresh crypts and/or after 8 days of culture of miniguts. Table I-B reports the main characteristics of primers used.
TABLE-US-00008 TABLE I-B Primers Gene Refseq Accession Band Size Reference Symbol UniGene# # (bp) Position LGR5 Hs.658889 NM_003667 91 1665 EPHB2 Hs.523329 NM_004442 68 2908 TERT Hs.492203 NM_198253 106 1072 ACTB Hs.520640 NM_001101 174 730 IGF-IR Hs.643120 NM_000875.3 64 2248 TMEM219 Hs.460574 NM_001083613.1 60 726 KRT20 Hs.84905 NM_019010.2 75 974 CHGA Hs.150793 NM_001275.3 115 521 EpcaM Hs.542050 NM_002354.2 95 784 LRP1 Hs.162757 NM_002332.2 64 656 TGFbR1 Hs.494622 NM_001130916.1 73 646 TGFbR2 Hs.604277 NM_001024847.2 70 1981 Caspase 8 Hs.599762 NM_001080124.1 124 648 Caspase 9 Hs.329502 NM_001229.4 143 1405
ELISA Assay
[0167] IGF-I and IGFBP3 levels in the pooled sera/plasma of all groups of subjects and in all groups of treated and untreated mice was assessed using commercially available ELISA kits, according to the manufacturer's instructions (R&D and Sigma).
[0168] Human immortalized hepatoma cell line HuH-7 was cultured for 5 days in DMEM 10% FBS at different glucose concentrations: 5.5 mM, 20 mM and 35.5 mM. Culturing supernatant was collected, and IGFBP3 was assessed using an IGFBP3 ELISA kit (Sigma) according to the manufacturer's instructions. Collected cells were separated by trypsin and counted with a hemacytometer.
[0169] Insulin levels were assayed with a microparticle enzyme immunoassay (Mercodia Iso-Insulin ELISA) with intra- and inter-assay coefficients of variation (CVs) of 3.0% and 5.0%.
Recombinant Proteins and Interventional Studies
[0170] Recombinant human IGF-I (Sigma, 13769), (IGF-I), recombinant human IGFBP3 (Life Technologies, 10430H07H5), (IGFBP3), and anti-IGF-IR (Selleckchem, Boston, OSI-906) were added to crypt cultures at day+2 from isolation. IGFBP3 (Reprokine, Valley Cottage, NY) was administered to nave and to STZ-treated B6 mice at 0.3 mg/mouse/day for 15 days; IGF-I (Reprokine) and ecto TMEM219 were administered in vivo to STZ-treated B6 mice after 2 weeks of diabetes at a dose of 5 g/mouse/day for 20 days and 100 g/mouse/day for 15 days respectively. Other molecules tested in in vitro mini-guts assay and added to crypt cultures at day+2 from isolation: Adiponectin (R&D), Thymosin (34 (Abcam), C-reactive protein (Merck Millipore), Cystatin C (Cell Signaling Technologies), Chromogranin A (Life Technologies), Fructosebisphosphate aldolase (Novoprotein), Osteopontin (R&D), Ribonuclease pancreatic (RNASE, Novoprotein), Serum amyloid A protein (Abcam), Mannan-binding lectin serine protease 1 (MASP1, Novoprotein), Tumor necrosis factor-alpha (TNF-alpha, R&D), FaS Ligand (FasL, R&D). Hydrogen peroxide (H.sub.2O.sub.2, 50 M) was also tested in the mini-guts assay.
Generation of Recombinant Human Ecto TMEM219
[0171] Recombinant human ecto-TMEM219 was generated using E. Coli as expression host for synthesis.
[0172] Briefly, gene sequence of extracellular TMEM219 was obtained:
TABLE-US-00009 (SEQIDNO:2) THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGS HVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQ LVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGA GNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR.
[0173] The DNA sequence of extracellular TMEM219 was cloned into a high copy-number plasmid containing the lac promoter, which is then transformed into the bacterium E. coli. Addition of IPTG (a lactose analog) activated the lac promoter and caused the bacteria to express extracellular TMEM219 (ecto TMEM219). SDS-P AGE and Western Blot were used to confirm purity higher than 90%. The molecular weight of the new generated protein recombinant human ecto TMEM219 was 80 kda.
[0174] Crypts from healthy subjects were isolated and cultured as previously described and ecto TMEM219 was added to the culture at three concentrations (260 ng/ml, 130 ng/ml and 75 ng/ml) as compared to IGFBP3 concentration used (2:1, 1:1 and 1:2) and appropriate controls were set up for each concentration. After 8 days of culture, caspase 8 and 9 expression, CoSCSC signature markers (EphB2 and LGR5) expression, number of developed mini-guts, were further assessed.
Small RNA Interference
[0175] Isolated crypts obtained from healthy subjects were grown to generate in vitro mini-guts in complete medium and in culturing medium modified by adding high glucose and long-standing T1D serum as previously described (see in vitro mini gut generation study in online methods). After 72h of culture, which allowed the crypts to recover, 750 ng of small interfering RNA (siRNA; Flexitube siRNA SI04381013, Qiagen, Valencia, Calif.) in 100 l culture medium without serum and with 6 l HiPerFect Transfection Reagent (Qiagen) were incubated at room temperature to allow for the formation of transfection complexes. Crypts were incubated with these transfection complexes under their normal growth conditions for 6h. Analysis of gene silencing was performed at 24, 48 and 72h by evaluating the percentage of normal mini-gut development. Control siRNA was used as a negative control to confirm the effect of gene silencing.
Proteomic Analysis
[0176] 8 l of pooled serum from 10 patients per group were depleted using a ProteoPrep 20 spin column (Sigma), thus allowing for the removal of the 20 highly abundant proteins. The procedure was twice repeated in order to obtain 99% depletion, according to the manufacturer's instructions. The recovered supernatant was analyzed to determine total protein concentration using the Direct Detect IR spectrophotometer and BSA as a standard. In order to obtain enough protein for proteomic analysis, 32 l from each pool were processed as above described. 40 g of total protein from each sample was in-solution digested using the Filter Aided Sample Preparation (FASP) protocol as reported in the literature (Wisniewski et al., 2009). Samples were desalted using C18 homemade tip columns (C18 Empore membrane, 3M) and injected into a capillary chromatographic system (EasyLC, Proxeon Biosystems, Thermo Scientific). Peptide separations were performed on a homemade 25 cm reverse phase spraying fused silica capillary column, packed with 3 m ReproSil Pur 120 C18-AQ. A gradient of eluents A (pure water with 2% v/v ACN, 0.5% v/v acetic acid) and B (ACN with 20% v/v pure water with 0.5% v/v acetic acid) was used to achieve separation (0.15 L/minute flow rate) (from 10 to 35% B in 230 minutes, from 35 to 50% B in 5 minutes and from 50 to 70% B in 30 minutes). Mass spectrometry analysis was performed using an LTQ-Orbitrap mass spectrometer (Thermo Scientific, Waltham, Mass.) equipped with a nanoelectrospray ion source (Proxeon Biosystems). Full scan mass spectra were acquired with the lock-mass option and resolution set to 60,000. The acquisition mass range for each sample was from m/z 300 to 1750 Da. The ten most intense doubly and triply charged ions were selected and fragmented in the ion trap using a normalized collision energy 37%. Target ions already selected for the MS/MS were dynamically excluded for 120 seconds. All MS/MS samples were analyzed using Mascot (v. 2.2.07, Matrix Science, London, UK) search engine to search the UniProt_Human Complete Proteome_cp _hum_2013_12. Searches were performed with trypsin specificity, two missed cleavages allowed, cysteine carbamidomethylation as fixed modification, acetylation at protein N-terminus, and oxidation of methionine as variable modification. Mass tolerance was set to 5 ppm and 0.6 Da for precursor and fragment ions, respectively. To quantify proteins, the raw data were loaded into the MaxQuant software version 1.3.0.5 (Cox et al., 2011). Label-free protein quantification was based on the intensities of precursors. Peptides and proteins were accepted with an FDR less than 1%, two minimum peptides per protein. The experiments were performed in technical triplicates. The complete dataset of proteins, obtained by proteomic analysis (Table I-C), was analyzed by Student's t-test using MeV software v. 4_8_1. 47 proteins, which were significantly different (p-value<0.01) in control pool versus T1D-ESDR pool, were further submitted to hierarchical clustering analysis.
TABLE-US-00010 TABLE I-C List of quantified proteins identified by proteomic analysis. The table reports correspondence between numbers and names of proteins detected by proteomic analysis and is shown as a heat-map in FIG. 10. Original row Protein names 1 14-3-3 protein zeta/delta 4 Actin, cytoplasmic 1; Actin, cytoplasmic 1, N-terminally processed; Actin, cytoplasmic 2; Actin, cytoplasmic 2, N-terminally processed 5 Adiponectin 6 Afamin 8 Alpha-1-antichymotrypsin; Alpha-1-antichymotrypsin His-Pro-less 9 Alpha-1-antitrypsin; Short peptide from AAT 12 Alpha-2-HS-glycoprotein; Alpha-2-HS-glycoprotein chain A; Alpha-2- HS-glycoprotein chain B 13 Alpha-2-macroglobulin 14 Alpha-actinin-1 16 Angiotensinogen; Angiotensin-1; Angiotensin-2; Angiotensin-3 17 Antithrombin-III 18 Apolipoprotein A-I; Truncated apolipoprotein A-I 20 Apolipoprotein A-IV 21 Apolipoprotein B-100; Apolipoprotein B-48 22 Apolipoprotein C-I; Truncated apolipoprotein C-I 23 Apolipoprotein C-II 24 Apolipoprotein C-III 25 Apolipoprotein C-IV 26 Apolipoprotein D 28 Apolipoprotein F 29 Apolipoprotein L1 31 Apolipoprotein(a) 34 Attractin 35 Basement membrane-specific heparan sulfate proteoglycan core protein; Endorepellin; LG3 peptide 36 Beta-2-glycoprotein 1 37 Beta-2-microglobulin; Beta-2-microglobulin form pI 5.3 39 Beta-Ala-His dipeptidase 42 C4b-binding protein beta chain 43 Cadherin-1; E-Cad/CTF 1; E-Cad/CTF2; E-Cad/CTF3 44 Cadherin-13 45 Cadherin-5 46 Calreticulin 50 Carboxypeptidase N subunit 2 51 Cartilage oligomeric matrix protein 54 CD44 antigen 57 Ceruloplasmin 59 Chromogranin-A; Vasostatin-1; Vasostatin-2; EA-92; ES-43; Pancreastatin; SS-18; WA-8; WE-14; LF-19; AL-11; GV-19; GR-44; ER- 37 60 Clusterin; Clusterin beta chain; Clusterin alpha chain; Clusterin 62 Coagulation factor V; Coagulation factor V heavy chain; Coagulation factor V light chain 63 Coagulation factor X; Factor X light chain; Factor X heavy chain; Activated factor Xa heavy chain 65 Cofilin-1 66 Collagen alpha-3(VI) chain 68 Complement C1r subcomponent; Complement C1r subcomponent heavy chain; Complement C1r subcomponent light chain 71 Complement C2; Complement C2b fragment; Complement C2a fragment 72 Complement C3; Complement C3 beta chain; Complement C3 alpha chain; C3a anaphylatoxin; Complement C3b alpha chain; Complement C3c alpha chain fragment 1; Complement C3dg fragment; Complement C3g fragment; Complement C3d fragment; Complement C3f fragment; Complement C3c alpha chain fragment 2 73 Complement C4-A; Complement C4 beta chain; Complement C4-A alpha chain; C4a anaphylatoxin; C4b-A; C4d-A; Complement C4 gamma chain 74 Complement C4-B; Complement C4 beta chain; Complement C4-B alpha chain; C4a anaphylatoxin; C4b-B; C4d-B; Complement C4 gamma chain 75 Complement C5; Complement C5 beta chain; Complement C5 alpha chain; C5a anaphylatoxin; Complement C5 alpha chain 76 Complement component C1q receptor 77 Complement component C6 78 Complement component C7 84 Complement factor D 88 Complement factor I; Complement factor I heavy chain; Complement factor I light chain 89 Corticosteroid-binding globulin 90 C-reactive protein; C-reactive protein (1-205) 91 Cystatin-C 92 Cystatin-M 95 EGF-containing fibulin-like extracellular matrix protein 1 96 Endothelial protein C receptor 97 Extracellular matrix protein 1 98 Extracellular superoxide dismutase [CuZn] 99 Fetuin-B 100 Fibrinogen alpha chain; Fibrinopeptide A; Fibrinogen alpha chain 101 Fibrinogen beta chain; Fibrinopeptide B; Fibrinogen beta chain 102 Fibrinogen gamma chain 103 Fibronectin; Anastellin; Ug1-Y1; Ug1-Y2; Ug1-Y3 104 Fibulin-1 105 Ficolin-3 106 Fructose-bisphosphate aldolase A; Fructose-bisphosphate aldolase 107 Galectin-3-binding protein 108 Gamma-glutamyl hydrolase 109 Gelsolin 111 Glyceraldehyde-3-phosphate dehydrogenase 112 Haptoglobin; Haptoglobin alpha chain; Haptoglobin beta chain 117 Heparin cofactor 2 122 Hypoxia up-regulated protein 1 123 Ig alpha-1 chain C region 125 Ig gamma-1 chain C region 126 Ig gamma-2 chain C region 127 Ig gamma-3 chain C region 129 Ig heavy chain V-II region SESS; Ig heavy chain V-II region OU 130 Ig heavy chain V-III region BRO; Ig heavy chain V-III region TEI; Ig heavy chain V-III region BUT; Ig heavy chain V-III region WEA 134 Ig heavy chain V-III region VH26 135 Ig kappa chain C region 136 Ig kappa chain V-I region EU; Ig kappa chain V-I region CAR 142 Ig kappa chain V-III region WOL; Ig kappa chain V-III region SIE; Ig kappa chain V-III region Ti; Ig kappa chain V-III region GOL 144 Ig kappa chain V-IV region Len 145 Ig lambda chain V-I region HA; Ig lambda chain V-I region WAH; Ig lambda chain V-II region MGC; Ig lambda chain V-II region WIN 146 Ig lambda chain V-III region LOI 148 Ig lambda-2 chain C regions; Ig lambda-3 chain C regions; Ig lambda-6 chain C region 153 Immunoglobulin lambda-like polypeptide 5; Ig lambda-1 chain C regions 154 Insulin-like growth factor-binding protein 2 155 Insulin-like growth factor-binding protein 3 156 Insulin-like growth factor-binding protein 6 158 Inter-alpha-trypsin inhibitor heavy chain H1 159 Inter-alpha-trypsin inhibitor heavy chain H2 160 Inter-alpha-trypsin inhibitor heavy chain H3 161 Inter-alpha-trypsin inhibitor heavy chain H4; 70 kDa inter-alpha-trypsin inhibitor heavy chain H4; 35 kDa inter-alpha-trypsin inhibitor heavy chain H4 164 Keratin, type I cytoskeletal 10 165 Keratin, type I cytoskeletal 9 166 Keratin, type II cytoskeletal 1 167 Kininogen-1; Kininogen-1 heavy chain; T-kinin; Bradykinin; Lysylbradykinin; Kininogen-1 light chain; Low molecular weight growth-promoting factor 168 Leucine-rich alpha-2-glycoprotein 171 L-lactate dehydrogenase B chain; L-lactate dehydrogenase 174 Lumican 175 Lymphatic vessel endothelial hyaluronic acid receptor 1 176 Lysozyme C 178 Mannan-binding lectin serine protease 1; Mannan-binding lectin serine protease 1 heavy chain; Mannan-binding lectin serine protease 1 light chain 180 Monocyte differentiation antigen CD14; Monocyte differentiation antigen CD14, urinary form; Monocyte differentiation antigen CD14, membrane- bound form 181 Multimerin-1; Platelet glycoprotein Ia*; 155 kDa platelet multimerin 183 Neudesin 185 Neural cell adhesion molecule L1-like protein; Processed neural cell adhesion molecule L1-like protein 187 Osteopontin 188 Peptidase inhibitor 16 189 Peptidyl-prolyl cis-trans isomerase A; Peptidyl-prolyl cis-trans isomerase 192 Phosphatidylethanolamine-binding protein 4 194 Pigment epithelium-derived factor 197 Plasminogen; Plasmin heavy chain A; Activation peptide; Angiostatin; Plasmin heavy chain A, short form; Plasmin light chain B 198 Platelet basic protein; Connective tissue-activating peptide III; TC-2; Connective tissue-activating peptide III(1-81); Beta-thromboglobulin; Neutrophil-activating peptide 2(74); Neutrophil-activating peptide 2(73); Neutrophil-activating peptide 2; TC-1; Neutrophil-activating peptide 2(1- 66); Neutrophil-activating peptide 2(1-63) 199 Platelet glycoprotein Ib alpha chain; Glycocalicin 200 Plexin domain-containing protein 2 203 Profilin-1 204 Proline-rich acidic protein 1 205 Properdin 206 Prostaglandin-H2 D-isomerase 207 Protein AMBP; Alpha-1-microglobulin; Inter-alpha-trypsin inhibitor light chain; Trypstatin 209 Prothrombin; Activation peptide fragment 1; Activation peptide fragment 2; Thrombin light chain; Thrombin heavy chain 212 Receptor-type tyrosine-protein phosphatase gamma 213 Retinol-binding protein 4; Plasma retinol-binding protein(1-182); Plasma retinol-binding protein(1-181); Plasma retinol-binding protein(1-179); Plasma retinol-binding protein(1-176) 214 Rho GDP-dissociation inhibitor 2 215 Ribonuclease pancreatic 216 Scavenger receptor cysteine-rich type 1 protein M130; Soluble CD163 217 Secreted and transmembrane protein 1 221 Serotransferrin 222 Serum albumin 223 Serum amyloid A protein 225 Serum amyloid P-component; Serum amyloid P-component (1-203) 226 Serum paraoxonase/arylesterase 1 228 SPARC-like protein 1 230 Talin-1 232 Tenascin-X 233 Tetranectin 234 Thrombospondin-1 235 Thrombospondin-4 236 Thymosin beta-4; Hematopoietic system regulatory peptide 237 Thyroxine-binding globulin 239 Transgelin-2 240 Trans-Golgi network integral membrane protein 2 242 Tropomyosin alpha-4 chain 243 Vascular cell adhesion protein 1 244 Vasorin 245 Vinculin 247 Vitamin K-dependent protein C; Vitamin K-dependent protein C light chain; Vitamin K-dependent protein C heavy chain; Activation peptide 248 Vitamin K-dependent protein S 249 Vitamin K-dependent protein Z 250 Vitronectin; Vitronectin V65 subunit; Vitronectin V10 subunit; Somatomedin-B 251 von Willebrand factor; von Willebrand antigen 2 254 Zinc-alpha-2-glycoprotein 258 Vitamin D-binding protein 259 Complement factor H 260 Fibulin-1 267 Mannan-binding lectin serine protease 1 270 Complement factor H-related protein 4
Strategy to Select Candidate Proteins
[0177] Among the 46 factors that segregated separately in long-standing T1D subjects and healthy controls, the inventors first selected those with a more significant difference in LFQ intensity in comparing the two groups (p>0.005), leading to the exclusion of 12 factors (
Animal Studies
[0178] C57BL/6 (B6) mice were obtained from the Jackson Laboratory, Bar Harbor, Me. All mice were cared for and used in accordance with institutional guidelines approved by the Harvard Medical School Institutional Animal Care and Use Committee. Mice were rendered diabetic with streptozotocin injection (225 mg/kg, administered i.p.; Sigma). Diabetes was defined as blood glucose levels >250 mg/dL for 3 consecutive measures. Diabetic enteropathy was assessed as follows: briefly, the entire intestine was extracted from sacrificed mice and flushed with PBS. The extreme part of the colon was then cut and divided in two pieces. One piece of colon tissue was directly submerged in formalin while the other was cut longitudinally to expose the lumen and the internal mucosa and then submerged in formalin. Tissue was then paraffin embedded and processed for H&E and immunostaining. In addition, colonic tissue was also cut and isolation of colonic stem cells was performed as previously described (Merlos-Suarez et al., 2011). Briefly, colon was cut into 2-4 mm pieces and the fragments were washed in 30 mL ice-cold PBS. Fragments were the transferred in 50 ml tubes containing pre-warmed 20 mM EDTA-PBS and incubated at 37 C. for 30 min. After incubation the suspended tissue was transferred into tube containing 30 ml cold PBS and centrifuged. Crypts were resuspended in 13 ml cold DMEMF12, washed with PBS and digested in 5-10 ml of trypsin/DNAse solution at 37 C. for 30 min. Crypts were then resuspended in DMEMF12/EDTA, filtered in 40 micron strainer twice and washed.
[0179] Finally, crypts were then resuspended in flow medium (DMEM+FBS+EDTA) and stained for anti EphB2-APC (R&D), mouse anti-CD45-PeRCP and mouse anti-CD1 lb-PE (BD Pharmingen). Samples were run using a FACSCalibur Analyzer and data analyzed with FlowJo.
[0180] Part of the tissue was also snap frozen and stored in Tryzol to perform RT-PCR studies for the following markers:
TABLE-US-00011 Gene Refseq Accession Band Size Reference Symbol: UniGene#: #: (bp): position: LGR5 Mm.42103 NM_010195.2 64 571 EPHB2 Mm.250981 NM_010142.2 85 1696 Casp8 Mm.336851 NM_001080126.1 96 1525 Casp9 Mm.88829 NM_001277932.1 68 377 GAPDH Mm.304088 NM_008084.2 107 75
[0181] Finally, plasma and serum were collected to perform analysis of IGF-I (IGF-I ELISA kit, R&D), IGFBP3 (IGFBP3 ELISA kit, R&D) and insulin levels (Mercodia Mouse Insulin ELISA kit). Blood glucose was monitored twice a week for the 8 weeks in order to confirm diabetes onset and permanence.
Statistical Analysis
[0182] Data are presented as mean and standard error of the mean (SEM) and were tested for normal distribution with the Kolmogorov-Smirnov test and for homogeneity of variances with Levene's test. The statistical significance of differences was tested with two-tailed t-test and the chi-square (x.sup.2) tests. Significance between the two groups was determined by two-tailed unpaired Student's t test. For multiple comparisons, the ANOVA test with Bonferroni correction was employed. All data were entered into Statistical Package for the Social Science (SPSS, IBM, SPSS Inc., Chicago, Ill.) and analyzed. Graphs were generated using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, Calif.). All statistical tests were performed at the 5% significance level.
Results
Intestinal Dysfunction and Clinical Symptoms are Present in Long-Standing TJD
[0183] The inventors first characterized intestinal morphology and function in a population of individuals with long-standing T1D and end stage renal disease (T1D+ESRD) and in healthy subjects (CTRL). Severe intestinal symptoms, such as diarrhea, abdominal pain and constipation, were evident in T1D+ESRD individuals as assessed using the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire (
CoSCs are Altered in Long-Standing TJD
[0184] The characterization of colonic crypts, revealed a significant reduction in EphB2.sup.+ expression and in the number of aldehyde dehydrogenase (Aldh).sup.+ immunoreactive cells, both markers of local stem cells (Carpentino et al., 2009; Jung et al., 2011), in T1D+ESRD individuals as compared to healthy subjects (
TABLE-US-00012 TABLE II List of up and down regulated stem cell target genes identified by transcriptomic profiling in CTRL vs. T1D + ESRD freshly isolated colonic crypts (at least p < 0.05). Down-regulated genes Up-regulated genes ACTC1 APC CD44 DVL1 BTRC SOX1 SOX2 WNT1 CCND2 FZD1 ADAR ACAN ALPI CD8A COL1A1 COL2A1 COL9A1 BMP1 BMP2 BMP3 CCNA2 CCNE1 CDC42 CDK1 CTNNA1 CXCL12 PARD6A CD3D CD8B MME CD4 DLL1 HDAC2 NOTCH1 DLL3 JAG1 NOTCH2 DTX2 KAT2A NUMB EP300 FGF2 FGF3 FGFRl GDF3 ISL1 KRT15 MSX1 MYOD1 T GJA1 GJBI GJB2 KAT8 RB1 h-TERT NCAM1 SIGMAR1 TUBB3 ABCG2 ALDH1A1 PDX1 IGF-I DHH BGLAP
[0185] Analysis of CoSC signature genes revealed that LGR5, EphB2 (Gracz et al., 2013; Merlos-Suarez et al., 2011), h-TERT (Breault et al., 2008) and other intestinal stem cell marker genes (Hughes et al., 2011; Munoz et al., 2012; Ziskin et al., 2013) were significantly underexpressed in T1D+ESRD as compared to healthy subjects as well (
In Vitro Generation of Mini-Guts is Altered in Long-Statnding T1D
[0186] In order to evaluate CoSC self-renewal properties, the inventors used the in vitro mini-gut assay. Indeed, crypts isolated from T1D+ESRD individuals and cultured in vitro for 8 days formed small spheroid mini-guts that failed to grow as compared to healthy subjects (
Serum Unbiased Proteomic Profiling Revealed Increased Levels of IGFBP3 in Long-Standing TJD
[0187] In order to identify potential circulating factors that may serve as enterotrophic hormones and may have a role in regulating the CoSCs, the inventors compared the serum proteome of healthy subjects with T1D+ESRD individuals using an unbiased proteomic array. A clear proteomic profile was evident in T1D+ESRD individuals as compared to healthy subjects, with more than 50% of the detected proteins segregating in either one group or the other (
Peripheral IGFBP3 and IGF-I Control CoSCs
[0188] To further elucidate the role of circulating IGF-I and IGFBP3 in the regulation of the CoSCs and of intestinal epithelial proliferation, the inventors demonstrated the expression of IGF-IR and of IGFBP3 receptor (TMEM219) on isolated crypts (
[0189] Other circulating proteins, which appeared altered in serum proteome of long-standing T1D individuals, were tested in the in vitro mini-gut assay and did not show any significant effect on mini-guts growth (
[0190] To further confirm that IGF-I/IGFBP3 dyad targets effectively CoSCs and not only crypts, the inventors tested its effect on single cell-derived mini-guts. The inventors flow sorted EphB2.sup.+ cells from isolated crypts and established that TMEM219 was highly expressed on their surface (
[0191] Moreover, expression of Caspase 8 and 9 was up regulated in IGFBP3-treated mini-guts and in those exposed to high glucose and long-standing T1D serum, while Ki67, marker of proliferation, was significantly under expressed (
Effect of the IGF-I/IGFBP3 Dyad on Previously Known Pathways that Control CoSCs
[0192] In order to clarify the effects of IGF-I/IGFBP3 dyad on pathways previously known to be involved in CoSC niche function (i.e. Wnt/Notch/BMP), the inventors obtained from their stem cell transcriptome profile the expression of niche specific gene transcripts. IGF-I restores significantly the expression of some factors associated with Wnt/Notch signaling pathways on mini-guts obtained from crypts of T1D+ESRD (
TABLE-US-00013 TABLE III List of up and down-regulated stem cell target genes identified by transcriptomic profiling in colonic crypts obtained from CTRL and from T1D + ESRD and cultured with/without IGFBP3 and IGF-I (at least p < 0.05). Down-regulated genes Up-regulated genes CTRL + IGF-I CD44, CDHl, COL9A1 ACAN, COL2Al, DLLl, FGF2, vs. FGF3, GDF3, GJAl, IGF-I, ISLl, CTRL MME, MSXl, NCAM1, NOTCH2, PDXI, SOXl, SOX2, h-TERT CTRL + IGFBP3 CD8B, COL9A1, RB1, SOX1, ASCL2, COL2Al, DHH, DLLl, vs. h-TERT DTXl, DVL1, FGF3, FGF4, CTRL FOXA2, FRAT1, GDF2, HSPA9, IGFl, KAT2A, MSXl, MYC, NEUROG2, S100B, WNTl T1D + ESRD + IGF-I ACTCI, CD3D, CD4, ABCG2, ADAR, BMPl, BMP2, vs. COL9Al, DTXl, FGFR1 BTRC, CDC42, CTNNAl, T1D + ESRD CXCL12, DLLl, DTX2, GDF3, HDAC2, ISLl, JAGl, NOTCH1, NOTCH2, NUMB, PARD6A, PDXI, RBI, SIGMAR1, h-TERT T1D + ESRD + IGFBP3 ABCG2, ALDHlAl, ALPI, ASCL2, KAT2A, MYC, NCAM1, vs. CD3D, CD4, CD44, CD8A, NEUROG2, SOX2 T1D + ESRD CDC42, FGF2, FGFRl, JAGl, SIGMARl, SOX1, TUBB3 Abbreviations: IGF-I, insulin-like growth factor 1; IGFBP3, insulin-like growth factor binding protein 3, CTRL, healthy subjects, T1D, type 1 diabetes, ESRD, end-stage renal disease.
[0193] This confirms that IGF-I preserves the expression of some genes involved in Wnt/Notch/BMP signaling, but also that IGFBP3 acts independently on CoSCs, without major alterations in the expression of key-target genes of the other previously known pathways.
Effect of IGF/IGFBP3 Dyad on Apoptotic Patheways in CoSCs
[0194] An extensive transcriptome analysis performed to clarify the IGFBP3 caspase-mediated effect on mini-guts, (
TABLE-US-00014 TABLE IV List of up and down-regulated pro/anti-apoptotic target genes identified by transcriptomic profiling in CTRL vs. T1D + ESRD freshly isolated colonic crypts and in those cultured with IGFBP3 and IGF-I (at least p < 0.05). Down-regulated genes Up-regulated genes T1D + ESRD BCL2, NOL3, FAS CASP1, CASP10, CASP14, vs. CASP5, CASP6, CASP7, CASP8, CTRL CASP9, CD27, CRADD, FADD, FASLG, HRK, TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF1A, TNFRSF1B, TNFRSF25, TNFRSF9, TNFSF8, TRADD, TRAF3 CTRL + IGF-I BNIPL3 CASPI4, CASP5, CD27, CRADD, vs. FASLG, TNFRSF25, TNFSF8, CTRL TRADD CTRL + IGFBP3 BAX, BCL2 CASP5, CASP8, CASP9, FAS, vs. TNFRSF1B, TNFSF8, TRADD, CTRL TRAF3 T1D + ESRD + IGF-I CASP1, CASP10, CASP5, BCL2 vs. CASP6, CASP7, CASP8, T1D + ESRD CASP9, CRADD, FADD, TNFRSF11B, TNFRSF9, TNFSF8, TRADD, TRAF3 T1D + ESRD + IGFBP3 BAX, BCL2, NOL3, CASP9, CD27 vs. TNFRSF1B T1D + ESRD Abbreviations: IGF-I, insulin-like growth factor 1; IGFBP3, insulin-like growth factor binding protein 3, CTRL, healthy subjects, T1D, type 1 diabetes, ESRD, end-stage renal disease.
[0195] Interestingly, anti-apoptotic genes (Bc12, Fas, No13) were significantly underexpressed in mini-guts grown from T1D+ESRD crypts as well, as compared to healthy subjects, while the majority of caspases related genes (Caspase 1, 5, 7, 8, 9, 14) were over expressed (
TABLE-US-00015 TABLE V List of up and down-regulated oxidative stress target genes identified by transcriptomic profiling in CTRL vs. T1D + ESRD freshly isolated colonic crypts and in those cultured with IGFBP3 and IGF-I (at least p < 0.05). Down-regulated genes Up-regulated genes T1D + ESRD DUOX1, PRDX4, STK25, CYBB, GPX5, KRT1, MT3, vs. GSS NOX4, OXR1, PTGS1, SFTPD CTRL CTRL + IGF-I DUOX1, TXNRD AOX1, FTH1, GPX7, GSS, KRT1, vs. LPO, MPO, NCF1, NOS2, NOX4, CTRL OXR1, PTGS1, PTGS2, SCARA3, SFTPD, TPO, TTN CTRL + IGFBP3 NCF1, SOD3 AOX1, GPX5, GPX7, HSPA1A vs. KRT1, MB, MPO, NOX5, OXR1, CTRL PTGS1, SFTPD, TPO, TTN, TXNRD2, UCP2 T1D + ESRD + IGF-I DUOX1, EPHX2, MB, MT3, MPO, PRDX4, PRNP, STK25 vs. NCF1, OXR1, PTGS1, T1D + ESRD SOD3, SRXN1 T1D + ESRD + IGFBP3 CYBB, DUOX1, EPHX2 NOS2, STK25 vs. GPX3, GSTP1, HSPA1A T1D + ESRD MGST3, NCF1, NQO1, PRDX6, RNF7, TXN Abbreviations: IGF-I, insulin-like growth factor 1; IGFBP3, insulin-like growth factor binding protein 3, CTRL, healthy subjects, T1D, type 1 diabetes, ESRD, end-stage renal disease.
Manipulation of the Circulating IGF-I/IGFBP3 Dyad Axis Alters the Course of Diabetic Enteropathy in a Preclinical Model
[0196] In order to further demonstrate the relevance of IGF-I/IGFBP3 circulating factors in vivo, the inventors tested the effects of IGF-I and IGFBP3 administration in a preclinical model of DE. After 8 weeks of chemically-induced diabetes (using streptozotocin [STZ]), C57BL/6 (B6) mice showed a reduced number of crypts in the colorectal tissue (
Treatment of Long-Standing T1D with Simultaneous Pancreas-Kidney Transplantation (SPK) Reverts Clinical and Morphological Features of DE
[0197] The gold standard treatment for long-standing T1D is SPK, which affords stable glycometabolic control, near-normalized risk factors and prolonged survival (Table VI) (Fiorina et al., 2004; Fiorina et al., 2005; Folli et al., 2010; Secchi et al., 1998; Smets et al., 1999).
TABLE-US-00016 TABLE VI Restoration of both normoglycemia and normal renal function in SPK is associated with stable glucose/lipid metabolism and blood pressure control over time at up to 8 years of follow-up as compared to K + T1D (data are shown at 8 years of follow-up). T1D + ESRD SPK K + T1D Parameters (n = 60) (n = 30) (n = 30) P value eGFR <15 65.6 20.2* 61.8 25.2.sup. *, .sup.<0.0001 (ml/min/1.73 m.sup.2) HbA1c (%) 8.4 1.5 5.4 0.3* 7.5 1.4.sup. *<0.0001; .sup.<0.001 EIR (UI) 37.4 2.3 0* 26.0 7.0.sup. *<0.0001; .sup.0.001 TG (mg/dl) 162.5 92.7 90.4 23.0* 147.1 98.0.sup. *0.01; .sup.0.04 Chol (mg/dl) 201.0 45.7 185 27.2 191.1 41.1.sup. Ns LDL (mg/dl) 116.3 40.3 119.5 34.0 97.8 2.1.sup. Ns HDL (mg/dl) 48.1 14.4 51.4 4.1 43.13 5.7.sup. Ns Systolic BP 146.3 18.7 133.1 14.2* 140.1 15.7.sup. 0.03; .sup.0.04 Diastolic BP 83.7 8.3 79.1 9.2 78.3 9.2.sup. Ns Abbreviations: T1D, type 1 diabetes; ESRD, end stage renal disease; SPK, simultaneous kidney-pancreas transplantation; K + T1D, kidney transplantation alone in type 1 diabetes; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; EIR, exogenous insulin requirement; TG, tryglycerides; Chol, total cholesterol; LDL, low density lipoprotein; HDL, high density lipoprotein; BP, blood pressure; UI, International Unit.
[0198] However, individuals with T1D+ESRD are also treated with kidney transplantation alone but remain diabetic (K+T1D) (Fiorina et al., 2001). A significant improvement in gastrointestinal symptoms was evident over time after SPK in inventors' cohort of transplanted individuals, while the K+T1D group did not report any benefit (
Treatment of Long-Standing T1D with SPK Re-Establishes Intestinal Mucosa Morphology and Local Self-Renewal Properties
[0199] Analysis of intestinal mucosa samples showed a significant recovery in the structure of the epithelial compartment, with compensatory epithelial hyperplasia in the SPK group (
Treatment of Long-Standing T1D Promotes Restoration of CoSCs
[0200] Treatment of long-standing TID with SPK is associated with an increase in Aldh.sup.+ cells (
TABLE-US-00017 TABLE VII List of up and down-regulated stem cell target genes identified by transcriptomic profiling in SPK as compared to T1D + ESRD freshly isolated colonic crypts (at least p < 0.05). Down-regulated genes Up-regulated genes DVL1 ACTC1 APC CCND2 WNT1 BTRC SOX1 SOX2 ACAN COL1A1 COL2A1 BMP3 CCNE1 CDK1 CXCL2 CD8B MME DLL3 HDAC2 JAG1 DTX2 FGF2 GDF3 ISL1 MSX1 MYO1 GJA1 RBI h-TERT NCA1 SIGMAR1 PDX1 DHH BGLA P Abbreviations: EGF, epithelial growth factor; FGF, fibroblast growth factor, BMP, bone morphogenetic protein.
[0201] It is concluded that treatment of long-standing T1D with SPK promotes restoration of CoSCs.
Treatment of Long-Standing T1D with SPK Restores Circulating IGF-I and IGFBP3
[0202] Broad proteomic analysis and targeted immunoassay, revealed a near-normalization of IGFBP3 and IGF-I serum levels after SPK (
The Ecto-TMEM219 Recombinant Protein Abrogates IGFBP3-Mediated Mini-Gut Destruction In Vitro and Preserves CoSCs In Vivo in a Murine Model of DE.
[0203] In order to further demonstrate the IGFBP3-mediated detrimental effects on CoSCs, the inventors generated a recombinant protein based on the TMEM219 extracellular domain (ecto-TMEM219). Addition of ecto-TMEM219 (2:1 molar ratio with IGFBP3) to crypts obtained from CTRL and cultured with IGFBP3 abrogated the pro-apoptotic effect of IGFBP3 on mini-guts and preserved the regenerative properties of crypts to generate mini-guts (
DISCUSSION
[0204] Diabetic enteropathy represents a clinically relevant complication in individuals with T1D, as it is associated with lower quality of life, malnutrition and malabsorbtion (Bytzer et al., 2002; Faraj et al., 2007; Talley et al., 2001). Particularly, in individuals with long-standing T1D (T1D+ESRD), intestinal disorders occur with high frequency and severity (Cano et al., 2007; Wu et al., 2004), resulting in body mass loss and cachexia (Pupim et al., 2005), indicating that enteropathy is an important complication of long-standing T1D (Atkinson et al., 2013; Pambianco et al., 2006). Inventors' results demonstrate that individuals with long-standing T1D experienced severe intestinal disorders (Table VIII) and that these clinical conditions are associated with alterations of the intestinal mucosa, with reduced proliferation of intestinal epithelial cells and with signs of neural degeneration.
TABLE-US-00018 TABLE VIII Overview of results of diabetic enteropathy assessment in T1D + ESRD individuals as compared to CTRL and SPK. T1D + ESRD vs. SPK vs. Results CTRL T1D + ESRD Metabolic Evaluation Glucose metabolism +++ Lipid metabolism + Blood pressure control + Intestinal Symptoms Diarrhea +++ Abdominal pain +++ Constipation ++ Anorectal Manometry Resting tone = = Contracting tone = Reflex response = Urgency volume ++ Mucosa Epithelial Renewal Proliferation +++ Differentiation +++ Neural Regeneration Nerves +++ Schwann cells +++ Colonic Stem Cell Turnover Colonic stem cells +++ Crypt growth +++ Arbitrary unit: +++ (high improvement); ++ (mild improvement); + (slight improvement); = no improvement; (severe worsening); (mild worsening), (slight worsening). Evaluations were performed as follows: T1D + ESRD vs. CTRL, SKP vs. T1D + ESRD, K + T1D vs. SKP. Abbreviations; TID, type 1 diabetes; ESRD, end stage renal disease; CTRL, healthy subjects; SPK, simultaneous kidney-pancreas transplantation.
[0205] Similar features have also been reported in rodent models of T1D and DE (Domenech et al., 2011). Inventors' data, for the first time, link DE to a defect in CoSCs and implicate IGFBP3 as having an important role in the maintenance of intestinal epithelium homeostasis. While hyperglycemia is a prominent feature of T1D, inventors' in vitro studies suggest that this feature cannot fully explain DE and that circulating factors may play an important role. Proteomic analysis led to the identification of IGF-I as an enterotrophic factor involved in the homeostasis of CoSCs. The inventors then confirmed that IGF-I and IGFBP3 control CoSCs and that this axis is dysfunctional in long-standing T1D. Inventors' data indicate that IGF-I acts as a circulating enterotrophic factor that promotes crypt growth and controls CoSCs through IGF-IR, while IGFBP3 can block IGF-I signaling by binding circulating IGF-I and reducing its bioavailability. In addition, and most importantly, the inventors showed that IGFBP3 acts through a pro-apoptotic IGF-I-independent mechanism on CoSCs, which the inventors demonstrated express TMEM219 (the IGFBP3 receptor), thereby inducing the failure of mini-gut growth. This latter effect is Caspase 8 and 9-mediated and TMEM219-dependent; indeed, the absence of the IGFBP3 receptor (TMEM219) on CoSCs greatly diminished high glucose-associated CoSC injuries. T1D together with starvation and cachexia are characterized by low circulating IGF-I levels (Bondy et al., 1994; Giustina et al., 2014) due to reduced hepatic IGF-I release, which is controlled and stimulated by endogenous insulin (Le Roith, 1997; Sridhar and Goodwin, 2009). More importantly, hyperglycemia appeared to have a direct effect on hepatic synthesis and release of IGFBP3. IGFBP3 may thus act as a hepatic hormone that reduces intestinal absorptive capacity during hyperglycemia. Interestingly, SPK provided a proof of concept to the inventors' hypothesis and supported their findings regarding the existence of circulating factors that control CoSCs. The striking improvement of clinical and functional features of DE that the inventors observed in their study, associated with replenishment of the CoSCs and with restoration of the circulating IGF-I and IGFBP3, strengthens inventors' hypothesis. Finally, the newly generated ecto-TMEM219 recombinant protein improved DE in diabetic mice in vivo and restored the ability of mini-guts to grow normally in vitro, thus confirming the role of IGFBP3 in controlling CoSCs and paving the way for a novel potential therapeutic strategy. In summary, inventors' study shows that an IGFBP3-mediated disruption of CoSCs linked to hyperglycemia is evident in DE. The inventors suggest that circulating IGF-I/IGFBP3 represent a hormonal dyad that controls CoSCs and a novel therapeutic target for individuals with intestinal disorders, in particular caused by diabetes mellitus of long duration (Bondy et al., 1994; Bortvedt and Lund, 2012; Boucher et al., 2010).
Example 2
Materials and Methods
Patients and Study Design
[0206] 60 individuals with T1D+ESRD registered on the waiting list for simultaneous pancreas-kidney transplantation (SPK) matched for (age 41 to 43 years old), gender, and duration of TID (29.41.8 years) were enrolled in the study. 20 subjects affected by type 1 diabetes (TID) from 10 to 20 years were enrolled as well. 20 healthy subjects matched for age and gender (CTRL), with normal renal function and normal glycometabolic parameters, were studied as well. TID+ESRD subjects were all on intensive insulin treatment at the time of enrollment in the study, while the CTRL group was not being administered any medication. All TID+ESRD subjects were on the same treatment as antiplatelet therapy (ASA) and anti-hypertension (angiotensin-converting-enzyme inhibitors), while 40 out of 60 received statins when enrolled in the study. Subjects with clear signs of inflammatory bowel diseases as well as celiac disease were not enrolled.
[0207] TID+ESRD individuals were followed up for 8 years (mean follow-up: 8.61.1 years) after receiving either SPK (n=30) or K+TID (n=30) transplantation according to the macroscopic surgical evaluation at the time of transplantation. Individuals taking an oral anticoagulant agent were not included. SPK individuals were all insulin-independent for the entire follow-up period, whereas K+TID individuals were on intensive subcutaneous insulin therapy. All subjects provided informed consent before study enrollment. Studies not included in the routine clinical follow-up were covered by an appropriate Institutional Review Board approval (Enteropatia-trapianto/01 Secchi/Fiorina).
IGFBP3 Assessment in Urine and Serum
[0208] Serum was collected from 3 ml of fresh blood after centrifugation. Urine samples were collected fresh, centrifuged and stored at 80 C. IGFBP3 levels of all groups of subjects were assessed in frozen samples of serum and urine using commercially available ELISA kits, according to the manufacturer's instructions (R&D).
Statistical Analysis
[0209] Correlation analysis and graphs were performed using Prism Graphpad software. Correlation analysis included assessment of IGFBP3 levels in serum vs. urine of individuals evaluated, IGFBP3 serum levels vs. estimated glomerular filtration rate (eGFR). Statistical significance was considered when p value was <0.05.
Measurement of Renal Function and Glycometabolic Parameters
[0210] MDRD formula was used to assess estimated glomerular filtration rate (eGFR) in ml/min/m2. Blood tests included assessment of Creatinine, blood glucose, glycated hemoglobin in all subjects enrolled in the study focusing on comparing CTRL with T1D individuals and individuals with longstanding T1D (T1D+ESRD).
Results
[0211] Serum IGFBP3 Levels Correlates with Urinary IGFBP3 Levels
[0212] Analysis of serum and urine levels of IGFBP3 in all subjects enrolled in the study documented a significant increase of both serum (
<350 pg/ml: normal levels (levels observed in healthy subjects)
350-500 pg/ml: altered levels (levels observed in T1D with a history of disease<5 years)
>500 pg/ml: indicative of enteropathy (levels observed in long-standing T1D, T1D subjects with other T1D complications, history of TID >5 years).
[0213] The inventors can also identify a normal range of urinary IGFBP3 levels (<350 pg/ml) by considering its correlation with serum IGFBP3 levels as represented in the gray area in
Example 3
[0214] Five individuals with long-term (>5 years) inflammatory bowel disease (IBD) were enrolled and screened for peripheral levels of IGFBP3, IGF-I and the ratio of the IGFBP-3/IGF-I, according to the same method described above for the analysis of diabetic samples.
[0215] It was found that while IGFBP3 was slightly increased, the levels of IGFI were severely reduced with an overall alteration of IGFBP3/IGF1 ratio (
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