U.S. patent application number 15/831235 was filed with the patent office on 2018-06-21 for inhibitor of igfbp3/tmem219 axis and diabetes.
The applicant listed for this patent is OSPEDALE SAN RAFFAELE SRL. Invention is credited to Francesca D'Addio, Paolo Fiorina.
Application Number | 20180169184 15/831235 |
Document ID | / |
Family ID | 56121063 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180169184 |
Kind Code |
A1 |
D'Addio; Francesca ; et
al. |
June 21, 2018 |
Inhibitor of IGFBP3/TMEM219 Axis and Diabetes
Abstract
The present invention relates to the role of the IGFBP3/TMEM219
axis in the onset of diabetes and the related use of IGFBP3/TMEM219
axis inhibitors for the treatment and/or prevention of diabetes.
The invention also relates to a method to identify a subject at
risk of developing Type 1 and/or Type 2 diabetes and relative
kit.
Inventors: |
D'Addio; Francesca; (Milan,
IT) ; Fiorina; Paolo; (Milan, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OSPEDALE SAN RAFFAELE SRL |
Milan |
|
IT |
|
|
Family ID: |
56121063 |
Appl. No.: |
15/831235 |
Filed: |
June 6, 2016 |
PCT Filed: |
June 6, 2016 |
PCT NO: |
PCT/EP2016/062792 |
371 Date: |
December 4, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/713 20130101;
A61K 38/1709 20130101; G01N 2800/042 20130101; A61K 47/60 20170801;
G01N 33/6893 20130101; G01N 2333/4745 20130101; G01N 2800/50
20130101; A61K 45/06 20130101; A61K 38/177 20130101; A61P 3/10
20180101; G01N 2800/52 20130101; C07K 2317/76 20130101; C07K 16/18
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 3/10 20060101 A61P003/10; A61K 47/60 20060101
A61K047/60; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 4, 2015 |
EP |
15170679.3 |
May 11, 2016 |
EP |
16169222.3 |
Claims
1.-19. (canceled)
20. A method of treating and/or preventing diabetes in a subject,
comprising: administering an effective amount of an inhibitor of
the IGFBP3/TMEM219 axis to a subject, wherein said inhibitor
comprises a fragment of the receptor TMEM219, said fragment
comprising an extracellular domain of TMEM219.
21. The method of claim 20, wherein said inhibitor is a fragment of
the receptor TMEM219.
22. The method of claim 20, wherein said inhibitor is
ecto-TMEM219.
23. The method of claim 20, wherein said inhibitor is soluble.
24. The method of claim 20, wherein said inhibitor is
pegylated.
25. The method of claim 20, wherein said inhibitor is a host cell
genetically engineered to express said fragment of the receptor
TMEM219.
26. The method of claim 20, wherein the diabetes is Type-1 or
Type-2 diabetes.
27. The method of claim 20, wherein the subject is selected from
the group consisting of: a subject at risk of developing Type-1
and/or Type-2 diabetes, and a subject with early stage Type-1
and/or Type-2 diabetes.
28. The method of claim 20, wherein said inhibitor of the
IGFBP3/TMEM219 axis is administered as a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
29. The method of claim 28, wherein said pharmaceutical composition
comprises a second therapeutic agent.
30. The method of claim 29, wherein the second therapeutic agent is
selected from the group consisting of: insulin in any form,
Pramlintide (Symlin), angiotensin-converting enzyme (ACE)
inhibitors or angiotensin II receptor blockers (ARBs), Aspirin,
Cholesterol-lowering drugs. Metformin (Glucophage, Glumetza,
others), Sulfonylureas (glyburide (DiaBeta, Glynase), glipizide
(Glucotrol) and glimepiride (Amaryl), Meglitinides (for instance
repaglinide (Prandin) and nateglinide (Starlix)),
Thiazolidinediones (Rosiglitazone (Avandia) and pioglitazone
(Actos) for examples), DPP-4 inhibitors (sitagliptin (Januvia),
saxagliptin (Onglyza) and linagliptin (Tradjenta)), GLP-1 receptor
agonists (Exenatide (Byetta) and liraglutide (Victoza)), SGLT2
inhibitors, examples include canagliflozin (Invokana) and
dapagliflozin (Farxiga).
31. A method of treating and/or preventing diabetes in a subject,
comprising: administering an effective amount of an inhibitor of
the IGFBP3/TMEM219 axis to a subject, wherein said inhibitor
comprises a polynucleotide coding for a fragment of the receptor
TMEM219, said fragment comprising an extracellular domain of
TMEM219.
32. The method of claim 31, wherein said polynucleotide codes for
ecto-TMEM219.
33. The method of claim 31, wherein said inhibitor is soluble.
34. The method of claim 31, wherein said inhibitor is a vector
comprising or expressing said polynucleotide.
35. The method of claim 31, wherein said inhibitor is a host cell
comprising said polynucleotide.
36. The method of claim 31, wherein the diabetes is Type-1 or
Type-2 diabetes.
37. The method of claim 31, wherein the subject is selected from
the group consisting of: a subject at risk of developing Type-1
and/or Type-2 diabetes, and a subject with early stage Type-1
and/or Type-2 diabetes.
38. The method of claim 31, wherein said inhibitor of the
IGFBP3/TMEM219 axis is administered as a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
39. The method of claim 38, wherein said pharmaceutical composition
comprises a second therapeutic agent.
40. The method of claim 39, wherein the therapeutic agent is
selected from the group consisting of: insulin in any form,
Pramlintide (Symlin), angiotensin-converting enzyme (ACE)
inhibitors or angiotensin II receptor blockers (ARBs), Aspirin,
Cholesterol-lowering drugs. Metformin (Glucophage, Glumetza,
others), Sulfonylureas (glyburide (DiaBeta, Glynase), glipizide
(Glucotrol) and glimepiride (Amaryl), Meglitinides (for instance
repaglinide (Prandin) and nateglinide (Starlix)), Thiazolidi
nediones (Rosiglitazone (Avandia) and pioglitazone (Actos) for
examples), DPP-4 inhibitors (sitagliptin (Januvia), saxagliptin
(Onglyza) and linagliptin (Tradjenta)), GLP-1 receptor agonists
(Exenatide (Byetta) and liraglutide (Victoza)), SGLT2 inhibitors,
examples include canagliflozin (Invokana) and dapagliflozin
(Farxiga).
41. A composition comprising an inhibitor of the IGFBP3/TMEM219
axis, wherein said inhibitor comprises a fragment of the receptor
TMEM219 or a polynucleotide coding for said fragment, wherein said
fragment comprises an extracellular domain of TMEM219.
42. The composition of claim 41, wherein said inhibitor is a
fragment of the receptor TMEM219.
43. The composition of claim 41, wherein said inhibitor is
ecto-TMEM219.
44. The composition of claim 41, wherein said polynucleotide codes
for ecto-TMEM219.
45. The composition of claim 41, wherein said inhibitor is a vector
comprising or expressing said polynucleotide.
46. The composition of claim 41, wherein said inhibitor is a host
cell genetically engineered to express said fragment of the
receptor TMEM219.
47. The composition of claim 41, wherein said inhibitor is a host
cell comprising said polynucleotide
48. The composition of claim 41, wherein said composition comprises
a pharmaceutical composition comprising said inhibitor and a
pharmaceutically acceptable carrier.
49. The composition of claim 48, wherein said pharmaceutical
composition comprises a second therapeutic agent.
Description
TECHNICAL FIELD
[0001] The present invention relates to the role of the
IGFBP3/TMEM219 axis in the onset of diabetes and the related use of
IGFBP3/TMEM219 axis inhibitors for the treatment and/or prevention
of diabetes. The invention also relates to a method to identify a
subject at risk of developing Type 1 and/or Type 2 diabetes and
relative kit.
BACKGROUND ART
[0002] Gastrointestinal disorders, consisting of gastroparesis,
abdominal distension, irritable bowel syndrome and fecal
incontinence, are common in individuals with type 1 diabetes
(T1D)(1993). Indeed up to 80% of individuals with long-standing
T1D, who are generally affected by several diabetic complications
including end stage renal disease (ESRD)(1993; Atkinson et al.,
2013; Fiorina et al., 2001), show intestinal symptoms. The presence
of these gastrointestinal symptoms, known as diabetic enteropathy
(DE), significantly reduces the quality of life (1993; Atkinson et
al., 2013; Camilleri, 2007; Talley et al., 2001) and has a largely
unknown pathogenesis (Feldman and Schiller, 1983). Preclinical
studies showed significant derangement of the intestinal mucosa
morphology in diabetic rodents (Domenech et al., 2011; Zhao et al.,
2003), suggesting that in T1D intestinal homeostasis may be
altered; however, little data are available in humans. The
intestinal epithelium is maintained by intestinal stem cells and
their niche, which respond to physiological stress and to
environmental injury (Barker, 2014; Medema and Vermeulen, 2011).
Colonic stem cells (CoSCs), located at the crypt base of the large
intestine and expressing the ephrin B receptor 2 (EphB2),
leucine-rich repeat containing G protein-coupled receptor 5 (LGR5),
h-TERT and aldehyde dehydrogenase (Aldh), among other markers
(Carlone and Breault, 2012; Carpentino et al., 2009; Jung et al.,
2011; Sato and Clevers, 2013), constitute with the local
microenvironment the CoSC niche (van der Flier and Clevers, 2009;
Zeki et al., 2011). Recent studies have established conditions that
recapitulate many features of intestinal homeostasis and generate
normal self-renewing large crypt organoids in vitro, or so-called
"mini-guts" (Sato and Clevers, 2013). Whether systemic factors,
such as circulating hormones, serve to control the CoSCs remains to
be established (Stange and Clevers, 2013).
[0003] The treatment of gastrointestinal disorders, in particular
diabetic enteropathy includes symptomatic drugs and reliever
medications for diarrhea, abdominal pain, constipation, and
dyspepsia. Up to date there is no specific treatment available for
diabetic enteropathy.
[0004] The diagnosis of gastrointestinal disorders, in particular
diabetic enteropathy includes colon endoscopy, gastric endoscopy,
anorectal manometry, esophageal manometry and analysis of fecal
samples, evaluation of peripheral cancer markers (i.e. CEA, Ca
19.9, alpha-fetoprotein, Ca125) and of celiac markers. None of the
aforementioned method is capable of providing a certain diagnosis
of diabetic enteropathy.
[0005] WO 2011133886 and WO2007024715 disclose a therapeutic
composite in the form of a IGFBP3 binding antibody.
[0006] WO0187238 relates to an anticancer pharmaceutical
composition comprising a therapeutically effective TMEM219, in
particular for the treatment of colon cancer.
[0007] WO 2014089262 discloses the use of IGFBP3 as a marker of
diagnosis of chronic inflammation (obesity) disorders (in
particular, inflammatory bowel disease such as UC and Crohn's
disease and colon cancer).
[0008] U.S. Pat. No. 6,066,464 relates to an immunoassay for the
detection of IGFBP3 on a solid support that is paper.
[0009] WO2013152989 relates to the use of IGFBP3 as a biomarker of
colorectal cancer.
[0010] WO0153837 discloses a method of monitoring or diagnosing
disease conditions that involve measuring a combination of tumor
markers and at least one component of the IGF axis. IGFBP3 is
proposed as a marker of colon tumors.
[0011] Type 1 diabetes (T1D) has historically been regarded as a T
cell-mediated autoimmune disease, resulting in the destruction of
insulin-producing pancreatic beta cells (Bluestone et al., 2010;
Eisenbarth, 1986). According to this perspective, an initiating
factor triggers the immune response against autoantigens, and the
subsequent newly activated autoreactive T cells target and further
destroy the pancreatic islets and insulin-producing beta cells
(Bluestone et al., 2010). Whether destruction of beta cells is
solely determined by the autoimmune attack or whether other
mechanisms such as paracrine modulation, metabolic deregulation,
non-immune beta cell apoptosis and halted beta cell regeneration
contribute to T1D pathogenesis is now a matter of debate (Atkinson
and Chervonsky, 2012; Atkinson et al., 2015). Recently, it has been
observed that environmental factors are required to initiate the
autoimmune response in T1D, particularly viral infections (Filippi
and von Herrath, 2008), and studies of the impact of gut microbiota
have revealed that enteroviruses are involved in activating
autoreactive T cells (McLean et al., 2015). Ongoing studies are
also focused on other environmental risk factors such as diet,
neonatal exposure to milk and gluten, and age at weaning,
suggesting that a new approach to study the pathogenesis of T1D is
gradually emerging (McLean et al., 2015), such that genetic factors
are no longer considered to be the sole determinant of T1D (Alper
et al., 2006) (Oilinki et al., 2012). Moreover, the efficacy of
immunotherapeutic strategies, which have been considered in the
last decade to be the principal prospect for establishing a cure
for T1D, is now being questioned (Ben Nasr et al., 2015a). While
targeting the autoimmune response using an immunosuppressive
treatment or a pro-regulatory regimen was shown to be satisfactory
in rodents, such strategies conversely achieved insulin
independence in a negligible number of T1D individuals (Atkinson et
al., 2015). In addition to underscoring the difference between
animal models and humans, these data also shed light on the fact
that investigation of the immune response primarily examined immune
events occurring in the periphery, while little is known with
respect to the disease process that occurs within islets and
particularly in beta cells. In this regard, the discovery of novel
factors involved in the initiation/facilitation of beta cell loss
in T1D will be of significant value. Such discoveries may pave the
way for novel therapeutic approaches capable of halting or delaying
the very first phase of the disease. Then, there is still the need
for alternative treatment for T1D and T2D.
[0012] WO2008153788 claims a method to inhibit or reduce IGFBP3
levels to treat insulin resistance or TD2, wherein the inhibitor is
a nucleic acid complementary to IGFBP3 mRNA or an antibody that
binds IGFBP3, anti IGFBP-3. The document is silent about the
IGFBP3/TMEM219 axis. Muzumdar et al. (Muzumdar et al., 2006)
discloses that IGFBP3 acts as an insulin antagonist through a
central mechanism leading to a reduced peripheral glucose uptake.
This document does not disclose the inhibition of the
IGFBP3/TMEM219 axis.
[0013] WO9739032 claims the use of an IGFBP3 inhibitor to treat
diabetes, wherein the inhibitor prevents IGFBP-3 binding to IGF-1.
Inhibition of IGFBP3/TMEM219 axis is not contemplated. D'Addio et
al., (2015) indicates that eco-TEM219 normalize circulating
IGF-I/IGFBP3 levels. WO2007024715 relates to the use of engineered
multivalent and multispecific binding proteins, namely dual
variable domain immunoglobulins, which bind two different antigens
or target peptides using a single middle linker and are bispecific.
The document mentions among the numerous target proteins, IGFBP3 in
combination with other members of the family.
[0014] WO2011133886: relates to a method of generating antibodies
and other multimeric protein complexes, namely heteromutlimeric
proteins, capable of specifically binding to more than one target.
IGFBP3 may represent a potential target.
SUMMARY OF THE INVENTION
[0015] Whether systemic factors serve to control the homeostasis of
colonic epithelium and of colonic stem cells (CoSCs) remains
unclear. The inventors hypothesize that a circulating "hormonal"
dyad controls CoSCs and is disrupted in long-standing type 1
diabetes (T1D) leading to diabetic enteropathy (DE). Individuals
with long-standing T1D exhibited abnormalities of intestinal mucosa
and CoSCs, and failure to generate in vitro mini-guts. Serum
proteomic profiling revealed altered circulating levels of
insulin-like growth factor 1 (IGF-I) and its binding protein-3
(IGFBP3) in long-standing T1D individuals, with evidences of an
increased hyperglycemia-mediated IGFBP3 hepatic release. IGFBP3
prevented mini-gut growth in vitro via a
TMEM219-dependent/caspase-mediated IGF-I-independent effect and
disrupted CoSCs in preclinical models in vivo. The restoration of
normoglycemia in long-standing T1D, with kidney-pancreas
transplantation, and the treatment with an ecto-TMEM219 recombinant
protein in diabetic mice, re-established CoSCs by restoring
appropriate IGF-I/IGFBP3 circulating levels. The peripheral
IGF-I/IGFBP3 dyad controls CoSCs and is dysfunctional in DE.
[0016] Here the inventors demonstrate that individuals with
long-standing T1D and DE have altered CoSCs and show increased
levels of IGFBP3. Administration of IGFBP3 alters CoSC regenerative
properties and mucosa morphology in vitro and in vivo, in a
preclinical model of DE, by quenching circulating IGF-I and by
exerting a TMEM219-dependent/caspase-mediated toxic effect on
CoSCs. Finally, a new ecto-TMEM219 recombinant protein, based on
the extracellular domain of the IGFBP3 receptor (TMEM219) was
generated. ecto-TMEM219 quenches peripheral IGFBP3 and prevents its
binding to IGFBP3 receptor, TMEM219. Then, targeting IGFBP3 with
such ecto-TMEM219 recombinant protein, expressed on CoSCs,
abrogates IGFBP3 deleterious effects in vitro and in vivo.
[0017] The present invention reports compelling data showing that
IGFBP3 release is increased in individuals at high-risk for T1D and
T2D. Interestingly, the inventors have discovered that the IGFBP3
receptor, TMEM219, is expressed in a beta cell line and on
murine/human islets, and that its ligation by IGFBP3 is toxic to
beta cells, raising the possibility of the existence of an
endogenous beta cell toxin. This suggests that beta cell toxin(s)
[betatoxin(s)] may be involved in the pathogenesis of TD1, in
particular in the early phase, when islet/beta cell injuries may
facilitate the exposure of autoantigens to immune cells, thus
creating a local inflamed environment and a sustained immune
reaction. Interestingly, authors have observed elevated levels of
IGFBP3 in pre-T2D and in T2D individuals as well, suggesting that a
potential role for this axis is also evident in T2D.
[0018] The inventors have also observed that IGFBP3 may induce
apoptosis of beta cells and of murine/human islets in vitro in a
caspase 8-dependent manner. Finally, the newly generated
recombinant ecto-TMEM219 protein, based on the TMEM219
extracellular domain, capable of quenching IGFBP3, prevents its
signaling via TMEM219 on pancreatic beta cells. Ecto-TMEM219
treatment reduces beta cell loss, improves islet insulin content
and glycometabolic control in murine models of diabetes (T1D and
T2D) in vivo, while in vitro it protects islets and beta cells from
IGFBP3-induced apoptosis. The inventors demonstrate that IGFBP3 is
an endogenous peripheral beta cell toxin (or betatoxin) that is
increasingly released in individuals at high-risk for diabetes (T1D
and T2D). Concomitant expression of the IGFBP3 receptor (TMEM219)
on beta cells initiates/facilitates beta cell death, thus favoring
diabetes onset/progression.
[0019] In other words, the invention is based on the finding that
TMEM219, the IGFBP3 receptor that mediates IGFBP3/IGF1 independent
detrimental effects, is expressed on pancreatic islets and beta
cells; moreover, targeting the IGFBP3/TMEM219 axis with
ecto-TMEM219 re-establishes appropriate IGFBP3 signaling in
diabetic mice and prevents beta cell loss and preserves islet
morphology, thereby confirming the critical role of the
IGFBP3/TMEM219 axis in favoring beta cell loss in diabetes.
[0020] The present therapeutic approach, based on the inhibition of
IGFBP3/TMEM219 axis, may overcome the limits of the current
therapies for T1D and T2D as it could prevent the beta cell damage
and the consequent reduced or abolished insulin secretion that
leads to the development of diabetes.
[0021] Then, the advantages of the present invention over prior art
treatments are: [0022] Prevention of beta cell and islets
destruction [0023] Protection of beta cell mass and of
insulin-producing cells [0024] Prevention of major diabetes
complications [0025] Limitation of autoimmune attack towards
pancreatic islets in T1D [0026] Prevention of insulin resistance in
T2D and [0027] No requirement for immunotherapy in T1D.
[0028] Then the invention provides an inhibitor of IGFBP3/TMEM219
axis for use in the treatment and/or prevention of diabetes in a
subject.
[0029] Preferably said inhibitor is selected from the group
consisting of: [0030] a) a polypeptide; [0031] b) a polynucleotide
coding for said polypeptide or a polynucleotide able to inhibit
IGFBP3/TMEM219 axis; [0032] c) a vector comprising or expressing
said polynucleotide; [0033] d) a host cell genetically engineered
expressing said polypeptide or said polynucleotide; [0034] e) a
small molecule; [0035] f) a peptide, a protein, an antibody, an
antisense oligonucleotide, a siRNA, antisense expression vector or
recombinant virus or any other agent able to inhibit or
IGFBP3/TMEM219 axis.
[0036] Preferably said inhibitor is the receptor TMEM219 or a
fragment thereof.
[0037] Preferably the fragment of TMEM219 is a fragment comprising
an extracellular domain of TMEM219.
[0038] In a preferred embodiment the inhibitor is ecto-TMEM219.
Preferably the inhibitor is soluble.
[0039] Preferably said inhibitor is a fusion protein TMEM219-Ig,
preferably said fusion protein quenches circulating IGFBP3 and
prevents its binding to TMEM219.
[0040] Preferably the inhibitor is an anti-IGFBP3 antibody,
preferably said antibody selectively blocks the TMEM219-binding
site;
[0041] Preferably said inhibitor is an anti-TMEM219 antibody,
preferably said antibody occupies the IGFBP3 binding site of
TMEM219 receptor thus preventing IGFBP3 binding.
[0042] More preferably said inhibitor is an oligonucleotide
complementary to IGFBP3 mRNA.
[0043] In a preferred embodiment the diabetes is Type-1 or Type-2
diabetes.
[0044] Still preferably the subject is selected from the group
consisting of: a subject at risk of developing Type-1 and/or Type-2
diabetes, a subject with early stage Type-1 and/or Type-2
diabetes.
[0045] The present invention also provides a pharmaceutical
composition for use in the treatment and/or prevention of diabetes
comprising the inhibitor of the invention and pharmaceutically
acceptable carriers. Preferably the pharmaceutical composition
further comprises a therapeutic agent.
[0046] Preferably the therapeutic agent is selected from the group
consisting of: insulin in any form, Pramlintide (Symlin),
angiotensin-converting enzyme (ACE) inhibitors or angiotensin II
receptor blockers (ARBs), Aspirin, Cholesterol-lowering drugs.
Metformin (Glucophage, Glumetza, others), Sulfonylureas (glyburide
(DiaBeta, Glynase), glipizide (Glucotrol) and glimepiride (Amaryl),
Meglitinides (for instance repaglinide (Prandin) and nateglinide
(Starlix)), Thiazolidinediones (Rosiglitazone (Avandia) and
pioglitazone (Actos) for examples), DPP-4 inhibitors (sitagliptin
(Januvia), saxagliptin (Onglyza) and linagliptin (Tradjenta)),
GLP-1 receptor agonists (Exenatide (Byetta) and liraglutide
(Victoza)), SGLT2 inhibitors, examples include canagliflozin
(Invokana) and dapagliflozin (Farxiga).
[0047] The present invention also provides a method to identify a
subject at risk of developing Type-1 and/or Type-2 or to monitor
the response to a therapeutic treatment in a subject comprising:
[0048] a) measuring the amount of the protein IGFBP3 or the amount
of the polynucleotide coding for said protein in a biological
sample obtained from the subject; [0049] b) comparing the measured
quantity of the protein IGFBP3 or measured quantity of the
polynucleotide coding for said protein to a control amount, wherein
if the measured quantity is higher than the control amount, the
subject is at risk of developing Type-1 and/or Type-2 diabetes.
[0050] Preferably the quantity of IGFBP3 is measured by an
antibody.
[0051] More preferably the biological sample is selected from the
group consisting of: serum, urine, cell culture supernatant.
[0052] The present invention also provides a kit comprising means
to measure the amount of the protein IGFBP3 and/or means to measure
the amount of the polynucleotide coding for said protein and
optionally, control means for use in the method of the
invention.
[0053] In the present invention inhibiting the IGFBP3/TMEM219 axis
means blocking IGFBP3 binding to TMEM219, for instance by quenching
IGFBP3 from the circulation, it also means blocking the
IGFBP3-binding site of TMEM219, blocking IGFBP3 binding site on
TMEM219. It further means inhibiting TMEM219 function and/or
expression and/or signaling, this may be achieved for instance by
silencing TMEM219 expression, in particular with SiRNA or
oligonucleotides. It also means inhibiting the function and/or
expression of IGFBP3.
[0054] According to the invention, an inhibitor of IGFBP3 binding
to TMEM219 can be one of the following molecules: [0055] Soluble
Ecto-TMEM219 (extracellular portion of TMEM219) which neutralizes
circulating IGFBP3; [0056] Fusion protein TMEM219-Ig, a Fc-based
fusion protein composed of an immunoglobulin Fc domain that is
directly linked to TMEM219 peptide or to its extracellular portion,
which quenches circulating IGFBP3 and prevents its binding to
TMEM219 expressed on beta cells; [0057] Anti-IGFBP3 antibody that
selectively blocks the TMEM219-binding site; [0058] Anti-TMEM219
antibody, which occupies the IGFBP3 binding site of TMEM219
receptor thus preventing IGFBP3 binding (having antagonistic
activity with respect to IGFBP3) [0059] Oligonucleotides
complementary to IGFBP3 mRNA
[0060] In the present invention the patient that may be treated are
individuals who are at risk for developing T1D (autoimmune
diabetes, based on the presence of peripheral anti-islet
autoantibodies or genetic predisposition or familiar predisposition
or altered beta cell function) or T2D (non autoimmune diabetes
based on the evidence of an impaired fasting glucose and/or
impaired glucose tolerance without fulfilling the criteria for the
diagnosis of diabetes), or individuals who develop T1D or T2D in
any stage of the disease, in particular a subject with early stage
Type-1 and/or Type-2 diabetes, with the purpose of protecting beta
cells from further destruction. The presence of any degree of
preserved beta cells is the only requirement for assessing the
successful therapy.
[0061] The expression of IGFBP3 may be measured by means of RT-PCR
on tissues and cells, Western blot on tissues and cells,
Immunohistochemistry on tissues, Immunofluorescence on tissue and
cells. Levels of IGFBP3 in biological fluids can be measured by
immune-targeted assays and proteomic analysis.
[0062] The function of IGFBP3 may be measured by means of detecting
Caspases 8 and 9 expression on target cells using RT-PCR,
microarrays, by co-culturing target cells/structures with Pan
Caspase inhibitor, Caspases 8 and 9 inhibitors and measuring live
cells/structures.
[0063] In the present invention "inhibit or block the interaction
of IGFBP3 with its receptor TMEM219" means quenching circulating
IGFBP3 and preventing its binding to TMEM219 receptor expressed on
pancreatic islets and beta cells. The IGFBP3-TMEM219 binding could
be prevented also by the use of an IGFBP3-blocking antibody. In
addition, a TMEM219 blocking antibody could bind TMEM219 receptor
thus rendering the receptor unavailable when IGFBP3 comes from the
circulation.
[0064] The inhibitor of the invention may be the receptor TMEM219
(MGNCQAGHNLHLCLAHHPPLVCATLILLLLGLSGLGLGSFLLTHRTGLRSPDIPQDWV
SFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMG
LKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEE
CVSVWSHEGLVLTKLLTSEELALCGSRLLVLGSFLLLFCGLLCCVTAMCFHPRRESHWS RTRL,
SEQ ID No. 1) or a fragment thereof.
[0065] In particular the fragment of TMEM219 is designed such as to
block/prevent IGFBP3 access and/or binding to TMEM219, it has a
smaller molecular weight, it contains five cysteins that form
disulfide bridges and a globular structure. Preferably the fragment
is at least 50 amino acid long, preferably 100 amino acid long,
still preferably 120 amino acid long, yet preferably 150 amino acid
long, preferably at least 160 amino acid long.
[0066] In a preferred embodiment the fragment is at least 162, 165,
170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,
235 amino acid long. Preferably the fragment has at least 65%
identity with the sequence of TMEM219, preferably at least 70%,
75%, 80%, 85%, 90%, 95% or 99% identity with the sequence of
TMEM219.
[0067] Preferably the fragment of TMEM219 is a fragment of an
extracellular domain of TMEM219 (ecto-TMEM219), in particular the
fragment comprises the sequence:
THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDR
NKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISC
SEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR (SEQ ID No. 2).
[0068] Preferably the fragment of TMEM219 is an extracellular
domain of TMEM219, in particular the fragment comprises the
sequence:
SFLLTHRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDG
PDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPP
ISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR (SEQ ID No. 3)
Preferably the fragment of TMEM219 consists of:
THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDR
NKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISC
SEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR (SEQ ID No. 2).
[0069] Preferably the fragment of TMEM219 consists of:
SFLLTHRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDG
PDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPP
ISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR (SEQ ID No. 3).
[0070] In the present invention TMEM219 is preferably eukaryote
TMEM219, preferably a mammal TMEM219, still preferably human
TMEM219.
[0071] The interaction of IGFBP3 with TMEM219 may be measured by
means of indirect assessment of the effects of IGFBP3 on target
cells (increased Caspase 8 and 9 expression with RT-PCR), direct
assessment of IGFBP3-IGFBP3-receptor (TMEM219) binding with Liquid
or Solid Phase Ligand Binding Assays (i.e. immunoprecipitation,
RT-PCR, immunoassays) and Non-radioactive Ligand Binding
Assays.
[0072] In the present invention "long-standing T1D" means a history
of type 1 diabetes longer than 15 years associated with the
development of diabetic complications.
[0073] In a preferred aspect of the invention, the inhibitor is an
antibody or synthetic or recombinant derivative thereof. Said
antibody is preferably a monoclonal or polyclonal antibody, or
synthetic or recombinant derivatives thereof, more preferably said
antibody being a humanized monoclonal antibody.
[0074] Preferably, said polynucleotide is a RNA or DNA, preferably
a siRNA, a shRNA, a microRNA or an antisense oligonucleotide.
[0075] In a preferred embodiment, the above vector is an expression
vector selected from the group consisting of: plasmids, viral
particles and phages.
[0076] Preferably, said host cell is selected from the group
consisting of: bacterial cells, fungal cells, insect cells, animal
cells, plant cells, preferably being an animal cell, more
preferably a human cell.
[0077] In a preferred embodiment, the inhibitor as above defined
(a) is combined with at least one therapeutic agent (b) to define a
combination or combined preparation. The therapeutic agent may be
an anti-diabetic agent, an agent used to prevent diabetes, an
anti-apoptotic agent, an anti-inflammatory agent, immune
suppressive agent, adjuvant therapy in organ transplantation,
protective agent in cell therapy approach a pain reliever.
[0078] Examples of therapeutic agent is insulin therapy, in any
form, Pramlintide (Symlin), angiotensin-converting enzyme (ACE)
inhibitors or angiotensin II receptor blockers (ARBs), Aspirin,
Cholesterol-lowering drugs. Metformin (Glucophage, Glumetza,
others), Sulfonylureas (glyburide (DiaBeta, Glynase), glipizide
(Glucotrol) and glimepiride (Amaryl), Meglitinides (for instance
repaglinide (Prandin) and nateglinide (Starlix)),
Thiazolidinediones (Rosiglitazone (Avandia) and pioglitazone
(Actos) for examples), DPP-4 inhibitors (sitagliptin (Januvia),
saxagliptin (Onglyza) and linagliptin (Tradjenta)), GLP-1 receptor
agonists (Exenatide (Byetta) and liraglutide (Victoza)), SGLT2
inhibitors, examples include canagliflozin (Invokana) and
dapagliflozin (Farxiga).
[0079] The terms "combination" and "combined preparation" as used
herein also define a "kit of parts" in the sense that the
combination partners (a) and (b) as defined above can be dosed
independently or by use of different fixed combinations with
distinguished amounts of the combination partners (a) and (b), i.e.
simultaneously or at different time points. The parts of the kit of
parts can then, e.g., be administered simultaneously or
chronologically staggered, that is at different time points and
with equal or different time intervals for any part of the kit of
parts. The ratio of the total amounts of the combination partner
(a) to the combination partner (b) to be administered in the
combined preparation can be varied, e.g. in order to cope with the
needs of a patient sub-population to be treated or the needs of the
single.
[0080] The combination therapy may result in unexpected improvement
in the treatment of diabetes. When administered simultaneously,
sequentially or separately, the inhibitor and the other therapeutic
agent may interact in a synergistic manner to reduce diabetes. This
unexpected synergy allows a reduction in the dose required of each
compound, leading to a reduction in the side effects and
enhancement of the clinical effectiveness of the compounds and
treatment. Determining a synergistic interaction between one or
more components, the optimum range for the effect and absolute dose
ranges of each component for the effect may be definitively
measured by administration of the components over different w/w
ratio ranges and doses to patients in need of treatment. For
humans, the complexity and cost of carrying out clinical studies on
patients renders impractical the use of this form of testing as a
primary model for synergy. However, the observation of synergy in
one species can be predictive of the effect in other species and
animal models exist, as described herein, to measure a synergistic
effect and the results of such studies can also be used to predict
effective dose and plasma concentration ratio ranges and the
absolute doses and plasma concentrations required in other species
by the application of pharmacokinetic/pharmacodynamic methods.
Established correlations between diabetes models and effects seen
in man suggest that synergy in animals may e.g. be demonstrated in
the models as described in the Examples below.
[0081] The above pharmaceutical compositions are preferably for
systemic, oral, locally, preferably rectally, or topical
administration.
[0082] Control amount is the amount measured in a proper
control.
[0083] Control means can be used to compare the amount or the
increase of amount of the compound as above defined to a proper
control. The proper control may be obtained for example, with
reference to known standard, either from a normal subject or from
normal population.
[0084] The above diagnosis method may also comprise a step of
treating the subject, in particular the treatment may be an
inhibitor of IGFBP3/TMEM219 axis as defined in the present
invention or an existing treatment for diabetes such as indicated
above.
[0085] The means to measure the amount of IGFBP3 as above defined
are preferably at least one antibody, functional analogous or
derivatives thereof. Said antibody, functional analogous or
derivatives thereof are specific for said compound.
[0086] In a preferred embodiment, the kit of the invention
comprises: [0087] a solid phase adhered antibody specific for said
compound; [0088] detection means of the ligand specific-biomarker
complex.
[0089] The kits according to the invention can further comprise
customary auxiliaries, such as buffers, carriers, markers, etc.
and/or instructions for use.
[0090] The proper control may be a sample taken from a healthy
patient or from a patient affected by a disorder other than
diabetes.
[0091] In the case of a method or a kit for monitoring the
progression of the diabetes, the progress of the disease is
monitored and the proper control may be a sample taken from the
same subject at various times or from another patient, and the
proper control amount may by the amount of the same protein or
polynucleotide measured in a sample taken from the same subject at
various times or from another patient.
[0092] In the case of a method or a kit for monitoring the efficacy
or response to a therapeutic treatment, the proper control may by a
sample taken from the same subject before initiation of the therapy
or taken at various times during the course of the therapy and the
proper control amount may be the amount of the same protein or
polynucleotide measured in a sample taken from the same subject
before initiation of the therapy or taken at various times during
the course of the therapy. The therapy may be the therapy with the
inhibitor of the present invention.
[0093] In the present invention, the expression "measuring the
amount" can be intended as measuring the amount or concentration or
level of the respective protein and/or mRNA thereof and/or DNA
thereof, preferably semi-quantitative or quantitative. Measurement
of a protein can be performed directly or indirectly. Direct
measurement refers to the amount or concentration measure of the
biomarker, based on a signal obtained directly from the protein,
and which is directly correlated with the number of protein
molecules present in the sample. This signal--which can also be
referred to as intensity signal--can be obtained, for example, by
measuring an intensity value of a chemical or physical property of
the biomarker. Indirect measurements include the measurement
obtained from a secondary component (e.g., a different component
from the gene expression product) and a biological measurement
system (e.g. the measurement of cellular responses, ligands, "tags"
or enzymatic reaction products).
[0094] The term "amount", as used in the description refers but is
not limited to the absolute or relative amount of proteins and/or
mRNA thereof and/or DNA thereof, and any other value or parameter
associated with the same or which may result from these. Such
values or parameters comprise intensity values of the signal
obtained from either physical or chemical properties of the
protein, obtained by direct measurement, for example, intensity
values in an immunoassay, mass spectroscopy or a nuclear magnetic
resonance. Additionally, these values or parameters include those
obtained by indirect measurement, for example, any of the
measurement systems described herein. Methods of measuring mRNA and
DNA in samples are known in the art. To measure nucleic acid
levels, the cells in a test sample can be lysed, and the levels of
mRNA in the lysates or in RNA purified or semi-purified from
lysates can be measured by any variety of methods familiar to those
in the art. Such methods include hybridization assays using
detectably labeled DNA or RNA probes (i.e., Northern blotting) or
quantitative or semi-quantitative RT-PCR methodologies using
appropriate oligonucleotide primers. Alternatively, quantitative or
semi-quantitative in situ hybridization assays can be carried out
using, for example, tissue sections, or unlysed cell suspensions,
and detectably labeled (e.g., fluorescent, or enzyme-labeled) DNA
or RNA probes. Additional methods for quantifying mRNA include RNA
protection assay (RPA), cDNA and oligonucleotide microarrays,
representation difference analysis (RDA), differential display, EST
sequence analysis, and serial analysis of gene expression
(SAGE).
[0095] If by comparing the measured amount of the protein IGFBP3 or
of the polynucleotide coding for said protein with the amount
obtained from a control sample, the amount of said compound in the
sample isolated from the subject corresponds to a higher value, the
subject may present the disease or go towards an aggravation of
said disease.
[0096] If by comparing the measured amount of the protein IGFBP3 or
of the polynucleotide coding for said protein with the amount
obtained from a control sample, the amount of said compound in the
sample isolated from the subject corresponds to a similar or lower
value, the subject may be not affected by the disease or go toward
an amelioration of the disease, respectively.
[0097] Alternatively, the expression "detection" or "measuring the
amount" is intended as measuring the alteration of the molecule.
Said alteration can reflect an increase or a decrease in the amount
of the compounds as above defined. An increase of the protein
IGFBP3 or of the polynucleotide coding for said protein can be
correlated to an aggravation of the disease. A decrease the protein
IGFBP3 or of the polynucleotide coding for said protein can be
correlated to an amelioration of the disease or to recovery of the
subject.
[0098] The expression "protein IGFBP3" or "IGFBP3" or "TMEM219" is
intended to include also the corresponding protein encoded from a
IGFBP3 or TMEM orthologous or homologous genes, functional mutants,
functional derivatives, functional fragments or analogues, isoforms
thereof. The expression "gene IGFBP3" or "IGFBP3" or "gene TMEM219"
or "TMEM219" is intended to include also the corresponding
orthologous or homologous genes, functional mutants, functional
derivatives, functional fragments or analogues, isoforms
thereof.
[0099] In the present invention "functional mutants" of the protein
are mutants that may be generated by mutating one or more amino
acids in their sequences and that maintain their activity for the
treatment of diabetes. Indeed, the protein of the invention, if
required, can be modified in vitro and/or in vivo, for example by
glycosylation, myristoylation, amidation, carboxylation or
phosphorylation, and may be obtained, for example, by synthetic or
recombinant techniques known in the art. The protein of the
invention "IGFBP3" or "TMEM219" may be modified to increase its
bioavailability or half-life by know method in the art. For
instance the protein may be conjugated to a polymer, may be
pegylated ect.
[0100] In the present invention the active ingredients may also be
entrapped in microcapsule prepared, for example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsule and
poly-(methylmethacylate) microcapsule, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0101] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0102] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-releabe matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and [gamma] ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide acetate, and poly-D-(-)-3-hydroxybutyric
acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated antibodies remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilization depending on the mechanism involved. For
example, if the aggregation mechanism is discovered to be
intermolecular S-- S bond formation through thio-disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0103] In the present invention "functional" is intended for
example as "maintaining their activity" e.g. therapeutic treatment
of diabetes.
[0104] The term "analogue" as used herein referring to a protein
means a modified peptide wherein one or more amino acid residues of
the peptide have been substituted by other amino acid residues
and/or wherein one or more amino acid residues have been deleted
from the peptide and/or wherein one or more amino acid residues
have been deleted from the peptide and or wherein one or more amino
acid residues have been added to the peptide. Such addition or
deletion of amino acid residues can take place at the N-terminal of
the peptide and/or at the C-terminal of the peptide.
[0105] The term "derivative" as used herein in relation to a
protein means a chemically modified peptide or an analogue thereof,
wherein at least one substituent is not present in the unmodified
peptide or an analogue thereof, i.e. a peptide which has been
covalently modified. Typical modifications are amides,
carbohydrates, alkyl groups, acyl groups, esters and the like. As
used herein, the term "derivatives" also refers to longer or
shorter polypeptides having e.g. a percentage of identity of at
least 41%, preferably at least 41.5%, 50%, 54.9%, 60%, 61.2%,
64.1%, 65%, 70% or 75%, more preferably of at least 85%, as an
example of at least 90%, and even more preferably of at least 95%
with IGFBP3, or with an amino acid sequence of the correspondent
region encoded from a IGFBP3 orthologous or homologous gene.
[0106] As used herein "fragments" refers to polypeptides having
preferably a length of at least 10 amino acids, more preferably at
least 15, at least 17 amino acids or at least 20 amino acids, even
more preferably at least 25 amino acids or at least 37 or 40 amino
acids, and more preferably of at least 50, or 100, or 150 or 200 or
250 or 300 or 350 or 400 or 450 or 500 amino acids. According to
the present invention, an "effective amount" of a composition is
one that is sufficient to achieve a desired biological effect, in
this case an amelioration or the treatment of diabetes.
[0107] It is understood that the effective dosage will be dependent
upon the age, sex, health, and weight of the recipient, kind of
concurrent treatment, if any, frequency of treatment, and the
nature of the effect desired. The provided ranges of effective
doses of the inhibitor or molecule of the invention (e.g. from 1
mg/kg to 1000 mg/kg, in particular systemically administered) are
not intended to limit the invention and represent preferred dose
ranges. However, the preferred dosage can be tailored to the
individual subject, as is understood and determinable by one of
skill in the art, without undue experimentation.
[0108] The administration of oligonucleotides of the present
invention may be carried out by known methods, wherein a nucleic
acid is introduced into a desired target cell in vitro or in
vivo.
[0109] An aspect of the present invention comprises a nucleic acid
construct comprised within a delivery vehicle. A delivery vehicle
is an entity whereby a nucleotide sequence can be transported from
at least one media to another. Delivery vehicles may be generally
used for expression of the sequences encoded within the nucleic
acid construct and/or for the intracellular delivery of the
construct. It is within the scope of the present invention that the
delivery vehicle may be a vehicle selected from the group of RNA
based vehicles, DNA based vehicles/vectors, lipid based vehicles,
virally based vehicles and cell based vehicles. Examples of such
delivery vehicles include: biodegradable polymer microspheres,
lipid based formulations such as liposome carriers, coating the
construct onto colloidal gold particles, lipopolysaccharides,
polypeptides, polysaccharides, pegylation of viral vehicles.
[0110] In one embodiment of the present invention may comprise a
virus as a delivery vehicle, where the virus may be selected from:
adenoviruses, retroviruses, lentiviruses, adeno-associated viruses,
herpesviruses, vaccinia viruses, foamy viruses, cytomegaloviruses,
Semliki forest virus, poxviruses, RNA virus vector and DNA virus
vector. Such viral vectors are well known in the art.
[0111] Commonly used gene transfer techniques include calcium
phosphate, DEAE-dextran, transfection, electroporation and
microinjection and viral methods. Another technique for the
introduction of DNA into cells is the use of cationic liposomes.
Commercially available cationic lipid formulations are e.g. Tfx 50
(Promega) or Lipofectamin 2000 (Life Technologies).
[0112] The compositions of the present invention may be in form of
a solution, e.g. an injectable solution, a cream, ointment, tablet,
suspension or the like. The composition may be administered in any
suitable way, e.g. by injection, particularly by intraocular
injection, by oral, topical, nasal, rectal application etc. The
carrier may be any suitable pharmaceutical carrier. Preferably, a
carrier is used, which is capable of increasing the efficacy of the
RNA molecules to enter the target-cells. Suitable examples of such
carriers are liposomes, particularly cationic liposomes.
[0113] The recombinant expression vector of the invention can be
any suitable recombinant expression vector, and can be used to
transform or transfect any suitable host. Suitable vectors include
those designed for propagation and expansion or for expression or
both, such as plasmids and viruses. The recombinant expression
vectors of the invention can be prepared using standard recombinant
DNA techniques. Constructs of expression vectors, which are
circular or linear, can be prepared to contain a replication system
functional in a prokaryotic or eukaryotic host cell. Replication
systems can be derived, e.g., from CoIE1, 2.mu. plasmid, .lamda.,
SV40, bovine papilloma virus, and the like.
[0114] Desirably, the recombinant expression vector comprises
regulatory sequences, such as transcription and translation
initiation and termination codons, which are specific to the type
of host (e.g., bacterium, fungus, plant, or animal) into which the
vector is to be introduced, as appropriate and taking into
consideration whether the vector is DNA- or RNA-based. The
recombinant expression vector can include one or more marker genes,
which allow for selection of transformed or transfected hosts.
Marker genes include biocide resistance, e.g., resistance to
antibiotics, heavy metals, etc., complementation in an auxotrophic
host to provide prototrophy, and the like. Suitable marker genes
for the inventive expression vectors include, for instance,
neomycin/G418 resistance genes, hygromycin resistance genes,
histidinol resistance genes, tetracycline resistance genes, and
ampicillin resistance genes. The recombinant expression vector can
comprise a native or normative promoter operably linked to the
nucleotide sequence encoding the PCYOX1 inhibitor (including
functional portions and functional variants thereof), or to the
nucleotide sequence which is complementary to or which hybridizes
to the nucleotide sequence encoding the RNA. The selection of
promoters, e.g., strong, weak, inducible, tissue-specific and
developmental-specific, is within the ordinary skill of the
artisan. Similarly, the combining of a nucleotide sequence with a
promoter is also within the skill of the artisan. The promoter can
be a non-viral promoter or a viral promoter, e.g., a
cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter
and a promoter found in the long-terminal repeat of the murine stem
cell virus.
[0115] The inventive recombinant expression vectors can be designed
for either transient expression, for stable expression, or for
both. Also, the recombinant expression vectors can be made for
constitutive expression or for inducible expression.
[0116] In the above IGFBP3 compositions further materials as well
as processing techniques and the like may be set out in Part 5 of
Remington's Pharmaceutical Sciences, 20th Edition, 2000, Marck
Publishing Company, Easton, Pa., which is incorporated herein by
reference. The compounds of this invention can also be administered
in sustained release forms or from sustained release drug delivery
systems. A description of representative sustained release
materials can also be found in the incorporated materials in
Remington's Pharmaceutical Sciences. Furthermore, pharmaceutical
formulations can be prepared using a process, which is generally
known in the pharmaceutical art.
[0117] In the present invention, when the molecule of the invention
is administered with another therapeutic agent, it may be
administered simultaneously or sequentially.
Sequences
Amino Acid Sequence of IGFBP3:
[0118] MQRARPTLWAAALTLLVLLRGPPVARAGASSAGLGPVVRCEPCDARALAQCAPPPAV
CAELVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVN
ASAVSRLRAYLLPAPPAPGEPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHS
KIIIIKKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVL
SPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYS MQSK (SEQ
ID No. 4)
Nucleotide Sequence of IGFBP3:
[0119] Homo sapiens insulin-like growth factor binding protein 3
(IGFBP3), RefSeqGene on chromosome 7, NCBI Reference Sequence:
NG_011508.1
mRNA Sequence of IGFBP3:
[0120] Homo sapiens insulin-like growth factor binding protein 3
(IGFBP3), transcript variant 1, mRNA, NCBI Reference Sequence:
NM_001013398.1
Amino Acid Sequence of TMEM219:
MGNCQAGHNLHLCLAHHPPLVCATLILLLLGLSGLGLGSFLLTHRTGLRSPDIPQDWVS
FLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDRNKTRTFQATVLGSQMGL
KGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEEC
VSVWSHEGLVLTKLLTSEELALCGSRLLVLGSFLLLFCGLLCCVTAMCFHPRRESHWSR TRL
(SEQ ID No. 2).
Nucleotide Sequence of TMEM219:
[0121] TMEM219 transmembrane protein 219 [Homo sapiens (human)],
Gene ID: 124446.
mRNA Sequence of TMEM219:
[0122] Homo sapiens transmembrane protein 219 (TMEM219), transcript
variant 1, mRNA, NCBI Reference Sequence: NM_001083613.1
[0123] The present invention will be illustrated by means of non
limiting examples referring to the following figures.
[0124] FIG. 1. Diabetic enteropathy in long-standing T1D is
characterized by intestinal mucosa abnormalities and impairment in
the colonic stem cells. A, B, C. Bar graphs depict the score of
diarrhea, abdominal pain and constipation according to the
administration of the GSRS questionnaire in healthy subjects (CTRL)
and long-standing T1D individuals (T1D+ESRD). Gray area indicates
normal range for the parameter. D, E, F. Bar graphs report the
measurements of anorectal sphincter contracting tone (mmHg), reflex
response (ml) and urgency volume (ml) by anorectal manometry in
healthy subjects (CTRL) and long-standing T1D individuals
(T1D+ESRD). Gray area indicates normal range for the parameter.
N=20 CTRL and n=60 T1D+ESRD individuals were included in the
evaluation. G1-G2, I1-I2, K1-K2, M1-M2, O1-O2, Q1-Q2.
Representative images of hematoxylin and eosin (H&E) histology
staining, immunostained MIB1.sup.+ cells, ultrastructural analysis
of neural structures with red arrows indicating localization and
presence of neuroendocrine vesicles, immunostained 5HT.sup.+,
aldehyde dehydrogenase (Aldh).sup.+ cells, and EphB2.sup.+
expression, on bioptic samples obtained from healthy subjects
(CTRL) and long-standing T1D individuals (T1D+ESRD).
Ultrastructural analysis scale bar: 2000 nm. Original
magnification: 100.times. in G1-G2; 400.times. in 11-12, K1-K2;
40.times. in O1-O2; 200.times., in Q1-Q2. Scale bar 80 micron. H,
J, L, N, P, R. Bar graphs reporting the measurement of crypts,
MIB1.sup.+ cells, of neuroendocrine vesicles of nerve terminals
(number of cases with >3 NE vesicles detected per nerve
terminal), of 5HT.sup.+, Aldh.sup.+ cells, and of EphB2.sup.+
expression (intensity score 0-5) in CTRL and long-standing T1D
subjects (T1D+ESRD). N=20 CTRL and n=60 T1D+ESRD individuals were
included in the evaluation. Data are expressed as mean.+-.standard
error of the mean (SEM) unless differently reported. *p<0.01;
**p<0.001; ***p<0.0001. Abbreviations: GSRS, Gastrointestinal
Symptom Rating Scale; CoSC, intestinal stem cell; T1D, type 1
diabetes; ESRD, end stage renal disease; CTRL, healthy subjects;
H&E, hematoxylin and eosin; MIB1, antibody against Ki67; EphB2,
Ephrin B receptor 2; Aldh, Aldehyde dehydrogenase; 5HT, serotonin;
NE, neuroendocrine vesicles.
[0125] FIG. 2. Diabetic enteropathy in long-standing T1D is
associated with a defect in CoSCs. A, B. Representative flow dot
plots of EphB2.sup.low, EphB2.sup.medium and EphB2.sup.hi cells in
healthy subjects (CTRL) and long-standing T1D individuals
(T1D+ESRD). C, D, E. Bar graphs depict results of flow cytometric
analysis of EphB2.sup.hi+, EphB2.sup.hi+LGR5.sup.+ and
EphB2.sup.+h-TERT.sup.+ cells in freshly isolated crypts (n=10 CTRL
and n=10 T1D+ESRD). F, G, H. Bar graphs depict expression data of
CoSC markers EphB2, LGR5, h-TERT as normalized mRNA expression
measured by quantitative RT-PCR on isolated intestinal crypts. All
samples were run in triplicate and normalized to expression of the
housekeeping gene ACTB (.DELTA..DELTA.Ct). I. Scatter plot
represents the CoSC signature markers and stem cell transcriptome
profiling examined in freshly isolated intestinal crypts of n=10
healthy subjects (CTRL) and n=10 long-standing T1D individuals
(T1D+ESRD). J1-J2. Representative images of mini-guts cultured for
8 days in vitro obtained from previously isolated crypts of
long-standing T1D individuals (T1D+ESRD) and healthy subjects
(CTRL). 10.times. magnification. Scale bar 50 micron. K. Bar graph
depicts the % of developed mini-guts of the total at 8 days of
culture of freshly isolated intestinal crypts from n=10 CTRL and
n=10 T1D+ESRD individuals. L1-L4. Representative images of
mini-guts obtained from previously isolated crypts of healthy
subjects (CTRL) and cultured for 8 days in the following
conditions: L1=normal (FBS) serum+normal glucose (5 mM);
L2=T1D+ESRD serum+normal glucose; L3=normal serum+high glucose (35
mM); L4=T1D+ESRD serum+high glucose. 10.times. magnification. Scale
bar 50 micron. M. Bar graph grouping % of developed mini-guts of
the total at 8 days of culture from freshly isolated intestinal
crypts cultured with the following conditions: normal (FBS)
serum+normal glucose (5 mM); T1D+ESRD serum+normal glucose; normal
serum+high glucose (35 mM); T1D+ESRD serum+high glucose.
Statistical significance has been calculated within each group
(normal glucose+normal serum, medium+high glucose,
medium+long-standing T1D serum, high glucose+long-standing T1D
serum) by comparing different culturing conditions. Comparison in
the bar graph refers to all conditions vs. normal serum+normal
glucose. N. Transcriptome profiling depicting CoSC signature
markers expression in isolated crypts obtained from healthy
subjects and cultured with/without high glucose and/or
long-standing T1D serum. N=10 subjects per group were evaluated.
Data are expressed as mean.+-.standard error of the mean (SEM)
unless differently reported. *p<0.01; **p<0.001;
***p<0.0001. Abbreviations: CoSC, colonic stem cell; T1D, type 1
diabetes; ESRD, end stage renal disease; CTRL, healthy subjects;
EphB2, Ephrin B receptor 2; LGR5, leucine-rich repeat containing G
protein-coupled receptor 5; RT-PCR, real-time polymerase chain
reaction; ACTB, beta actin; FBS, fetal bovine serum.
[0126] FIG. 3. Circulating IGF-I and IGFBP3 are altered in
long-standing T1D and its manipulation in vitro induces profound
effects on CoSC growth and self-renewal. A. Heat map represents the
proteomic profile in long-standing T1D (T1D+ESRD) as compared to
healthy subjects (CTRL). The complete dataset of identified and
quantified proteins was subjected to statistical analysis
(p<0.01). Significantly differentially expressed proteins were
further analyzed through hierarchical clustering. Sera of n=10 CTRL
and n=10 T1D+ESRD individuals were analyzed. B. Bar graph depicts
LFQ intensity for a single protein extrapolated from the untargeted
proteomic analysis, insulin-like growth factor binding protein 3
(IGFBP3). C1-C2. Representative images (40.times. magnification) of
IGFBP3 expression in the liver. IGFBP3 is mildly and diffusely
expressed in the liver parenchyma from healthy subjects (C1), while
it is more zonally positive in long-standing diabetic individuals
(C2). D. Bar graph represents IGFBP3 levels measured by ELISA in
the supernatants of immortalized human hepatoma cell line (HuH-7)
cultured for 5 days at different glucose concentrations (35 mM:
high glucose; 20 mM: intermediate glucose; 5 mM: normal glucose).
Experiments were run in triplicate. E. Bar graph represents
insulin-like growth factor 1 (IGF-I) levels measured by ELISA in
serum of healthy subjects and long-standing T1D (T1D+ESRD). F.
Western blot analysis (cropped blots) confirmed IGF-IR and TMEM219
expression on the intestinal crypt surface. Evaluation of total
IGF-IR expression by WB includes the detection of IGF-IRu, a
subunit of IGF-IR whole protein. Representative pictures of TMEM219
in situ hybridization (G1 negative control, G2 TMEM219 staining)
performed on rectal mucosa biopsy samples obtained from CTRL.
20.times. magnification. G1-G2. Representative pictures of TMEM219
in situ hybridization (G1 negative control, G2 TMEM219 staining)
performed on rectal mucosa biopsy samples obtained from CTRL.
Magnification 400.times.. H. Bar graph depicts normalized mRNA
expression of TMEM219 (IGFBP3 receptor) using the .DELTA..DELTA.Ct
method. N=5 subjects per group were evaluated. I. Bar graph
grouping % of developed mini-guts of the total obtained from
long-standing T1D individuals in different conditions and showing
the effect of IGF-I, IGFBP3 and anti-IGF-IR. The p values are
relative to baseline conditions and addition of IGF-I to culture.
J. Bar graph representing normalized mRNA expression of Caspase 8
and 9 in crypts isolated from healthy subjects cultured in the
presence of IGFBP3 and IGF-I+IGFBP3, performed in triplicate. K.
Bar graph grouping % of developed mini-guts of the total at 8 days
of culture, obtained from healthy subjects and cultured in the
presence of a Pan-Caspase inhibitor, selective inhibitors of
Caspase 8, 9 and 3, and IGFBP3. Assay was performed in triplicate.
L. Bar graphs grouping % of developed mini-guts of the total
obtained from healthy subjects and cultured in different conditions
(normal glucose+normal serum, high glucose+normal serum, T1D+ESRD
serum+normal glucose, T1D+ESRD serum+high glucose) and showing the
effect of IGF-I, IGFBP3 and anti-IGF-IR. The p values are relative
to baseline condition (medium alone, medium+high glucose,
medium+long-standing T1D serum, high glucose+long-standing T1D
serum). Additional p values have been calculated to compare the
difference in mini-gut growth among the following conditions:
medium alone vs. medium+high glucose, vs. medium+high
glucose+long-standing T1D serum). Assay was performed in
triplicate. M. Bar graph grouping % of developed mini-guts of the
total obtained from healthy subjects, cultured for 8 days, exposed
to TMEM219 targeting with siRNA and finally compared to
TMEM219-expressing crypts in medium alone and in medium+high
glucose+long-standing T1D serum. Assay was performed in triplicate.
Data are expressed as mean.+-.standard error of the mean (SEM)
unless differently reported. *p<0.01; **p<0.001;
***p<0.0001. Abbreviations: IGF-I, insulin-like growth factor 1;
IGFBP3, insulin-like growth factor binding protein 3; IGF-IR,
insulin-like growth factor 1 receptor; CoSC, colonic stem cell;
T1D, type 1 diabetes; ESRD, end stage renal disease; CTRL, healthy
subjects; RT-PCR, real-time polymerase chain reaction; ACTB, beta
actin; LFQ, Label-free quantitation; SEM, standard error of the
mean; siRNA, small RNA interference; inhib, inhibitor.
[0127] FIG. 4. Effects of the peripheral IGF-I/IGFBP3 dyad on
single-cell derived in vitro mini-guts and on caspase cascade.
Manipulating the peripheral IGF-I/IGFBP3 dyad alters the
progression of diabetic enteropathy in a preclinical model of
diabetic enteropathy, while the treatment of long-standing T1D with
simultaneous pancreas-kidney transplantation (SPK) ameliorates
intestinal symptoms, motility and morphology. A. Bar graph
representing normalized mRNA expression of TMEM219, LRP1,
TGF-.beta. type I and II, in EphB2.sup.+ sorted single cells
obtained from crypts of healthy subjects. Experiments were
performed in triplicate. B. Bar graphs showing % of developed
single cell-derived mini-guts (of the total) obtained from
EphB2.sup.+ cells sorted from freshly isolated crypts of healthy
subjects and cultured in different conditions (normal
glucose+normal serum, high glucose+normal serum, T1D+ESRD
serum+normal glucose, T1D+ESRD serum+high glucose) and showing the
effect of IGF-I and IGFBP3. The p values are relative to baseline
condition. C, D. Scatter plot representing the apoptosis
transcriptome profiling examined in freshly isolated intestinal
crypts of healthy subjects (CTRL) and long-standing T1D individuals
(T1D+ESRD) cultured with/without IGFBP3 and IGF-I. Experiments were
run in triplicate. E. Schematic attempt to represent the effect of
circulating IGF-I and IGFBP3 on the CoSCs. F, G, I. Line graphs
reporting the number of crypts (B), depth of crypts (C) and width
of crypts (E) assessed on intestinal lower tract sections harvested
at baseline and after 8 weeks from STZ-treated B6 mice developing
diabetic enteropathy (B6+STZ), naive B6 (WT), and naive B6 treated
with IGFBP3 (WT+IGFBP3). WT: wild type, STZ:
streptozoticin-treated. N=3 mice per group were evaluated. H1-H3.
Representative images of intestinal crypts on H&E sections of
WT, B6+STZ mice developing diabetic enteropathy, and naive B6
treated with IGFBP3 (WT+IGFBP3). Histology magnification,
400.times.. J. Bar graph representing the number of Aldh.sup.+
cells/mm.sup.2 in immunostained sections of STZ-treated B6 mice
developing diabetic enteropathy, WT, and naive B6 treated with
IGFBP3 (WT+IGFBP3). K1-K3. Representative images of Aldh.sup.+
cells on immunostained sections of intestinal lower tract harvested
from STZ-treated B6 mice developing diabetic enteropathy, WT, and
naive B6 treated with IGFBP3 (WT+IGFBP3). Histology magnification,
400.times.. L, N, P. Bar graphs report the measurement of
MIB1.sup.+ and Aldh.sup.+ cells, and EphB2.sup.+ expression
(intensity score 0-5) in the four groups of subjects (n=20 CTRL,
n=30 SPK, n=K+T1D and n=60 T1D+ESRD). M1-M2, O1-O2, Q1-Q2.
Representative images of MIB1.sup.+ and Aldh.sup.+ cells, and
EphB2.sup.+ expression in immunostained rectal mucosa bioptic
samples of T1D+ESRD who underwent kidney alone (K+T1D) or
simultaneous pancreas-kidney (SPK) transplantation at 8 years of
follow-up. Histology 400.times. in M1-M2 and O1-O2, 20.times. in
Q1-Q2. Scale bar 80 micron. Data are expressed as mean.+-.standard
error of the mean (SEM) unless differently reported. *p<0.01;
**p<0.001; ***p<0.0001. Abbreviations: WT, wild type; STZ,
streptozoticin-treated; B6, C57BL/6J mice; IGF-I, insulin-like
growth factor 1; IGFBP3, insulin-like growth factor binding protein
3; IGF-IR, insulin-like growth factor 1 receptor; CoSC, colonic
stem cell; T1D, type 1 diabetes; ESRD, end stage renal disease;
CTRL, healthy subjects; SPK, simultaneous kidney-pancreas
transplantation; K+T1D, kidney transplantation alone in type 1
diabetes; H&E, hematoxylin and eosin; MIB1, antibody against
Ki67; EphB2, Ephrin B receptor 2; Aldh, Aldehyde dehydrogenase;
SEM, standard error of the mean.
[0128] FIG. 5. Treatment of long-standing T1D with SPK replenishes
CoSCs and restores the CoSC signature profile and mini-gut
development through restoration of circulating IGF-I and IGFBP3. A,
B, C. Bar graphs depict results of flow cytometric analysis of
EphB2.sup.hi+, EphB2.sup.hi+LGR5.sup.+, EphB2.sup.+h-TERT.sup.+
cells obtained from isolated crypts in long-standing T1D
(Baseline), T1D+ESRD who underwent kidney pancreas (SPK) or kidney
alone (K+T1D) transplantation at 8 years of follow-up. N=10
subjects per group were evaluated. D, E, F. Bar graphs depict
normalized mRNA expression of intestinal stem cell markers EphB2,
LGR5, h-TERT, measured by quantitative RT-PCR on isolated
intestinal crypts obtained from long-standing T1D (Baseline),
T1D+ESRD who underwent kidney pancreas (SPK) or kidney alone
(K+T1D) transplantation at 8 years of follow-up. All samples were
run in triplicate and normalized to expression of the housekeeping
gene ACTB using the .DELTA..DELTA.Ct method. N=10 subjects per
group were evaluated. G. Western blot analysis depicts the
expression of EphB2, LGR5, h-TERT in isolated intestinal crypts of
the four groups at 8 years of follow-up. N=5 subjects per group
were evaluated. H. Bar graph depicts the % of developed mini-guts
of the total at 8 days of culture of freshly isolated intestinal
crypts obtained from long-standing T1D individuals (Baseline), SPK
and K+T1D subjects at 8 years of follow-up. N=10 subjects per group
were evaluated. I. Heat map represents the CoSC signature marker
transcriptomic profiling examined in freshly isolated intestinal
crypts of CTRL, long-standing T1D individuals (T1D+ESRD), SPK and
K+T1D subjects at 8 years of follow-up. N=10 subjects per group
were evaluated. J. Bar graph represents IGF-I levels measured by
ELISA in serum of the four groups of subjects at 8 years of
follow-up. N=10 subjects per group were evaluated. K. Bar graph
depicts IGFBP3 levels measured by ELISA in serum of the four groups
of subjects. N=20 subjects per group were evaluated. L, M
Correlation between IGFBP3 serum levels and intestinal symptoms
assessed using the GSRS questionnaire (0-7) in n=20 subjects of
K+T1D (L) and SPK (M) group. Analysis was conducted using ANOVA
(p<0.05) in comparing all groups. Data are expressed as
mean.+-.standard error of the mean (SEM) unless differently
reported. *p<0.01; **p<0.001; ***p<0.0001. Abbreviations:
CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stage
renal disease; CTRL, healthy subjects; SPK, simultaneous
kidney-pancreas transplantation; EphB2, Ephrin B receptor 2; LGR5,
leucine-rich repeat containing G protein-coupled receptor 5;
RT-PCR, real-time polymerase chain reaction; ACTB, beta actin;
K+T1D, kidney transplantation alone in type 1 diabetes; IGF-I,
insulin-like growth factor 1; IGFBP3, insulin-like growth factor
binding protein 3; SEM, standard error of the mean.
[0129] FIG. 6. Treatment with the newly generated recombinant
protein ecto-TMEM219 (ecto-TMEM219) abrogates IGFBP3-mediated
mini-gut destruction and preserves CoSCs in preclinical model. A.
Bar graph grouping % of developed mini-guts of the total obtained
from healthy subjects in different conditions and showing the
effect of ecto-TMEM219 at various concentrations (1:2, 1:1 and 2:1
molar ratio as compared to IGFBP3) in IGFBP3-treated mini-guts and
in those exposed to high glucose. The p values are relative to
baseline conditions. B. Bar graph representing normalized mRNA
expression of EphB2 in crypts isolated from healthy subjects
cultured in the presence of IGFBP3 and ecto-TMEM219+IGFBP3,
performed in triplicate. C. D. Bar graph representing normalized
mRNA expression of Caspase 8 and 9 in crypts isolated from healthy
subjects cultured in the presence of IGFBP3 and
ecto-TMEM219+IGFBP3, performed in triplicate. E, F, G. Line graphs
reporting the number of crypts (E), depth of crypts (F) and width
of crypts (G) assessed on intestinal lower tract sections harvested
at baseline and after 8 weeks from STZ-treated B6 mice developing
diabetic enteropathy (B6+STZ), naive B6 (WT), and STZ-B6 mice
treated with ecto-TMEM219. WT: wild type, STZ:
streptozoticin-treated. N=3 mice per group were evaluated. H. Line
graph reporting the weight at baseline and after 8 weeks of
STZ-treated B6 mice developing diabetic enteropathy (B6+STZ), naive
B6 (WT), and of STZ-treated B6 mice developing diabetic enteropathy
treated with ecto-TMEM219. WT: wild type, STZ:
streptozoticin-treated. N=3 mice per group were evaluated. I. Bar
graph representing results of flow cytometric analysis of
EphB2.sup.+ cells isolated from intestinal samples collected from
naive B6 mice, STZ-treated B6 mice and in STZ-B6 mice treated with
ecto-TMEM219 at 8 weeks. J. Representative flow histograms of
EphB2.sup.+ cells isolated from crypts isolated from naive B6 mice,
STZ-treated B6 mice and in STZ-B6 mice treated with ecto-TMEM219 at
8 weeks. N=3 to 5 mice per group were evaluated. K. Bar graph
representing normalized mRNA expression of EphB2 in intestinal
samples collected from naive B6 mice, STZ-treated B6 mice and in
STZ-B6 mice treated with ecto-TMEM219 at 8 weeks. L, M. Bar graph
representing normalized mRNA expression of Caspase 8 (K) and
Caspase 9 (L) in intestinal samples collected from naive B6 mice,
STZ-treated B6 mice and in STZ-B6 mice treated with ecto-TMEM219 at
8 weeks. N. Bar graph representing IGFBP3 circulating levels
measured in naive B6 mice (WT) and STZ-treated B6 mice (B6+STZ) and
in B6+STZ mice treated with ecto-TMEM219 at 8 weeks. Data are
expressed as mean.+-.standard error of the mean (SEM) unless
differently reported. *p<0.01; **p<0.001; ***p<0.0001.
Abbreviations: WT, wild type; STZ, streptozoticin-treated; B6,
C57BL/6J mice; IGF-I, insulin-like growth factor 1; IGFBP3,
insulin-like growth factor binding protein 3; CoSC, colonic stem
cell; H&E, hematoxylin and eosin; EphB2, Ephrin B receptor 2;
SEM, standard error of the mean, T1D, type 1 diabetes; ESRD, end
stage renal disease; CTRL, healthy subjects; RT-PCR, real-time
polymerase chain reaction; ACTB, beta actin.
[0130] FIG. 7. Assessment of IGFBP3 levels in serum (A) and urine
(B) of CTRL, T1D and T1D+ESRD individuals. (C) Correlation between
serum and urine IGFBP3 levels in all subjects of the cohort
evaluated for this study. (D-E) Correlation between IGFBP3 serum
levels and eGFR calculated with MDRD formula in subjects with
T1D+ESRD on dialysis (D) and with T1D with eGFR>15 ml/min/m2
(E). (F) Correlation between serum and urine IGFBP3 levels in all
subjects of the cohort evaluated for this study. The gray area
indicates the normal range within urinary and serum levels of
IGFBP3.
[0131] FIG. 8. CoSC profile, in vitro generation of mini-guts,
expression of IGFBP3 in the liver and of IGF-IR on CoSCs in
long-standing T1D and healthy subjects. A-B. Representative flow
dot plots of PI.sup.- cells gating strategy in healthy subjects
(CTRL) and long-standing T1D individuals (T1D+ESRD). C. Bar graphs
depict results of flow cytometric analysis of PI.sup.- cells in
freshly isolated crypts (n=10 CTRL and n=10 T1D+ESRD). D-E.
Representative flow dot plots of EphB2.sup.hiLGR5.sup.+ (D) and
EphB2.sup.+h-TERT.sup.+ cells in healthy subjects (CTRL) and
long-standing T1D individuals (T1D+ESRD). F. Western blot analysis
(cropped blots) confirms low expression of EphB2, LGR5, h-TERT in
in vitro isolated intestinal crypts of long-standing T1D
individuals (T1D+ESRD). Full-length blots are presented in FIG. 5.
N=5 subjects per group were evaluated. G. Scatter plot representing
the stem cell transcriptome profiling examined in freshly isolated
intestinal crypts of healthy subjects (CTRL) and long-standing T1D
individuals (T1D+ESRD). A table summarizes genes and pathways
analyzed (Table S1). N=10 subjects per group were evaluated. H-I.
Representative images of freshly isolated crypts obtained from
healthy subjects and long-standing T1D individuals stained with
DAPI. 20.times. magnification. J. Bar graph representing percentage
of mini-guts forming efficiency of plated crypts obtained from
healthy subjects and long-standing T1D individuals at 12 hours.
N=10 subjects per group were evaluated. K. Bar graph representing
the calculated combined score of IGFBP3 intensity/diffusion (0-6)
upon immunohistochemical evaluation in liver samples obtained from
healthy subjects and long-standing T1D individuals. N=3 subjects
per group were evaluated. L1-L6. Representative images (63.times.
magnification) of IGFBP3 expression in the liver.
Immunofluorescence confirmed the colocalization of Hep Par-1.sup.+
cells and IGFBP3 expression (L1-L3), while no colocalization was
observed between IGFBP3 and CD163.sup.+ cells (L4-L6). M. Bar graph
depicts normalized mRNA expression of the IGF-I receptor (IGF-IR)
measured by quantitative RT-PCR on isolated intestinal crypts. All
samples were run in triplicate and normalized to the housekeeping
gene ACTB using the .DELTA..DELTA.Ct method. N1-N2. Representative
pictures of IGF-IR.sup.+ cells on rectal mucosa samples obtained
from CTRL and from T1D+ESRD individuals. Black arrow indicates
positive cells at the crypt base. Magnification 200.times.. O1-O2.
Representative pictures of TMEM219 in situ hybridization performed
on rectal mucosa biopsy samples obtained from CTRL and from
T1D+ESRD individuals. Magnification 400.times.. Data are expressed
as mean.+-.standard error of the mean (SEM) unless differently
reported. *p<0.01. Abbreviations: PI, propidium iodide; IGF-I,
insulin-like growth factor 1; IGFBP3, insulin-like growth factor
binding protein 3; IGF-IR, insulin-like growth factor 1 receptor;
CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stage
renal disease; CTRL, healthy subjects; EphB2, Ephrin B receptor 2;
LGR5, leucine-rich repeat containing G protein-coupled receptor 5;
RT-PCR, real-time polymerase chain reaction; ACTB, beta actin; SEM,
standard error of the mean.
[0132] FIG. 9. Caspases expression in IGF-I/IGFBP3 cultured
mini-guts and the lack of effect of other circulating factors
confirmed IGFBP3 major pro-apoptotic effect on mini-guts
development. A. Bar graph representing normalized mRNA expression
of Caspase 8 in crypts isolated from individuals with T1D+ESRD
cultured in the presence of IGFBP3, IGF-I+IGFBP3 and IGF-I,
performed in triplicate. B. Bar graph representing normalized mRNA
expression of Caspase 9 in crypts isolated from individuals with
T1D+ESRD cultured in the presence of IGFBP3, IGF-I+IGFBP3 and
IGF-I, performed in triplicate. C, D. Bar graph grouping % of
mini-guts developed from healthy subjects (C) and from
long-standing T1D individuals (D), cultured in the presence of
medium with FBS and medium with serum obtained from healthy
subjects, "CTRL serum". Assay was run in triplicate. E. Bar graph
grouping % of developed mini-guts of the total obtained from
healthy subjects, cultured for 8 days, exposed to TMEM219 targeting
with siRNA and anti-IGF-IR, and finally compared to
TMEM219-expressing crypts in medium alone and in medium+high
glucose+long-standing T1D serum. Assay was performed in triplicate.
F, G. Bar graph grouping % of developed mini-guts at 8 days of
culture, obtained from healthy subjects (F) and long-standing T1D
individuals (G) cultured in the presence of medium alone and
various molecules identified with proteomic analysis (Table S7).
Assay was performed in triplicate. H. Bar graph grouping % of
mini-guts obtained from healthy subjects and cultured in the
presence of medium alone, medium+high glucose, medium+high glucose
and long-standing T1D serum, IGF-I, IGFBP3 with/without insulin.
Assay was performed in triplicate. Data are expressed as
mean.+-.standard error of the mean (SEM) unless differently
reported. *p<0.01; **p<0.001. Abbreviations: IGF-I,
insulin-like growth factor 1; IGFBP3, insulin-like growth factor
binding protein 3; IGF-IR, insulin-like growth factor 1 receptor;
CoSC, colonic stem cell; T1D, type 1 diabetes; ESRD, end stage
renal disease; CTRL, healthy subjects; RT-PCR, real-time polymerase
chain reaction; ACTB, beta actin; SEM, standard error of the mean;
siRNA, small RNA interference; ALDOA, Fructose-bisphosphate
aldolase A; RNASE, Ribonuclease pancreatic; MASP, Mannan-binding
lectin serine protease 1.
[0133] FIG. 10. Effect of IGF-I/IGFBP3 dyad on single cell derived
mini-guts, on stem cell transcriptome profile and on apoptotic
pathways. A1-A3. Representative images of single cell-derived
mini-guts, cultured for 8 days in vitro obtained from previously
isolated EphB2.sup.+ sorted cells of healthy subjects and cultured
with medium alone, medium+IGFBP3, medium+Glucose 35
mM+long-standing T1D serum. Images are shown at 10.times.
magnification. Scale bar 50 micron. B, C, D. Bar graph representing
normalized mRNA expression of Caspase 8, Caspse 9 and Ki67 in
single cell-derived mini-guts grown from flow sorted EphB2.sup.+
cells isolated from healthy subjects and cultured in different
conditions. Assay was performed in triplicate. E, F. Scatter plot
representing the stem cell transcriptome profiling examined in
freshly isolated intestinal crypts of healthy subjects (CTRL) and
long-standing T1D individuals (T1D+ESRD) cultured with/without
IGFBP3 and IGF-I. Assays were run in triplicate. G, H. Scatter plot
representing the apoptosis transcriptome profiling examined in
freshly isolated intestinal crypts of healthy subjects (CTRL) and
long-standing T1D individuals (T1D+ESRD) cultured with/without
IGF-I. A table summarizes genes and pathways analyzed (Table S3).
Assays were run in triplicate. I, J. Bar graph grouping % of
mini-guts developed from crypts obtained from healthy subjects (I)
and long-standing T1D (J) and then cultured in the presence of
medium alone, Fas Ligand (FasL), hydrogen peroxide (H.sub.2O.sub.2)
and Tumor Necrosis Factor alpha (TNF-.alpha.). Assay was performed
in triplicate. Data are expressed as mean.+-.standard error of the
mean (SEM) unless differently reported. *p<0.01; **p<0.001;
***p<0.0001. Abbreviations: IGF-I, insulin-like growth factor 1;
IGFBP3, insulin-like growth factor binding protein 3; CoSC, colonic
stem cell; T1D, type 1 diabetes; ESRD, end stage renal disease;
CTRL, healthy subjects; RT-PCR, real-time polymerase chain
reaction; ACTB, beta actin; SEM, standard error of the mean; FasL,
Fas Ligand; H.sub.2O.sub.2, hydrogen peroxide; TNF-.alpha., Tumor
Necrosis Factor alpha.
[0134] FIG. 11. Manipulating IGF-I/IGFBP3 dyad in preclinical
models of diabetic enteropathy. A. Bar graph representing IGFPB3
circulating levels measured in naive B6 mice (WT) and STZ-treated
B6 mice (B6+STZ). B. Bar graph representing IGF-I circulating
levels measured in naive B6 mice (WT) and STZ-treated B6 mice
(B6+STZ). C. Bar graph representing insulin serum levels measured
in naive B6 mice (WT) and STZ-treated B6 mice (B6+STZ). D, E, F:
Line graphs reporting the number of crypts (D), depth of crypts (E)
and width of crypts (F) assessed on intestinal lower tract sections
harvested at baseline and after 8 weeks from STZ-treated B6 mice
developing diabetic enteropathy (B6+STZ), naive B6 (WT), and STZ-B6
mice treated with IGFBP3 (B6+STZ+IGFBP3) or with IGF-I
(B6+STZ+IGF-I). WT: wild type, STZ: streptozoticin-treated. N=3
mice per group were evaluated. G. Bar graph representing the number
of Aldh.sup.+ cells/mm.sup.2 in immunostained sections of
STZ-treated B6 mice developing diabetic enteropathy, WT, and STZ-B6
mice treated with IGFBP3 (B6+STZ+IGFBP3) or with IGF-I
(B6+STZ+IGF-I). H1-H2: Representative images of intestinal crypts
on H&E sections of STZ-B6 mice treated with IGFBP3
(B6+STZ+IGFBP3), (H1) or with IGF-I (B6+STZ+IGF-I), (H2). Histology
magnification, 400.times.. I. Line graph reporting the weight of
STZ-treated B6 mice developing diabetic enteropathy (B6+STZ), naive
B6 (WT), STZ-treated B6 mice developing diabetic enteropathy
treated with IGFBP3 (B6+STZ+IGFBP3). WT: wild type, STZ:
streptozoticin-treated. N=3 mice per group were evaluated. J. Bar
graph representing results of flow cytometric analysis of
EphB2.sup.+ cells in intestinal samples collected from naive B6
mice, STZ-treated B6 mice and in STZ-B6 mice treated with IGFBP3
(B6+STZ+IGFBP3). K, L. Bar graph representing normalized mRNA
expression of EphB2 (K) and LGR5 (L) in intestinal samples
collected from naive B6 mice, STZ-treated B6 mice and in STZ-B6
mice treated with IGFBP3 (B6+STZ+IGFBP3). M, N. Bar graph
representing normalized mRNA expression of Caspase 8 (M) and
Caspase 9 (N) in intestinal samples collected from naive B6 mice,
STZ-treated B6 mice and in STZ-B6 mice treated with IGFBP3
(B6+STZ+IGFBP3). Data are expressed as mean.+-.standard error of
the mean (SEM) unless differently reported. *p<0.01;
**p<0.001; ***p<0.0001. Abbreviations: WT, wild type; STZ,
streptozoticin-treated; B6, C57BL/6J mice; IGF-I, insulin-like
growth factor 1; IGFBP3, insulin-like growth factor binding protein
3; CoSC, colonic stem cell; H&E, hematoxylin and eosin; EphB2,
Ephrin B receptor 2; Aldh, Aldehyde dehydrogenase; SEM, standard
error of the mean.
[0135] FIG. 12. The treatment of long-standing T1D with SPK
ameliorates diabetic enteropathy. A, B, C. Bar graphs depict the
score of abdominal pain, diarrhea and constipation according to the
GSRS questionnaire in healthy subjects (CTRL), long-standing T1D
individuals (Baseline), T1D+ESRD who underwent kidney pancreas
(SPK) or kidney alone (K+T1D) transplantation. Gray area indicates
normal range for all the parameters. Statistics are expressed as
mean.+-.SEM. D1-D2, E1-E2, G1-G2, J1-J2. Representative pictures of
hematoxylin and eosin (H&E) staining and ultrastructural
analysis of neural structures (red arrows indicate localization and
presence of neuroendocrine vesicles), Schwann cells (red arrows
indicate cytoplasm derangements), and 5HT.sup.+ cells performed on
rectal mucosa biopsy samples obtained from T1D+ESRD who underwent
kidney pancreas (SPK) or kidney alone (K+T1D) transplantation at 8
years of follow-up. Magnification 400.times.. F, H, I, K. Bar
graphs report the measurements of neuroendocrine vesicles (% of
cases with >3 NE vesicles detected per nerve terminal), % of
Schwann cells with picnotic nuclei and cytoplasm derangements (% of
positive cases) using electron microscopy, 5HT.sup.+ cells,
performed on bioptic samples obtained from rectal mucosa of CTRL,
long-standing T1D individuals (Baseline), T1D+ESRD who underwent
kidney pancreas (SPK) or kidney alone (K+T1D) over an 8-year
follow-up period. Statistics are expressed as mean.+-.SEM. N=20
CTRL, n=30 SPK, n=30 K+T1D and n=60 T1D+ESRD subjects were
evaluated. Statistics are expressed as mean.+-.SEM. All parameters
examined were statistically significantly different when comparing
different groups as following: *p<0.01; **p<0.001;
***p<0.0001. N=10 subjects per group were evaluated.
Abbreviations: GSRS, Gastrointestinal Symptom Rating Scale; SPK,
simultaneous kidney-pancreas transplantation; K+T1D, kidney
transplantation alone in type 1 diabetes; CTRL, healthy subjects;
T1D, type 1 diabetes; ESRD, end stage renal disease; 5HT,
serotonin; H&E, hematoxylin and eosin; NGF, neural growth
factor; SEM, standard error of the mean; NE, neuroendocrine
vesicles.
[0136] FIG. 13. Analysis of colonic stem cells, IGF-IR and
proteomic profile of circulating factors in diabetic enteropathy in
SPK and K+T1D groups. A1-A6. Representative images of mini-guts,
cultured for 8 days in vitro obtained from previously isolated
crypts of long-standing T1D individuals, T1D+ESRD who underwent
kidney pancreas (SPK) or kidney alone (K+T1D) transplantation at 8
years of follow-up. Images are shown at 5.times. and 10.times.
magnification. Scale bar 10 micron. B. Scatter plot representing
the stem cell transcriptome profiling examined in freshly isolated
intestinal crypts of SPK individuals. N=3 subjects were evaluated.
C. Bar graphs depict relative expression levels of IGF-I receptor
(IGF-IR) on isolated crypts of healthy subjects (CTRL),
long-standing T1D individuals (T1D+ESRD), SPK and K+T1D measured by
quantitative RT-PCR. All samples were run in triplicate and
normalized to the ACTB relative expression level using the
.DELTA..DELTA.Ct method. Results are expressed as mean.+-.SEM. D.
Heat map represents the proteomic profile of long-standing T1D as
compared to CTRL and SPK subjects at 8 years of follow-up. The
complete dataset of identified and quantified proteins was
subjected to statistical analysis (p<0.05). Significantly
differentially expressed proteins were further analyzed through
hierarchical clustering. Statistics are expressed as mean.+-.SEM.
Sera of n=10 subjects per group were evaluated. All parameters
examined were statistically significantly different when comparing
different groups as following: *p<0.01. Abbreviations: T1D, type
1 diabetes; ESRD, end stage renal disease; CTRL, healthy subjects;
SPK, simultaneous kidney-pancreas transplantation; K+T1D, kidney
transplantation alone in type 1 diabetes; RT-PCR, real-time
polymerase chain reaction; ACTB, beta actin; IGF-I, insulin-like
growth factor 1; IGFBP3, insulin-like growth factor binding protein
3; IGF-IR, insulin-like growth factor 1 receptor; SEM, standard
error of mean.
[0137] FIG. 14. Correlation of intestinal symptoms with levels of
insulin, HbA1C and blood glucose in SPK and K+T1D groups. A, B.
Correlation between insulin serum levels and intestinal symptoms
assessed using the GSRS questionnaire and considering the item with
the highest score (0-7) in n=20 subjects of K+T1D (A) and SPK (B)
group. Analysis was conducted using ANOVA (p<0.05) in comparing
all groups. C. Insulin serum levels measured using the Free-insulin
method in n=20 subjects of K+T1D (A) and SPK (B) group. Data are
expressed as mean.+-.standard error of the mean (SEM). D, E.
Correlation between glycated hemoglobin (HbA1C) serum levels and
intestinal symptoms assessed using the GSRS questionnaire (0-7) in
n=20 subjects of K+T1D (A) and SPK (B) group. Analysis was
conducted using ANOVA (p<0.05) in comparing all groups. F, G.
Correlation between blood glucose levels (Glycemia) and intestinal
symptoms assessed using the GSRS questionnaire (0-7) in n=20
subjects of K+T1D (A) and SPK (B) group. Analysis was conducted
using ANOVA (p<0.05) in comparing all groups. Abbreviations:
T1D, type 1 diabetes; ESRD, end stage renal disease; CTRL, healthy
subjects; SPK, simultaneous kidney-pancreas transplantation; K+T1D,
kidney transplantation alone in type 1 diabetes; IGF-I,
insulin-like growth factor 1; IGFBP3, insulin-like growth factor
binding protein 3.
[0138] FIG. 15. Expression of cell lineages markers in mini-guts
exposed to different culturing conditions. A1-A4, B1-B4, C1-C4,
D1-D4, E1-E4. Representative images (O1.times. magnification) of
citokeratin 20 (KRT20), vimentin, Synaptofisin and Aldehyde
Dehydrogenase (ALDH) expression in mini-guts obtained from crypts
isolated from healthy subjects, CTRL (A1-A4), and T1D+ESRD
individuals (B1-B4), cultured with IGFBP3 (C1-C4), Glucose 35 mM
(D1-D4), and Glucose 35 mM)+long-standing T1D serum (T1D+ESRD
serum)+IGF-I (E1-E4). Immunofluorescence confirmed that expression
of all lineages markers is reduced in mini-guts obtained from
T1D+ESRD individuals as compared to CTRL (A1-A4, B1-B4), with ALDH
being the least expressed marker (B4). Decreased ALDH expression
was also detected in IGFBP3-treated mini-guts (C4), while mini-guts
exposed to high glucose and long-standing T1D serum and treated
with IGF-I showed evident ALDH expression recovery. F. Bar graph
representing expression of TMEM219, KRT20, Epithelial-cell adhesion
molecule (EpCam) and Chromogranin A (CHGA) on non-stem cells
(EphB2.sup.- cells) measured by quantitative RT-PCR. All samples
were run in triplicate and normalized to the ACTB relative
expression level using the .DELTA..DELTA.Ct method. Results are
expressed as mean.+-.SEM. Abbreviations: T1D, type 1 diabetes;
ESRD, end stage renal disease; CTRL, healthy subjects; IGF-I,
insulin-like growth factor 1; IGFBP3, insulin-like growth factor
binding protein 3; IF, immunofluorescence; KRT20, citokeratin 20,
ALDH, Aldehyde Dehydrogenase, EpCam, epithelial cell adhesion
molecule; CHGA, Chromogranin A; RT-PCR, real-time polymerase chain
reaction; ACTB, beta actin.
[0139] FIG. 16. Selection strategy to test candidate proteins in in
vitro mini-guts assay. Flow chart depicting the strategy used to
select protein candidates based on proteomic profile to be tested
in in vitro mini-guts assay.
[0140] FIG. 17. Analysis of developed mini-guts using the crypt
domain quantitative criteria. A-P. Bar graphs grouping % of
developed mini-guts with at least 1 crypt domain detectable in
different conditions already reported throughout the paper.
[0141] FIG. 18. Peripheral IGFBP3 levels are increased in
individuals with inflammatory bowel disease as compared to healthy
subjects.
[0142] FIG. 19. IGFBP3 peripheral levels are increased in
pre-diabetic and diabetic conditions in T1D (A) and T2D human
subjects (B). *p<0.05, ** p<0.01, *** p<0.001.
Abbreviations: IGFBP3, insulin-like growth factor binding protein
3; CTRL, healthy subjects; T1D, type 1 diabetes; T2D, type 2
diabetes; AutoAb positive: non diabetic subjects at risk for
developing T1D with detected positivity of Antibodies against
islets peptides; IGT: impaired glucose tolerance measured at the
OGTT (oral glucose tolerance test) in fasting and non-fasting
condition. NGT: normal glucose tolerance measured at the OGTT. IFG:
impaired fasting glucose tolerance measured at OGTT and resulting
positive only in fasting conditions.
[0143] FIG. 20. IGFBP3 peripheral levels increase in pre-diabetic
and diabetic conditions in murine models of T1D (A) and T2D (B).
*p<0.05, ** p<0.01, *** p<0.001. Abbreviations: C57BL6/J,
B6 mice; B6, naive mice; NOD, non-obese diabetic mice; HFD,
high-fat diet, IGFBP3, insulin-like growth factor binding protein
3.
[0144] FIG. 21. IGFBP3 production in primary human hepatocytes
increases during glucose exposure (11 mM, 20 mM, 35 mM) (A) and
inflammation (IFN.gamma. 1,000 U/ml, plus Il-1.beta. 2 ng/ml) (B).
*p<0.05, ** p<0.01, *** p<0.001. Abbreviations: INF,
inflammation (IFN.gamma.+Il-1.beta.); mM, millimolar; IGFBP3,
insulin-like growth factor binding protein 3.
[0145] FIG. 22. TMEM219 is expressed on human islets (A-C).
*p<0.05, ** p<0.01, *** p<0.001. Abbreviations:
.beta.-ACT, beta actin.
[0146] FIG. 23. TMEM219 is expressed on murine islets (A) and on a
murine beta cell line (B-D). *p<0.05, ** p<0.01, ***
p<0.001. Abbreviations: .beta.-TC, murine beta cell line;
.beta.-ACT, beta actin.
[0147] FIG. 24. IGFBP3 (50 ng/ml) increases apoptosis and caspase8
expression (A-B) and reduces insulin release and expression (C,
D1-D2, E) to a greater extent as compared to pro-inflammatory
stimuli (IFN.gamma.+Il-1.beta.) in a murine beta cell line in
vitro. *p<0.05, ** p<0.01, *** p<0.001. Abbreviations:
IFN.gamma., interferon gamma; IL-1.beta., interleukin beta; IGFBP3,
insulin-like growth factor binding protein 3; .beta.-TC, murine
beta cell line.
[0148] FIG. 25. IGFBP3 (50 ng/ml) increases apoptosis (A) and
caspase8 expression in murine islets (B with a reduction of insulin
(C) in vitro. *p<0.05, ** p<0.01, *** p<0.001.
[0149] FIG. 26. IGFBP3 (50 ng/ml) increases apoptosis and caspase8
expression in human islets (A-B and C1-C2) and reduces insulin
expression (D1-D2, E) in vitro. *p<0.05, ** p<0.01, ***
p<0.001. Abbreviations: Pi, Propidium Iodide; M30, monoclonal
antibody M30 that recognizes caspase-cleaved cytokeratin 18; INS,
insulin, IGFBP3, insulin-like growth factor binding protein 3.
[0150] FIG. 27. IGFBP3 injection (150 .mu.g/day for 15 days) in
C57BL/6 mice alters islet morphology in vivo after 8 weeks of
diabetes (A1-A6). Abbreviations: STZ, streptozotocin; B6, naive
C57BL6/J mice.
[0151] FIG. 28. Ecto-TMEM219 (130 ng/ml) prevents IGFBP3-associated
apoptotic effects on murine beta cell line (A-B, C1-C3).
*p<0.05, ** p<0.01, *** p<0.001. Abbreviations: 3-TC,
murine beta cell line; INS, insulin, IGFBP3, insulin-like growth
factor binding protein 3.
[0152] FIG. 29. Ecto-TMEM219 treatment (130 ng/ml) near-normalizes
casapse 8 and insulin expression in murine islets in vitro.
*p<0.05, ** p<0.01, *** p<0.001. Abbreviations: IGFBP3,
insulin-like growth factor binding protein 3.
[0153] FIG. 30. Ecto-TMEM219 (130 ng/ml) prevents IGFBP3-associated
apoptotic effects on human islets (A-B, C1-C3). *p<0.05, **
p<0.01, *** p<0.001. Abbreviations: M30, monoclonal antibody
M30 that recognizes caspase-cleaved cytokeratin 18; INS, insulin;
IGFBP3, insulin-like growth factor binding protein 3.
[0154] FIG. 31. Ecto-TMEM219 treatment (130 ng/ml) in diabetic mice
rescues serum insulin (A, C) and blood glucose levels (B).
*p<0.05, ** p<0.01, *** p<0.001.
[0155] FIG. 32. Working Hypothesis. Abbreviations: IGFBP3,
insulin-like growth factor binding protein 3; IGF-I, insulin-like
growth factor 1; IGF-IR, insulin-like growth factor 1 receptor.
DETAILED DESCRIPTION OF THE INVENTION
Example 1
Material and Methods
[0156] 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 (FIG. 15). Broad
serum proteomic was used to detect circulating factors that may
regulate CoSCs and candidate factors were then tested in the in
vitro mini-gut assay (FIG. 16). Detailed methods and statistical
analysis are described below. The Study was approved by the
Institutional Review Board of Istituto di Ricovero e Cura a
Carattere Scientifico Ospedale San Raffaele, Milano, Italy
(Enteropathy-Pancreas KidneyTransplantation/01 Secchi/Fiorina).
Patients and Study Design
[0157] 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 (29.4.+-.1.8
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.
[0158] T1D+ESRD individuals were followed up for 8 years (mean
follow-up: 8.6.+-.1.1 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
[0159] 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)
[0160] Gastrointestinal symptoms were evaluated by GSRS
questionnaire in healthy subjects, in long-standing 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
[0161] 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 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
[0162] 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 .mu.m-thick sections of each enrolled case were stained with
Hematoxylin & Eosin (H&E) for morphological evaluations.
For immunohistochemistry, 3 .mu.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 650 W 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.degree. C. for 18-20 hours, followed by the avidin-biotin complex
procedure (Hsu et al., 1981). Immunoreactions were developed using
0.03% 3,3'diaminobenzidine tetrahydrochloride, and then sections
were counterstained with Harris' hematoxylin. The following
antibodies were used: Ki67 (monoclonal, clone MIB1, 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-1R (polyclonal, 1:100, Cell
Signaling Technologies), (Fiorina et al., 2003). For
ultrastructural studies, samples were fixed for 2 hours at
4.degree. 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 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 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
[0163] 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, Jena,
Germany) with a 63.times. 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).
[0164] 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 Synaptofisin
for immunofluorescence analysis to assess expression of cell
lineages markers (FIG. 15: A1-A4, B1-B4, C1-C4, D1-D4, E1-E4). The
following primary antibodies were used: mouse vimentin (1:80,
monoclonal, clone: V9 Dako) mouse Aldheyde (1:1000, monoclonal,
clone: 44, BD), mouse citocherain 20 (1:100, monoclonal,
clone:Ks20.8, Dako) and Synaptofisin (1:100, monoclonal, clone:
syn88, BioGenex).
In Situ Hybridization
[0165] 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.degree. C. in proteinase K (30 .mu.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.degree. C. in hybridization
solution (50% formamide, 5.times.SSC, pH4.5, 2% Blocking Reagent
(Roche), 0.05% CHAPS (Sigma), 5 mM EDTA, 50 .mu.g/ml Heparin
(Sigma) and 50 .mu.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.degree. C.
Post-hybridization washes were performed 3.times.20 min in 50%
Formamide/2.times.SSC at 65.degree. C. Sections were rinsed in
TBS-T buffer (0.1M TrisHCl 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.degree. 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
[0166] 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.degree. 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 40.times.g 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).
[0167] 200-300 isolated human colonic crypt units were mixed with
50 .mu.l matrigel and plated on pre-warmed 24-well culture dishes
as already described. After solidification (15-20 minutes at
37.degree. C.), crypts were overlaid with 600 .mu.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 [1.times.], B-27 without retinoic acid
[1.times.], 10 mM Nicotinamide, 1 mM N-Acetyl-L-cysteine, 50 ng/ml
human EGF (Life Technologies, Grand Island, N.Y.), 1 .mu.g/ml RSPO1
(Sino Biological, Beijing, China), 100 ng/ml human Noggin
(Peprotech, Rocky Hill, N.J., USA), 1 .mu.g/ml Gastrin
(Sigma-Aldrich, St. Louis, Mo.), 500 nM LY2157299 (Axon MedChem,
Groningen, The Netherlands), 10 .mu.M SB202190 (Sigma) and 0.01
.mu.M PGE2 (Sigma)]. Medium was replaced every other day. Rock
inhibitor Y-27632 (10 VM, 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 compromized 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 (FIG. 17: A-P). Insulin and
glucose levels measured on long-standing T1D (T1D+ESRD) and CTRL
serum are reported below:
[0168] Glucose levels (T1D+ESRD vs. CTRL, 178.+-.47.5 vs 90.+-.5.5
mg/dl, p0.0001);
[0169] Insulin levels (T1D+ESRD vs. CTRL, 12.9.+-.4.6 vs 5.8.+-.1.6
.mu.IU/ml, p=0.009).
Flow Cytometry
[0170] 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 CD11b-positive cells (V450
anti-human CD45 and CD1 b, BD Biosciences, San Jose, Calif.).
Propidium iodide (PI) was added (10 .mu.g/ml) to exclude dead
cells. EphB2.sup.+ 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
[0171] 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.
[0172] 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/F12 medium in order to obtain the crypts culture
medium as previously described (see Crypt purification).
[0173] To assess the properties of sorted EphB2.sup.+ cells in
generating mini-guts, 2000 sorted cells were mixed with 50 .mu.l
matrigel and plated on pre-warmed 24-well culture dishes. After
solidification of the matrigel (10-15 min at 37.degree. 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
[0174] Total proteins of intestinal bioptic samples were extracted
in Laemmli buffer (Tris-HCl 62.5 mmol/l, pH 6.8, 20% glycerol, 2%
SDS, 5% .beta.-mercaptoethanol) and their concentration was
measured (Lowry et al., 1951). 35 .mu.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.degree. 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 TBS-5% milk at 4.degree. 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
[0175] 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 S100 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.degree. 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
[0176] 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.times. magnification.
Transcriptome Profiling
[0177] 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 .mu.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
[0178] 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 .DELTA..DELTA.Ct
method. Quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR) data were normalized for the expression of ACTB,
and .DELTA..DELTA.Ct 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-00001 TABLE I-B Primers Gene Refseq Band Size Reference
Symbol UniGene # Accession # (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
[0179] IGF-I and IGFBP3 levels in the pooled sera/palsma 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).
[0180] 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.
[0181] 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
[0182] 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, N.Y.) was administered to naive 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 .mu.g/mouse/day for 20 days and 100
.mu.g/mouse/day for 15 days respectively.
[0183] Other molecules tested in in vitro mini-guts assay and added
to crypt cultures at day +2 from isolation: Adiponectin (R&D),
Thymosin J34 (Abcam), C-reactive protein (Merck Millipore),
Cystatin C (Cell Signaling Technologies), Chromogranin A (Life
Technologies), Fructose-bisphosphate 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 (H2O2, 50 .mu.M) was also tested in the mini-guts
assay.
Generation of Recombinant Human Ecto TMEM219
[0184] Recombinant human ecto-TMEM219 was generated using E. coli
as expression host for synthesis. Briefly, gene sequence of
extracellular TMEM219 was obtained:
THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTLNFGDGPDR
NKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCLYFSAVPGILPSSQPPISC
SEEGAGNATLSPRMGEECVSVWSHEGLVLTKLLTSEELALCGSR (SEQ ID No. 2).
[0185] 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-PAGE 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.
[0186] 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
[0187] 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 72 h of culture, which allowed the crypts to
recover, 750 ng of small interfering RNA (siRNA; Flexitube siRNA
SI04381013, Qiagen, Valencia, Calif.) in 100 .mu.l culture medium
without serum and with 6 .mu.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 6 h. Analysis of gene silencing was performed at 24, 48 and 72
h 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
[0188] 8 .mu.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 .about.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 .mu.l
from each pool were processed as above described. 40 .mu.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 .mu.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 .mu.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-00002 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/CTF1; 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 [Cu--Zn] 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;
Lysyl-bradykinin; 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 266 Fibulin-1 267
Mannan-binding lectin serine protease 1 270 Complement factor
H-related protein 4
Strategy to Select Candidate Proteins
[0189] 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 (FIG. 16). Next, the inventors evaluated
whether altered factors may be associated with intestinal disorders
and/or with the development of diabetes by searching for already
reported studies and publications in the field. This led us to
exclude other 12 factors. The inventors also excluded those factors
mainly related to the lymphoid compartment (n=5). The inventors
ended up with 17 factors. The inventors excluded cell-membrane
proteins (n=4) and proceeded with testing the remaining (n=13) in
the mini-gut assay. Two factors were not available to be tested in
vitro. The inventors tested n=1 proteins in total.
Animal Studies
[0190] 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.degree. 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.degree. C. for
30 min. Crypts were then resuspended in DMEMF12/EDTA, filtered in
40 micron strainer twice and washed. 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-CD11b-PE
(BD Pharmingen). Samples were run using a FACSCalibur Analyzer and
data analyzed with FlowJo. Part of the tissue was also snap frozen
and stored in Tryzol to perform RT-PCR studies for the following
markers:
TABLE-US-00003 Gene Refseq Band Size Reference Symbol: UniGene #:
Accession #: (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
[0191] 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
[0192] 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 (.chi..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.RTM.,
IBM.RTM., 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 T1D
[0193] 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 (FIG. 1: A-C). Symptoms were associated with
abnormalities in anorectal sphincter function (FIG. 1: D-F). The
intestinal mucosa was altered in individuals with T1D+ESRD as
compared to healthy subjects, with lower number of crypts,
distortion and zonal sclerosis of the lamina propria (FIG. 1:
G1-G2, H). A significant reduction in epithelial cell proliferation
as assessed by Ki67 (MIB1 antibody) staining (FIG. 1: I1-I2, J),
signs of neural degeneration (FIG. 1: K1-K2, L) and reduction in
serotonin expression in intestinal neuroendocrine cells (FIG. 1:
M1-M2, N) were observed, confirming the presence of DE in these
individuals.
CoSCs are Altered in Long-Standing T1D
[0194] 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
(FIG. 1: O1-O2, P, Q1-Q2, R). A profound decrease was evident, upon
gating on PI.sup.- cells at FACS analysis (FIG. 8: A-C), in the
percentage of EphB2.sup.hi, EphB2.sup.hi+LGR5.sup.+ and
EphB2.sup.+h-TERT.sup.+ cells isolated from intestinal crypts
obtained from T1D+ESRD individuals as compared to healthy subjects
(FIG. 2: A-B, C-E, FIG. 8: D-E) and was confirmed by RT-PCR (FIG.
2: F-H) and western blot (WB) analysis (FIG. 8F). Transcriptome
profiling of crypts obtained from T1D+ESRD documented a decreased
expression of Notch pathway (Notch1 and 2, JAG1, Dll1, Sox1 and 2),
Wnt pathway (APC, FZD1, DKC1, ETS2, FAM84A, GPX2, RNF43) and BMP
pathway (BMP1, BMP2, BMP3) genes, previously known pathways that
control CoSCs, as compared to the expression of these genes in
healthy subjects (FIG. 8G and Table II).
TABLE-US-00004 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 FGFR1 GDF3 ISL1 KRT15 MSX1 MYOD1 T GJA1 GJB1 GJB2
KAT8 RB1 h-TERT NCAM1 SIGMAR1 TUBB3 ABCG2 ALDH1A1 PDX1 IGF-I DHH
BGLAP
[0195] 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 (FIG. 2I), confirming that the CoSCs are altered
in individuals with DE.
In Vitro Generation of Mini-Guts is Altered in Long-Standing
T1D
[0196] 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 (FIG. 2: J1, J2, K), despite a comparable viability (FIG.
8: H-I) and efficiency of forming mini-guts in both groups (FIG.
8J). To begin to elucidate the effect of circulating factors and
high glucose on CoSCs, the inventors cultured isolated intestinal
crypts obtained from healthy subjects in high glucose with/without
serum obtained from long-standing T1D individuals in vitro for 8
days (FIG. 2: L1-L4, M). High glucose partially prevented the
generation of fully mature mini-guts and synergized with serum of
long-standing T1D individuals in altering CoSC self-renewal
properties, such that mini-guts appeared collapsed (FIG. 2: L2-L4).
Analysis of gene expression also revealed changes in the CoSC
signature (FIG. 2N), thus suggesting that hyperglycemia and
circulating factors act together to alter CoSC regenerative
properties in long-standing T1D.
Serum Unbiased Proteomic Profiling Revealed Increased Levels of
IGFBP3 in Long-Standing T1D
[0197] 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
(FIG. 3A). Some proteins were associated with diabetes, and some
were growth factors or stem cell-related proteins or were
potentially involved in intestinal functions (FIG. 3A). In
particular, the levels of IGF-I binding proteins (IGFBP2 and 3)
were detectable in long-standing T1D individuals as compared to
healthy subjects, with IGFBP3 almost 5-fold increased (FIG. 3B),
while IGFBP1, 4, 5 and 6 remained almost undetectable.
Interestingly, in the liver of individuals with long-standing T1D,
hepatocytes, but not Kuppfer cells, showed a higher IGFBP3
immunohistochemical expression as compared to healthy subjects
(FIG. 3: C1-C2, FIG. 8: K, L1-L6), suggesting an increase in IGFBP3
hepatic synthesis and release. The effect of high glucose on IGFBP3
hepatic release was confirmed by the detection of increased IGFBP3
levels in the supernatant of human immortalized hepatocytes exposed
to high glucose (FIG. 3D). Finally, serum levels of free IGF-I
appeared significantly reduced in long-standing T1D as compared to
healthy subjects (FIG. 3E), indicating that circulating IGF-I and
IGFBP3 levels are altered in long-standing T1D.
Peripheral IGFBP3 and IGF-I Control CoSCs
[0198] 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 (FIG. 3: F, H,
FIG. 8: M, N1-N2) using RT-PCR and WB (FIG. 3: F, H, FIG. 8M), and
confirmed the expression of IGF-IR on CoSCs with immunostaining
(FIG. 8: N1-N2), and of TMEM219 with in situ hybridization (FIG. 3:
G1-G2). In order to mechanistically confirm the role of IGF-I and
IGFBP3 on CoSC, the inventors tested the effect of several
molecules, identified by proteomic profiling, in their in vitro
mini-gut assay. Inventors' strategy to select potential targets is
reported in Supplemental Information. The severely altered
mini-guts generated from intestinal samples obtained from T1D+ESRD
individuals were rescued by the addition of recombinant human IGF-I
(IGF-I) to the culture medium (FIG. 31), while the addition of
recombinant human IGFBP3 (IGFBP3) resulted in the abrogation of the
positive effect observed with IGF-I, with a decreased development
of mini-guts and increased formation of collapsed and distorted
organoids (FIG. 31). Because IGFBP3 has been recently shown to act
independently of IGF-I (Williams et al., 2007) via the IGFBP3
receptor (TMEM219)(Baxter, 2013), it was necessary to clarify
whether IGFBP3 exerts its effects on CoSCs by binding IGF-I or by
directly targeting TMEM219 on CoSCs. The inventors first confirmed
that IGFBP3 has a direct pro apoptotic effect on CoSCs by
demonstrating increased Caspase 8 and 9 expression in mini-guts
obtained from healthy subjects and long-standing T1D individuals
and cultured with IGFBP3 (FIG. 3J, FIG. 9: A-B), while the addition
of a Pan-Caspase inhibitor (Z-VAD-FMK) or the addition of both
selective inhibitors of caspases 8 and 9, but not that of other
caspase cascade inhibitors (Caspase 3 inhibitor) abrogated the
IGFBP3 effect (FIG. 3K). The inventors then demonstrated that the
addition of IGF-I did not rescue the development of mini-guts
obtained from healthy subjects and exposed to IGFBP3 (FIG. 3L),
confirming that IGFBP3 may act through both a direct and indirect
IGF-I mechanism. Interestingly, high glucose alone was unable to
completely disrupt mini-gut growth, and anti-IGF-IR did not worsen
growth and morphology of mini-guts formed from healthy subjects
(FIG. 3L). The addition of IGF-I to mini-guts generated from
healthy subjects, but cultured with high glucose and serum from
long-standing T1D individuals, rescued mini-gut morphology, while
IGFBP3 abolished the positive effect of IGF-I when added to the
mini-gut culture (FIG. 3L). Interestingly, the use of healthy
subjects "CTRL" serum in culturing crypts obtained from
long-standing T1D nearly restored mini-guts development/morphology,
indicating that circulating factors, and in particular IGF-I/IGFBP3
dyad, control CoSCs (FIG. 9: C-D). The inventors then genetically
modulated TMEM219 expression by using siRNA in vitro in mini-guts
obtained from healthy subjects. Knockdown of TMEM219 in mini-guts
preserved their ability to grow and self-renew, despite the
addition of IGFBP3 and high glucose with long-standing T1D serum
(FIG. 3M). Concomitant blockade of TMEM219 by SiRNA and IGF-IR by
blocking antibody did not result in any additional beneficial
effect on mini-guts development despite using serum from healthy
subjects or from long-standing T1D (FIG. 9E).
[0199] 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 (FIG. 9: F-G). C-peptide and insulin, whose levels
are commonly altered in T1D and which may interfere with
IGF-I/IGFBP3 dyad by binding IGF-IR (FIG. 9H), were tested as well
and did not show any effect.
[0200] 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 (FIG. 4A). The inventors then
cultured EphB2.sup.+ cells in the in vitro single cell-derived
mini-gut assay and confirmed that high glucose and long-standing
T1D serum exposure as well as addition of IGFBP3 significantly
abrogate single cell-derived mini-guts growth, thus recapitulating
the main features reported in their previous observations on
crypt-derived mini-guts (FIG. 4B, FIG. 10: A1-A3). 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 (FIG. 10: B-D).
Effect of the IGF-I/IGFBP3 Dyad on Previously Known Pathways that
Control CoSCs
[0201] 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 (FIG. 10E, Table III), while
IGFBP3 poorly affects Wnt/Notch/BMP gene expression in mini-guts
obtained from crypts of healthy subjects or from those of T1D+ESRD
(FIG. 10F, Table III).
TABLE-US-00005 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, CDH1,
COL9A1 ACAN, COL2A1, DLL1, FGF2, vs. FGF3, GDF3, GJA1, IGF-I, ISL1,
CTRL MME, MSX1, NCAM1, NOTCH2, PDX1, SOX1, SOX2, h-TERT CTRL +
IGFBP3 CD8B, COL9A1, RB1, SOX1, h-TERT ASCL2, COL2A1, DHH, DLL1,
vs. DTX1, DVL1, FGF3, FGF4, CTRL FOXA2, FRAT1, GDF2, HSPA9, IGF1,
KAT2A, MSX1, MYC, NEUROG2, S100B, WNT1 T1D + ESRD + IGF-I ACTC1,
CD3D, CD4, COL9A1, DTX1, ABCG2, ADAR, BMP1, BMP2, vs. FGFR1 BTRC,
CDC42, CTNNA1, T1D + ESRD CXCL12, DLL1, DTX2, GDF3, HDAC2, ISL1,
JAG1, NOTCH1, NOTCH2, NUMB, PARD6A, PDX1, RB1, SIGMAR1, h-TERT T1D
+ ESRD + IGFBP3 ABCG2, ALDH1A1, ALPI, CD3D, CD4, ASCL2, KAT2A, MYC,
NCAM1, vs. CD44, CD8A, CDC42, FGF2, FGFR1, NEUROG2, SOX2 T1D + ESRD
JAG1, SIGMAR1, SOX1, TUBB Abbreviations: IGF-I, insulin-like growth
factor 1; IGFBP3, insulin-like grwth factor binding protein 3,
CTRL, healthy subjects, T1D, type 1 diabetes, ESRD, end-stage renal
disease.
[0202] 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 Pathways in CoSCs
[0203] An extensive transcriptome analysis performed to clarify the
IGFBP3 caspase-mediated effect on mini-guts, (FIG. 4: C-D, FIG. 10:
G-H, Table IV), showed that addition of IGFBP3 to mini-guts grown
from healthy subjects crypts, was associated with a significant up
regulation of caspase-cascade activators (Caspases 8 and 9) and
proapoptotic genes, while the anti-apoptotic gene Bcl2 was down
regulated (FIG. 4C).
TABLE-US-00006 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, CASP5, vs. CASP6, CASP7, CASP8, CASP9, CTRL
CD27, CRADD, FADD, FASLG, HRK, TNFRSF10A, TNFRSF10B, TNFRSF11B,
TNFRSF1A, TNFRSF1B, TNFRSF25, TNFRSF9, TNFSF8, TRADD, TRAF3 CTRL +
IGF-I BNIPL3 CASP14, 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 grwth factor binding protein 3, CTRL, healthy
subjects, T1D, type 1 diabetes, ESRD, end-stage renal disease.
[0204] Interestingly, anti-apoptotic genes (Bcl2, Fas, Nol3) 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 (FIG. 10G). Moreover, the expression of genes involved in
other pro apoptotic pathways was either not altered (i.e. Fas
Ligand, FADD, TNF) or inhibited (TRADD) in T1D+ESRD mini-guts. The
opposite effect was observed by adding IGF-I (FIG. 4D, FIG. 10H).
The absence of alterations in the expression of oxidative stress
target genes (Table V) and of any effect of oxidative stress
factors (FIG. 10: I-J), confirmed the main apoptotic-related
caspase-mediated IGFBP3 mechanism whereby circulating IGFBP3
directly controls CoSCs (FIG. 4E).
TABLE-US-00007 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, GSS CYBB, GPX5, KRT1, MT3, NOX4, vs. 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 grwth factor binding protein
3, CTRL, healthy subjects, T1D, type 1 diabetes, ESRD, end-stage
renal disease.
Manipulation of the Circulating IGF-I/IGFBP3 Dyad Alters the Course
of Diabetic Enteropathy in a Preclinical Model
[0205] 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.
[0206] After 8 weeks of chemically-induced diabetes (using
streptozotocin [STZ]), C57BL/6 (B6) mice showed a reduced number of
crypts in the colorectal tissue (FIG. 4F), which displayed
increased depth and width in more than 70% of cases (FIG. 4: G,
H1-H2, I) and a reduced number of Aldh.sup.+ cells (FIG. 4: J,
K1-K2). Interestingly, those mice showed increased serum levels of
IGFBP3 and low levels of IGF-I, with lower murine insulin levels as
compared to naive B6 (FIG. 11: A-C). Intraperitoneal (i.p.)
administration of IGFBP3 in naive B6 mice resulted in a reduction
in local crypt numbers (FIG. 4: F, H3), with the majority of crypts
showing increased depth and width (FIG. 4: G, H3, I) and
significant reduction in Aldh.sup.+ cells as compared to untreated
mice (FIG. 4: J, K3). Those features were aggravated by IGFBP3
administration to STZ-treated B6 mice (FIG. 11: D-G, H1-H2), with
evidences of weight decrease (FIG. 11J), CoSCs loss (FIG. 11: J-L)
and up regulated expression of Caspase 8 and 9 (FIG. 11: M-N).
Administration of IGF-I i.p in STZ-treated B6 mice only partially
improved mucosa morphology increased the number of normal crypts,
which remained abnormal (FIG. 11D), and only partially restored the
number of Aldh.sup.+ cells (FIG. 11: G, H1-H2).
Treatment of Long-Standing T1D with Simultaneous Pancreas-Kidney
Transplantation (SPK) Reverts Clinical and Morphological Features
of DE
[0207] The gold standard treatment for long-standing T1D is SPK,
which affords stable glycometabolic control, near-normalize 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-00008 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..sctn. *, .sup..sctn.<0.0001 (ml/min/1.73 m.sup.2)
HbA1c (%) 8.4 .+-. 1.5 5.4 .+-. 0.3* 7.5 .+-. 1.4.sup..sctn.
*<0.0001; .sup..sctn.<0.001 EIR (UI) 37.4 .+-. 2.3 0* 26.0
.+-. 7.0.sup..sctn. *<0.0001; .sup..sctn.0.001 TG (mg/dl) 162.5
.+-. 92.7 90.4 .+-. 23.0* 147.1 .+-. 98.0.sup..sctn. *0.01;
.sup..sctn.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..sctn. 0.03; .sup..sctn.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, try glycerides; Chol, total cholesterol; LDL, low
density lipoprotein; HDL, high density lipoprotein; BP, blood
pressure; UI, International Unit.
[0208] 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 (FIG. 12: A-C), suggesting that DE is reversible.
Treatment of Long-Standing T1D with SPK Re-Establishes Intestinal
Mucosa Morphology and Local Self-Renewal Properties
[0209] Analysis of intestinal mucosa samples showed a significant
recovery in the structure of the epithelial compartment, with
compensatory epithelial hyperplasia in the SPK group (FIG. 12:
D1-D2). Recovery of normal crypt histology and number was evident
in the SPK group when long-standing T1D was successfully treated
while none of these features were evident in individuals who
received kidney transplant only and remained diabetic (FIG. 12:
D1-D2). Epithelial cell proliferation (MIB1.sup.+ cells) increased
after SPK over time as compared to baseline and to K+T1D at each
timepoint (FIG. 4: L, M1-M2), with near-normalization of intestinal
morphology, epithelial renewal and neural features (FIG. 12: E1-E2,
F, G1-G2, H-I, J1-J2, K). This demonstrates that treatment of
long-standing T1D with SPK promoted recovery of intestinal
epithelial repair and of self-renewing properties.
Treatment of Long-Standing T1D Promotes Restoration of CoSCs
[0210] Treatment of long-standing T1D with SPK is associated with
an increase in Aldh.sup.+ cells (FIG. 4: N, O1-O2) and EphB2.sup.+
expression in the intestinal crypt (FIG. 4: P, Q1-Q2) and nearly
normalizes the percentage of EphB2.sup.hi+, EphB2.sup.+hTERT.sup.+
and EphB2.sup.hi+LGR5.sup.+ cells in isolated intestinal crypts as
compared to baseline (FIG. 5: A-C). CoSC marker expression (FIG. 5:
D-G) and growth/morphology of mini-guts obtained from SPK
individuals were nearly normalized as well (FIG. 5H, FIG. 13:
A1-A6). Transcriptome analysis revealed that SPK nearly restored
the expression of stem cell and CoSC markers and of pathways
involved in preserving CoSCs (FIG. 5I, FIG. 13B, Table VII).
TABLE-US-00009 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 RB1
h-TERT NCA1 SIGMAR1 PDX1 DHH BGLA P Abbreviations: EGF, epithelial
growth factor; FGF, fibroblast growth factor, BMP, bone
morphogenetic protein.
[0211] 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
[0212] Broad proteomic analysis and targeted immunoassay, revealed
a near-normalization of IGFBP3 and IGF-I serum levels after SPK
(FIG. 5: J-K) in association with a nearly re-established
expression of IGF-IR (FIG. 13C). These results were not evident in
the K+T1D group, who showed low levels of IGF-I (FIG. 5J) and
IGF-IR expression (FIG. 13C) and only a partial recovery in their
IGFBP profile (FIG. 13D). A significant correlation between IGFBP3
serum levels and intestinal symptoms in both SPK and K+T1D groups,
but more evident in the latter, confirmed that the restoration of
IGFBP3 levels is associated with an improvement in diabetic
enteropathy (FIG. 5: L-M, FIG. 14: A-G). Treatment of long-standing
T1D with SPK ameliorates diabetic enteropathy via a
glucose-associated restoration of the circulating IGF-I/IGFBP3
dyad.
The Ecto-TMEM219 Recombinant Protein Abrogates IGFBP3-Mediated
Mini-Gut Destruction In Vitro and Preserves CoSCs In Vivo in a
Murine Model of DE.
[0213] 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 (FIG. 6A).
The expression of CoSC signature markers, EphB2 and LGR5,
significantly recovered in mini-guts cultured with IGFBP3 and
ecto-TMEM219, emphasizing a favorable effect in preserving CoSCs
(FIG. 6B), which was also confirmed in high glucose-cultured
mini-guts (FIG. 6A). Moreover, Analysis of Caspase 8 and 9 by
RT-PCR documented a net decrease in their expression when
ecto-TMEM219 was added to IGFBP3-cultured mini-guts as compared to
IGFBP3 alone (FIG. 6: C-D). The inventors then treated STZ-B6 mice
with ecto-TMEM219 and observed improved mucosa morphology with
recovered number, depth and width of crypts (FIG. 6: E, F, G).
Administration of ecto-TMEM219 was associated with an increase in
mice body weight as compared to STZ-treated B6 (FIG. 6H), with
significant regain of CoSCs (FIG. 6: I-K), a decreased expression
of caspase 8 and 9 (FIG. 6: L-M) and a re-establishment of
circulating IGFBP3 levels (FIG. 6N).
Discussion
[0214] 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-00010 TABLE VIII Overview of results of diabetic
enteropathy assessment in T1D + ESRD individuals as compared to
CTRL and SPK. TID + ESRD SPK vs. vs. Results CTRL TID + 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; T1D, type 1 diabetes;
ESRD, end stage renal disease; CTRL, healthy subjects; SPK,
simultaneous kidney-pancreas transplantation.
[0215] 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
[0216] 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 (29.4.+-.1.8
years) were enrolled in the study. 20 subjects affected by type 1
diabetes (T1D) 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. 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.
[0217] T1D+ESRD individuals were followed up for 8 years (mean
follow-up: 8.6.+-.1.1 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).
IGFBP3 Assessment in Urine and Serum
[0218] Serum was collected from 3 ml of fresh blood after
centrifugation. Urine samples were collected fresh, centrifuged and
stored at -80.degree. 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
[0219] 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
[0220] 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
[0221] enrolled in the study focusing on comparing CTRL with T1D
individuals and individuals with longstanding T1D (T1D+ESRD).
Results
[0222] Serum IGFBP3 Levels Correlates with Urinary IGFBP3
Levels
[0223] Analysis of serum and urine levels of IGFBP3 in all subjects
enrolled in the study documented a significant increase of both
serum (FIG. 7A) and urine (FIG. 7B) levels of IGFBP3 in T1D+ESRD
subjects as compared to CTRL and to a lesser extent to T1D
individuals. A significant correlation between urine levels and
serum levels of IGFBP3 was observed in all subjects evaluated (FIG.
7C). Higher levels of serum IGFBP3 correlate with higher levels of
urinary IGFBP3. In order to exclude that this might be related to
renal function, a correlation between IGFBP3 serum levels and renal
function (eGFR) was performed. IGFBP3 serum levels were
significantly higher in subjects with ESRD (eGFR<15 ml/min/m2)
(FIG. 7D). However, subjects with an eGFR>15 ml/min/m2, thus not
affected by ESRD, regardless the presence and history of T1D, did
not show any statistical significant correlation between eGFR and
IGFBP3 serum levels (FIG. 7E). Considering the correlation between
IGFBP3 urinary vs. serum levels in CTRL and comparing their means
and medians values within the 250 and 750 percentiles, inventors
may set up a range for urinary IGFBP3 as following:
[0224] <350 pg/ml: normal levels (levels observed in healthy
subjects)
[0225] 350-500 pg/ml: altered levels (levels observed in T1D with a
history of disease <5 years)
[0226] >500 pg/ml: indicative of enteropathy (levels observed in
long-standing T1D, T1D subjects with other T1D complications,
history of T1D>5 years).
[0227] 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 FIG. 7F.
Example 3
[0228] Five individuals with long-term inflammatory bowel disease
(IBD) were enrolled and screened for peripheral levels of IGFBP3,
IGF-1 and the ratio of the IGFBP-3/IGF-1, according to the same
method described above for the analysis of diabetic samples.
[0229] It was found that while IGFBP3 was slightly increased, the
levels of IGF1 were severely reduced with an overall alteration of
IGFBP3/IGF1 ratio (FIG. 18). Thus in inflammatory bowel disease, a
large amount of IGFBP3 is free and available to exert its toxic
effect on the intestinal stem cells.
[0230] Consequently, an inhibitor of IGFBP3 is also beneficial for
the treatment and/or prevention of inflammatory bowel diseases.
Example 4
Material and Methods
Patients and Study Design
[0231] Sixty serum samples from individuals with type 1 (T1D), with
T1D of long (>15 years) duration (long-standing T1D) and healthy
volunteers (CTRL) matched for age and gender were obtained from
blood collection at the San Raffaele Hospital. Twenty serum samples
from individuals screened positive for islets Autoantibodies test
were collected at the collaborating site of Gainsville (Florida).
235, 200 and 81 serum samples from normal glucose tolerant (NGT),
impaired glucose tolerant (IGT) and type 2 diabetes (T2D)
individuals were collected from University of Pisa (Italy) under
the Genfiev protocol study. NGT, IGT, and T2D were determined based
on the results of OGTT test according to the ADA 2003 criteria.
[0232] T1D and long-standing T1D 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
subjects were on the same treatment as antiplatelet therapy (ASA)
and anti-hypertension (angiotensin-converting-enzyme inhibitors).
Concomitant treatment, inclusion and exclusion criteria have been
already described (Diabetes Care 2015) and reported at the
following website
https://clinicaltrials.gov/ct2/show/record/NCT00879801?term=GENFIEV.
[0233] 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).
Pancreatic Islets
[0234] The human islets used in this study were isolated from
cadaveric organ donors according to the procedure already described
(Petrelli et al., 2011) in conformity to the ethical requirements
approved by the Niguarda Ca Granda Ethics Board. Briefly, islets
were isolated using the automated method already described (D'Addio
et al., 2014). Two types of enzymes were used: collagenase type P
(1-3 mg/ml) and liberase (0.5-1.4 mg/ml) (Roche, Indianapolis,
Ind., USA). Islets were purified by discontinuous gradient in
syringes (density gradient: 1,108; 1,096; 1,037: Euroficoll,
(Sigma-Aldrich, Milan, Italy), or by continuous gradient with
refrigerated COBE processor as previously described (Nano et al.,
2005). After isolation, islets were cultured at 22.degree. C. in a
humidified atmosphere (5% CO.sub.2), in M199 medium (Euroclone,
Celbio, Milan, Italy) or CMRL (Mediatech, Cellgro, Va., USA)
supplemented with 10% FCS, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin sulphate (Euroclone, Celbio) and 2 mmol/l glutamine
(Mediatech, Cellgro, Va., USA). In vitro characterisation and
culture of islets was performed on islet material processed within
72 h after isolation. Islets were cultured in CMRL 10% FCS,
supplemented with 100 .mu.g/ml streptomycin sulphate (Euroclone,
Celbio) and 2 mmol/l glutamine (Mediatech, Cellgro, Va., USA) with
a glucose concentration of 5 mM for 4 days. Murine islets were
kindly provided by Prof. James Markmann (Transplantation Unit,
Department of Surgery, Massachusetts General Hospital, Harvard
Medical School, Boston) (Ben Nasr et al., 2015b; Vergani et al.,
2010). Pancreatic islets were isolated from C57B16/J mice by
collagenase digestion followed by density gradient separation and
then hand-picking, as described previously (Forbes et al., 2010).
Islets were then plated and cultured in RPMI 1640 medium
supplemented with L-glutamine, penicillin and 10% as already
described, with a glucose concentration of 5 mM for 4 days.
Beta Cell Lines
[0235] Mouse .beta.TC3 and .alpha.TC1 cells were kindly provided by
Carla Perego, University of Milan, with the permission of Prof.
Douglas Hanahan (Department of Biochemistry and Biophysics,
University of California, San Francisco, Calif.)(Di Cairano et al.,
2011). .beta.TC3 were cultured in RPMI 1640 medium (Sigma)
containing 0.1 mM glutamic acid and supplemented with 0.7 mM
glutamine as described (Di Cairano et al., 2011). The glucose
concentration was 11 mM for cell lines.
Pathology and Immunohistochemistry
[0236] 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 650 W 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.degree. C. for 18-20 hours, followed by the avidin-biotin complex
procedure. Immunoreactions were developed using 0.03%
3,3'diaminobenzidine tetrahydrochloride, and then sections were
counterstained with Harris' hematoxylin. The following antibodies
were used: insulin (Dako, A0564), anti-IGFBP3 primary antibody
(polyclonal, 1:50 dilution, Sigma Aldrich HPA013357) and
anti-TMEM219 primary antibody (polyclonal, 1:100, Sigma HPA059185).
These antibodies were immunohistochemically tested in pancreatic
tissues of healthy subjects, B6 and NOD mice and in liver biopsies
of patients with T1D/T2D, islet transplanted patients who did not
achieve insulin independence. Tissues 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
[0237] Immunofluorescence samples were observed using a confocal
system (Leica TCS SP2 Laser Scanning Confocal). Images were
acquired in multitrack mode, using consecutive and independent
optical pathways. The following primary antibodies were used for
staining of human tissues/cells: mouse monoclonal anti-caspase
cleavage product of cytokeratin 18 M30 (clone M30,
Hoffmann-LaRoche, Basel, Switzerland), rabbit polyclonal IGFBP3
(1:250, Sigma, HPA013357), rabbit polyclonal TMEM219 (1:250, Sigma,
HPA059185) and Guinea Pig polyclonal insulin (1:50, DAKO, A0564).
The following primary antibodies were used for staining of murine
tissues/cells: rabbit polyclonal IGFBP3 (1:250, Sigma, SAB4501527),
goat polyclonal TMEM219 (1:50, Santa Cruz, 244405), Guinea Pig
polyclonal insulin (1:50, DAKO, A0564). The following secondary
antibodies were used for staining of human tissues/cells: donkey
anti-rabbit FITC (Jackson) and donkey anti-guinea pig TRITC
(Jackson). The following antibody was used for staining of murine
tissues/cells: donkey anti-goat FITC (Jackson).
[0238] Human and murine pancreatic islets co-cultured with/without
IGFBP3 (Life Technologies, 10430H07H5), with/without ecto-TMEM219
(generated by us in collaboration with Genscript, 130 ng/ml),
with/without high glucose (20 mM Glucose), with/without IFN-.gamma.
and IL-1.beta. (R&D Systems, Minneapolis, Minn. 201-LB-005, 2
ng/ml and PeProTech, 300-O2, 1,000 U/ml), were stained with
TMEM219, insulin and M30 for immunofluorescence for co-localization
studies. Murine beta cells co-cultured in the same conditions as
pancreatic islets, were fixed in 10% neutral buffered for 30 min,
washed with PBS three times and permeabilized with PBS--BSA 2%
triton .times.100 0.3% for 20 min, blocked with serum 10%, and
finally incubated with primary antibodies over-night at 4.degree.
C. and subsequently labeled with fluorescent secondary antibodies
for 2 hour at room temperature. Primary and secondary antibodies
were selected as described above.
Islets and Beta Cells In Vitro Studies and Characterization
Culturing Conditions
[0239] Human and murine islets were cultured at different glucose
concentration (5 mM, 20 mM, Sigma), with/without inflammatory
stimuli/cocktail (IFN-.gamma.+IL-1.beta., 2 ng/ml R&D Systems
and 1,000 U/ml Peprotech, respectively), with/without IGFBP3 (Life
Technologies, 50 ng/ml), with/without ecto-TMEM219 (130 ng/ml, see
Recombinant proteins and interventional studies) and islets were
collected for immunofluorescence studies, RNA extraction, apoptosis
detection, and protein analysis. Supernatants were collected for
assessment of insulin, IGFBP3 and IGF-I secretion.
[0240] .beta.-TC were cultured as previously described,
with/without inflammatory stimuli/cocktail
(IFN-.gamma.+IL-1.beta.), with/without IGFBP3, with/without
ecto-TMEM219 (see Recombinant proteins and interventional studies)
and cells were collected as for islets studies.
Immunoblotting
[0241] Total proteins of intestinal bioptic samples were extracted
in Laemmli buffer (Tris-HCl 62.5 mmol/l, pH 6.8, 20% glycerol, 2%
SDS, 5% b-mercaptoethanol) and their concentration was measured. 35
mg 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.degree. C., incubated
for 12 h with a rabbit polyclonal IGFBP3 antibody (Sigma,
HPA013357) diluted 1:250, or goat polyclonal TMEM219 (Santa Cruz
Biotechnology, 244404 or 244405) diluted 1:200 or with a monoclonal
mouse anti-b-actin antibody (Santa Cruz Biotechnology) diluted
1:500 in TBS-5% milk at 4.degree. C., washed four times with
TBS-0.1% Tween-20, then incubated with a peroxidase-labeled mouse
anti-rabbit IgG secondary antibody (DAKO) (for IGFBP3) or rabbit
anti-goat (for TMEM219) or rabbit anti mouse for b-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).
qRT-PCR Analysis
[0242] 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 .DELTA..DELTA.Ct
method. Quantitative reverse transcriptase polymerase chain
reaction (qRT-PCR) data were normalized for the expression of ACTB,
and .DELTA.Ct 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 before/after 3 days of
culture. Below are reported the main characteristics of primers
used for human genes:
TABLE-US-00011 Gene Refseq Band Size Reference Symbol UniGene #
Accession # (bp) Position INS Hs.272259 NM_000207.2 126 252 IGF-IR
Hs.643120 NM_000875.3 64 2248 TMEM219 Hs.460574 NM_001083613.1 60
726 LRP1 Hs.162757 NM_002332.2 64 656 TGFbR1 Hs.494622
NM_001130916.1 73 646 TGFbR2 Hs.604277 NM_001024847.2 70 1981 CASP8
Hs.599762 NM_001080124.1 124 648 ACTB Hs.520640 NM_001101 174
730
[0243] Below are reported the main characteristics of primers used
for murine genes:
TABLE-US-00012 Gene Refseq Band Size Reference Symbol UniGene #
Accession # (bp) Position INS Mm.4626 NM_008386.3 80 533 IGF-IR
Mm.275742 NM_010513.2 106 3901 TMEM219 Mm.248646 NM_026827,1 78 677
LRP1 Mm.271854 NM_032538.2 104 2995 TGFbR1 Mm.197552 NM_009370.2 85
90 TGFbR2 Mm.172346 NM_033397.3 132 1656 Casp8 Mm.336851
NM_001080126.1 96 1525 GAPDH Mm. 304088 NM_008084.2 107 75
ELISA Assay
[0244] IGF-I and IGFBP3 levels in the pooled sera/plasma of all
groups of subjects and in all groups of treated and untreated mice
were assessed using commercially available ELISA kits, according to
the manufacturer's instructions (R&D SG300, and Sigma
RAB0235).
[0245] Human primary hepatocytes (HEP10.TM. Pooled Human
Hepatocytes, ThermoFisher Scientific) were cultured for 3 days in
Williams Medium as per manufacturer's instructions at different
glucose concentrations: 11 mM, 20 mM and 35 mM. Culturing
supernatant was collected, and IGFBP3 was assessed using an IGFBP3
ELISA kit (Sigma, RAB0235) according to the manufacturer's
instructions. Collected cells were separated by trypsin and counted
with a hemacytometer.
[0246] Insulin levels were assayed with a microparticle enzyme
immunoassay (Mercodia Iso-Insulin ELISA, 10-1113-01) with intra-
and inter-assay coefficients of variation (CVs) of 3.0% and
5.0%.
Recombinant Proteins and Interventional Studies
[0247] Recombinant human IGF-I (Sigma, 13769), 100 ng/ml (IGF-I),
recombinant human IGFBP3 (Life Technologies, 10430H07H5), 50 ng/ml
(IGFBP3), anti-IGF-IR (Selleckchem, Boston, OSI-906), 1 .mu.M/L and
ecto-TMEM219 (D'Addio et al., 2015), 130 ng/ml were added to
islets/cell cultures at day +1 from islets collection/cell culture.
Pancreatic islets and beta cells were also exposed to complex
diabetogenic conditions: 20 mM glucose, the mixture of 2 ng/ml
recombinant human IL-1.beta. (R&D Systems, Minneapolis, Minn.
201-LB-005), and 1,000 U/ml recombinant human IFN-.gamma.
(PeProTech, 300-O2) for 72 h.
[0248] IGFBP3 (Reprokine, Valley Cottage, N.Y.) was administered to
naive B6 mice at 150 .mu.g /mouse/day for 15 days intraperitoneally
(i.p.); ecto-TMEM219 was administered in vivo to STZ-treated B6, to
10 weeks old NOD and to B6 fed a high fat diet (HFD-B6) mice
intraperitoneally (i.p.) at a dose of 150 .mu.g/mouse/day for 15
days in STZ-treated B6 and in NOD, and 100 .mu.g/mouse every other
day for 8 weeks in HFD-B6 mice.
Animal Studies
[0249] Male C57BL/6 (B6) mice and female non-obese diabetic (NOD)
mice (4 weeks old and 10 weeks old) 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. B6 mice
were rendered diabetic using a chemical approach with
streptozotocin (STZ) injection (225 mg/kg, administered i.p.; Sigma
S0130) this model is accepted and validated as a model of T1D
diabetes (Carvello et al., 2012; Petrelli et al., 2011; Vergani et
al., 2013). Diabetes was defined in both STZ-treated B6 and NOD as
blood glucose levels >250 mg/dL for 3 consecutive measures.
[0250] To study the onset and progression of T2D, B6 mice (6 weeks
old) were housed in a germfree Animal house in accordance with the
Principles of Laboratory Animal Care (NIH Publication No 85-23,
revised 1985) and received water and food ad libitum. The study
protocol was approved by the local ethics committee. Mice were fed
with either a High Fat Diet (HFD) (DIO diet D12492, 60% of total
calories from fat) or a normal-fat diet (NFD; DIO diet D12450B; 10%
of total calories from fat), purchased from Research Diets
(Mucedola, Settimo Milanese, Italy). Each group of treatment or
control consisted of 10 animals. After 16 weeks, glycemia was
measured and IV glucose tolerance test (IVGTT) was performed. The
next day, mice were anaesthetized and then a blood sample was
obtained and pancreas was harvested for histology studies. A
portion of the tissue was also snap-frozen and stored in Trizol to
perform RT-PCR studies.
[0251] Finally, plasma and serum were collected to perform analysis
of IGF-I (IGF-I ELISA kit, R&D MG100), IGFBP3 (IGFBP3 ELISA
kit, R&D MGB300) and insulin levels (Mouse Insulin ELISA kit,
Mercodia, 10-1247-O1). Blood glucose was monitored twice per week
up to 12 weeks in HFD-B6 in order to confirm diabetes onset and
permanence.
Statistical Analysis
[0252] 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 (.chi..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.RTM.,
IBM.RTM., 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
IGFBP3 Peripheral Levels are Increased in Pre-Diabetic and Diabetic
Mice.
[0253] In order to identify potential circulating factors that may
have a role in inducing beta cell death, the inventors profiled the
serum proteome of healthy subjects and individuals at risk for T1D,
based on the presence of one or more anti-islets autoantibodies,
using an unbiased proteomic approach. Proteins, which were
significantly different (p-value<0.01) in control pool versus
individuals at risk for T1D pool, were further submitted to
hierarchical clustering analysis. A clear proteomic profile was
evident in individuals at risk for T1D (and in overtly T1D as well)
as compared to healthy subjects, with more than 50% of the detected
proteins segregating in either one group or the other. In
particular, the levels of IGF-I binding proteins 3 (IGFBP3) were
increased in individuals at risk for T1D using an immune-targeted
assay (FIG. 19A), and thus preceded the onset of hyperglycemia.
Interestingly, IGFBP3 levels were also altered in samples obtained
from the Genfiev Study, which enrolled more than 800 individuals,
and classified them based on the results of the OGTT test in three
main categories: normal glucose tolerant (NGT), impaired glucose
tolerant (IGT) subjects and T2D individuals (T2D). The inventors
observed that IGFBP3 levels were increased in IGT and T2D as
compared to NGT subjects, confirming that high peripheral levels of
IGFBP3 mainly characterized pre-diabetic conditions (FIG. 19B). To
demonstrate the detrimental effect of IGFBP3 on islets and beta
cells, the inventors first demonstrated that pre-diabetic NOD mice
as well as diabetic NOD mice and streptozotocin-induced diabetic
C57BL/6 mice (STZ-B6) exhibited increased peripheral IGFBP3 levels
as compared to naive B6 (FIG. 20A). The inventors then confirmed
this in a murine model of T2D, the HFD model. C57B16/J (B6) mice
fed a high fat diet, which develop T2D in 16 weeks, showed
increased levels of peripheral IGFBP3 as compared to B6 mice fed a
normal fat diet (FIG. 20B).
Increased IGFBP3 Production by Hepatocytes in Inflamed Environment
and in T1D.
[0254] Liver is known to be a site of IGFBP3 production. In order
to explore if inflammatory stimuli could influence hepatic IGFBP3
production, the inventors cultured human primary hepatocytes with
various cytokines and with different glucose concentrations (11, 20
and 35 mM) and demonstrated that IGFBP3 levels in the supernatants
increased rapidly following different pro-inflammatory stimuli and
increased glucose levels (FIG. 21: A-B).
TMEM219 is Expressed in Human Islets.
[0255] In order to evaluate the effect of IGFBP3/TMEM219 axis on
islets and beta cells, the inventors first assessed TMEM219
expression by using immunofluorescence and its co-localization with
insulin at the confocal microscopy (FIG. 22: A1-A2). Human islets
obtained from cadaver donors whose pancreas were not suitable for
organ donation were studied. TMEM219 (green staining) is diffusely
expressed within islets and co-localize with insulin (red staining)
(FIG. 22: A1-A2). The inventors further evaluated the expression of
the other known receptors for IGFBP3 (i.e. LPR1, TGF-.beta.R1 and
TGF-.beta.R2) but none appeared expressed (FIG. 22B). The inventors
then confirmed TMEM219 expression by using RT-PCR and WB (FIG. 22:
B-C).
[0256] The inventors further proved expression of TMEM219 in murine
islets using RT-PCR and excluded that of other known IGFBP3
receptors (LRP1, TGF-beta type 1 and TGF-beta type 2) already
described in other cells and models (Baxter, 2013; Forbes et al.,
2010) (FIG. 23A). Finally, the inventors made use of the
availability of murine beta and alpha cell lines (.alpha.TC and
.beta.TC), and determined by RT-PCR that expression of TMEM219 is
restricted to beta cells while other islet cells, such as alpha
cells, do not express it (FIG. 23B) and further confirm TMEM219
expression by WB (FIG. 23C). Immunofluorescence staining of TMEM219
(green) and its co-localization with insulin was also confirmed on
beta cell line at the confocal microscope (FIG. 23D).
IGFBP3 Damages a Beta Cell Line In Vitro.
[0257] To demonstrate that IGFBP3 targets beta cells within the
islets, the inventors cultured a beta cell line (.beta.TC) for 3
days with/without IGFBP3. By using a viability/apoptosis assay, the
inventors were able to demonstrate a reduced percentage of viable
beta cells in IGFBP3-treated conditions as compared to untreated
(FIG. 24A). Interestingly, IGFBP3-treated beta cells also showed a
significant increase in caspase8 expression (FIG. 24B) and a
reduction in insulin expression by both immunofluorescence and
RT-PCR (FIG. 24: C, D1-D2, E). Interestingly, IGFBP3-induced
apoptosis was markedly higher than that induced by the
pro-inflammatory stimuli IL-1.beta. and IFN-.gamma. (FIG. 24: A-B)
and insulin expression and release were only slightly reduced (FIG.
24: C-E).
IGFBP3 Damages Murine Islets In Vitro.
[0258] To further demonstrate the IGFBP3-mediated detrimental
effect on islets, the inventors cultured murine islets isolated
from C57BL/6 mice for 4 days with/without IGFBP3. The appearance of
extensive apoptosis as assessed by FACS (Annexin V.sup.+7AAD.sup.-)
documented that IGFBP3-treated islets undergo early apoptosis
(87.+-.2 vs. 67.+-.2%, p=0.004), associated with an increase in
caspase 8 expression and with a decrease in insulin expression by
RT-PCR (FIG. 25: A-C).
IGFBP3 Damages Human Islets In Vitro.
[0259] The inventors finally confirmed the IGFBP3-mediated
detrimental effects in human islets by demonstrating that in vitro
cultured human islets, obtained from cadaver donors whose pancreata
were not suitable for organ donation, exposed to IGFBP3 for 4 days
underwent greatly to apoptosis (FIG. 26A), showed an increase in
caspase 8 expression (FIG. 26B) and an increased expression of M30
(FIG. 8: C1-C2), a marker for apoptosis, associated with a decrease
in insulin expression at immunostaining (FIG. 26:D1-D2) and using
RT-PCR (FIG. 26E).
IGFBP3 Injection in C57BL/6 Mice Alters Islet Morphology In
Vivo.
[0260] In order to confirm that IGFBP3 alters islet morphology, the
inventors injected recombinant IGFBP3 (Reprokine) in naive B6 and
STZ-treated B6 mice (150 .mu.g every day for 15 days). Histology
(H&E) analysis of collected pancreata demonstrated an increased
derangement in islets of STZ-B6 IGFBP3-treated mice as compared to
islets of naive and STZ-B6 mice, confirmed by scattered insulin
expression upon immunostaining (FIG. 27: A1-A6).
The Recombinant Protein Ecto-TMEM219 Prevents IGFBP3-Associated
Damage in a Beta Cell Line In Vitro.
[0261] To demonstrate that ecto-TMEM219 prevents IGFBP3-associated
detrimental effects specifically on beta cells, the inventors
cultured a beta cell line with IGFBP3 and ecto-TMEM219 and observed
that beta cell apoptosis was greatly reduced by the addition of
ecto-TMEM219. The effect was also confirmed by the analysis of
caspase 8 expression which appeared reduced in
IGFBP3+ecto-TMEM219-treated beta cells as compared to those
cultured with IGFBP3 only (FIG. 28: A-B). Insulin expression, as
assessed by RT-PCR and immunofluorescence (red), was consistently
increased by the addition of ecto-TMEM219 to IGFBP3-cultured beta
cells (FIG. 28: C1-C3).
The Recombinant Protein Ecto-TMEM219 Prevents IGFBP3-Associated
Detrimental Effects in Murine Islets In Vitro.
[0262] In order to further confirm the therapeutic properties of
ecto-TMEM219 in preventing IGFBP3-associated damage, the inventors
tested the effect of ecto-TMEM219 in cultured murine islet in
vitro. The addition of ecto-TMEM219 (2:1 molar ratio with IGFBP3)
to isolated C57BL/6 islets co-cultured with IGFBP3 abrogated the
pro-apoptotic effect of IGFBP3. Moreover, caspase 8 expression was
significantly reduced in islets cultured with IGFBP3 and
ecto-TMEM219 (FIG. 29A). Insulin expression was increased by the
addition of ecto-TMEM219 to murine islets cultured with IGFBP3
(FIG. 29B), emphasizing a favorable effect of ecto-TMEM219 on
preserving islet function.
The Recombinant Protein Ecto-TMEM219 Prevents IGFBP3 Detrimental
Effects on Human Islets In Vitro.
[0263] To demonstrate the beneficial effects of ecto-TMEM219 in
preventing islets destruction, the inventors cultured human islets
with IGFBP3 and ecto-TMEM219 for 4 days and the inventors
demonstrated a rescue of IGFBP3-mediated islets damaging by
ecto-TMEM219, associated with an increase of insulin expression and
a decrease of caspase 8 expression at RT-PCR (FIG. 30: A-B).
[0264] Interestingly, the co-staining of insulin (red) and M30
(green), a marker for apoptosis, confirmed that insulin-producing
cells were protected by ecto-TMEM219 during the co-cultured with
IGFBP3 (FIG. 30: C1-C3).
The Recombinant Protein ectoTMEM219 Prevents IGFBP3-Associated
Islet Alterations.
[0265] In order to prove the effect of ecto-TMEM219 in the
treatment of diabetes, the inventors measured insulin serum levels
in STZ-treated diabetic mice at 8 weeks and observed that insulin
was significantly increased in those mice that were treated with
ecto-TMEM219 (i.p. 150 .mu.g every other day for 2 weeks) as
compared to untreated STZ-B6 (FIG. 31A). Finally, in another model
of islet injury in vivo, B6 mice fed with a high fat diet (B6-HFD)
showed altered blood glucose and insulin levels, while B6-HFD
treated with ecto-TMEM219 (i.p. 100 .mu.g every other day for 6
weeks) maintained near-normal glucose and insulin levels (FIG.
31B), thus suggesting a curative effect of ecto-TMEM219 in type-1
and type-2 diabetes.
Discussion
[0266] Type 1 diabetes (T1D) has historically been regarded as a T
cell-mediated autoimmune disease, resulting in the destruction of
insulin-producing pancreatic beta cells (Bluestone et al., 2010;
Eisenbarth, 1986). According to this perspective, an initiating
factor triggers the immune response against autoantigens, and the
subsequent newly activated autoreactive T cells target and further
destroy insulin-producing beta cells (Bluestone et al., 2010).
Whether destruction of beta cells is solely determined by the
autoimmune attack or whether other mechanisms such as paracrine
modulation, metabolic deregulation and non-immune beta cell
apoptosis contribute to T1D pathogenesis is now a matter of debate
(Atkinson and Chervonsky, 2012; Atkinson et al., 2015). Recently,
it has been observed that environmental factors (e.g.; viral
infections, diet, neonatal exposure to milk and microbiota) may be
required to initiate the autoimmune response in T1D (Filippi and
von Herrath, 2008; McLean et al., 2015). Thus a new approach to
study the pathogenesis of T1D is gradually emerging (McLean et al.,
2015), such that immunological and genetic factors are no longer
considered to be the sole determinant of T1D (Alper et al., 2006;
Oilinki et al., 2012). Moreover, the efficacy of immunotherapeutic
strategies, which have been considered in the last decade to be the
principal prospect for establishing a cure for T1D, is now being
questioned (Ben Nasr et al., 2015a). While targeting the autoimmune
response using an immunosuppressive treatment or a pro-regulatory
regimen was shown to be satisfactory in rodents, such strategies
conversely achieved insulin independence in a negligible number of
T1D individuals (Atkinson et al., 2015). In addition to
underscoring the difference between animal models and humans, these
data also shed light on the fact that investigation of the immune
response primarily examined immune events occurring in the
periphery, while little is known with respect to the disease
process that occurs within islets and particularly in beta cells.
In this regard, the discovery of novel factors involved in the
initiation/facilitation of beta cell loss in T1D will be of
significant value. Such discoveries may pave the way for novel
therapeutic approaches capable of halting or delaying the very
first phase of the disease. In the present invention it was found
that in individuals at high-risk for T1D and in those with overt
T1D, IGFBP3 peripheral levels are increased. Interestingly a
similar pattern was also observed in individuals at risk of
developing T2D (IGT, IFG), where glucose intolerance was already
detectable, and in those with established T2D, confirming that,
despite a different etiology, the mediator of beta cell loss, which
occurs in both types of diabetes, may be the same, a betatoxin
called IGFBP3. In fact, T1D and T2D are both characterized by a
loss of beta cells, which results in a reduced secretion of
insulin, failure to control blood glucose levels and hyperglycemia
(Brennand and Melton, 2009; Yi et al., 2014). Despite different
etiological mechanisms, either autoimmune response in T1D or
insulin resistance/inflammation in T2D, lead to a progressive
reduction of beta cell mass. Several approaches are currently
available to treat T1D and T2D, but none of them aims to target
beta cell loss, protect from beta cell injury and preserve beta
cell mass, thus preventing diabetes onset. IGFBP3 may also be a
mechanism to explain the decompensation observed in patients with
T2D, which slowly but steadily lose their beta cell function and
stop producing insulin. The chronic IGFBP3 overproduction observed
in T2D may favor the destruction of beta cells and lead to the
failure for instance of oral anti-diabetic agent. The inventors
have also observed that the IGFBP3 receptor (TMEM219) is expressed
in murine/human islets, and that its ligation by IGFBP3 is toxic to
beta cells, raising the possibility of the existence of an
endogenous beta cell toxin (betatoxin) that may be involved in the
early phase of T1D and in diabetes in general. A non-immunological
factor may determine islet/beta cell injuries, and facilitate the
exposure of autoantigens to immune cells, thus creating a local
inflamed environment and a sustained immune reaction. Liver has
been already documented to be the primary source for IGFBP3, and
its exposure to inflammation and high glucose levels significantly
increases IGFBP3 release in the circulation. As a result, IGFBP3
targets islets and beta cells thus favoring their damage and loss.
Therefore, neutralization of IGFBP3-mediated beta cell injury
through the use of newly generated inhibitors of IGFBP3/TMEM219
axis, such as recombinant ecto-TMEM219, may prevent beta cell loss
by quenching peripheral IGFBP3, thus blocking its signaling via
TMEM219 and halting/delaying T1D progression (FIG. 32). This may
lead to clinical application in the field of diabetes prevention,
resulting in the use of ecto-TMEM219 in individuals at high-risk
for T1D and eventually T2D. Inhibitors of IGFBP3/TMEM219 axis may
thus prevent early beta cell injuries associated with the early
phase of T1D, by inhibiting binding of IGFBP3 to TMEM219 expressed
on the target tissue. Considering its role in preventing early loss
of beta cells, inhibitors of IGFBP3/TMEM219 axis may also be
considered of benefit in the early treatment of T2D. Therefore,
inhibitors of IGFBP3/TMEM219 axis may represent a therapeutic
strategy that prevent diabetes onset and protect beta cell from
loss and damage thus becoming a relevant clinical option for
individuals at risk of developing diabetes, both T1D and T2D, and
in those with diabetes in the early stages. Individuals at risk of
developing T1D are mainly characterized by the early detection in
the serum of multiple autoantibodies against islet peptides, which
are usually absent in healthy subjects (Ziegler et al., 2013).
These individuals are usually relatives (brothers, sisters) of
individuals with T1D, but do not have any sign or symptom related
to T1D. The probability of progressing to T1D in these subjects
within 10 years is high, with the majority of them (70%) developing
T1D in the next 15 years, but are often underestimated (Ziegler et
al., 2013). Individuals at risk for developing T2D are difficult to
identify, especially in the early phase. Prevention consists mainly
of lifestyle modifications, which may delay the onset of the
disease but could not prevent it (Schwarz et al., 2012). Various
screening methods (genetic analysis, metabolomics profile, obesity
and risk factors assessment) to early detect alterations in glucose
metabolism are underway, but therapeutic agents capable of
preventing or protecting from T2D onset are not available and
current options only include anti-diabetic agents that control
hyperglycemia and delay T2D progression (metformin), or agents that
control other risk factors (lipid-lowering and blood
pressure-lowering agents) (Nathan, 2015). Therefore, treatments
aiming to reduce the burden of diabetes in the general population,
both T1D and T2D, should focus on these high-risk populations. This
invention is intended as a new clinical therapeutic agent to be
used in individuals at risk for developing diabetes to prevent its
onset and in those who are in the early stages of the disease
(new-onset) to protect from progression into established diabetes,
by counteracting beta cell loss and preserving beta cell mass.
Given its role in preventing beta cells loss and damage, inhibitors
of IGFBP3/TMEM219 axis are of use in individuals at risk for
developing T1D or T2D, and in those with the disease in its early
stages.
REFERENCES
[0267] (1993). The effect of intensive treatment of diabetes on the
development and progression of long-term complications in
insulin-dependent diabetes mellitus. The Diabetes Control and
Complications Trial Research Group. N Engl J Med 329, 977-986.
[0268] Alper, C. A., Husain, Z., Larsen, C. E., Dubey, D. P.,
Stein, R., Day, C., Baker, A., Beyan, H., Hawa, M., Ola, T. O., et
al. (2006). Incomplete penetrance of susceptibility genes for
MHC-determined immunoglobulin deficiencies in monozygotic twins
discordant for type 1 diabetes. Journal of autoimmunity 27, 89-95.
[0269] Atkinson, M. A., and Chervonsky, A. (2012). Does the gut
microbiota have a role in type 1 diabetes? Early evidence from
humans and animal models of the disease. Diabetologia 55,
2868-2877. [0270] Atkinson, M. A., Eisenbarth, G. S., and Michels,
A. W. (2013). Type 1 diabetes. Lancet. [0271] Atkinson, M. A., von
Herrath, M., Powers, A. C., and Clare-Salzler, M. (2015). Current
concepts on the pathogenesis of type 1 diabetes-considerations for
attempts to prevent and reverse the disease. Diabetes care 38,
979-988. [0272] Barker, N. (2014). Adult intestinal stem cells:
critical drivers of epithelial homeostasis and regeneration. Nat
Rev Mol Cell Biol 15, 19-33. [0273] Baxter, R. C. (2013).
Insulin-like growth factor binding protein-3 (IGFBP-3): Novel
ligands mediate unexpected functions. Journal of cell communication
and signaling 7, 179-189. [0274] Ben Nasr, M., D'Addio, F.,
Usuelli, V., Tezza, S., Abdi, R., and Fiorina, P. (2015a). The
rise, fall, and resurgence of immunotherapy in type 1 diabetes.
Pharmacological research: the official journal of the Italian
Pharmacological Society 98, 31-38. [0275] Ben Nasr, M., Vergani,
A., Avruch, J., Liu, L., Kefaloyianni, E., D'Addio, F., Tezza, S.,
Corradi, D., Bassi, R., Valderrama-Vasquez, A., et al. (2015b).
Co-transplantation of autologous MSCs delays islet allograft
rejection and generates a local immunoprivileged site. Acta
diabetologica 52, 917-927. [0276] Bluestone, J. A., Herold, K., and
Eisenbarth, G. (2010). Genetics, pathogenesis and clinical
interventions in type 1 diabetes. Nature 464, 1293-1300. [0277]
Bondy, C. A., Underwood, L. E., Clemmons, D. R., Guler, H. P.,
Bach, M. A., and Skarulis, M. (1994). Clinical uses of insulin-like
growth factor I. Ann Intern Med 120, 593-601. [0278] Bortvedt, S.
F., and Lund, P. K. (2012). Insulin-like growth factor 1: common
mediator of multiple enterotrophic hormones and growth factors.
Curr Opin Gastroenterol 28, 89-98. [0279] Boucher, J., Macotela,
Y., Bezy, O., Mori, M. A., Kriauciunas, K., and Kahn, C. R. (2010).
A kinase-independent role for unoccupied insulin and IGF-1
receptors in the control of apoptosis. Sci Signal 3, ra87. [0280]
Breault, D. T., Min, I. M., Carlone, D. L., Farilla, L. G.,
Ambruzs, D. M., Henderson, D. E., Algra, S., Montgomery, R. K.,
Wagers, A. J., and Hole, N. (2008). Generation of mTert-GFP mice as
a model to identify and study tissue progenitor cells. Proc Natl
Acad Sci USA 105, 10420-10425. [0281] Brennand, K., and Melton, D.
(2009). Slow and steady is the key to beta-cell replication.
Journal of cellular and molecular medicine 13, 472-487. [0282]
Bytzer, P., Talley, N. J., Hammer, J., Young, L. J., Jones, M. P.,
and Horowitz, M. (2002). GI symptoms in diabetes mellitus are
associated with both poor glycemic control and diabetic
complications. Am J Gastroenterol 97, 604-611. [0283] Camilleri, M.
(2007). Clinical practice. Diabetic gastroparesis. N Engl J Med
356, 820-829. [0284] Cano, A. E., Neil, A. K., Kang, J. Y.,
Barnabas, A., Eastwood, J. B., Nelson, S. R., Hartley, I., and
Maxwell, D. (2007). Gastrointestinal symptoms in patients with
end-stage renal disease undergoing treatment by hemodialysis or
peritoneal dialysis. Am J Gastroenterol 102, 1990-1997. [0285]
Carlone, D. L., and Breault, D. T. (2012). Tales from the crypt:
the expanding role of slow cycling intestinal stem cells. Cell Stem
Cell 10, 2-4. [0286] Carpentino, J. E., Hynes, M. J., Appelman, H.
D., Zheng, T., Steindler, D. A., Scott, E. W., and Huang, E. H.
(2009). Aldehyde dehydrogenase-expressing colon stem cells
contribute to tumorigenesis in the transition from colitis to
cancer. Cancer Res 69, 8208-8215. [0287] Carrington, E. V.,
Brokjaer, A., Craven, H., Zarate, N., Horrocks, E. J., Palit, S.,
Jackson, W., Duthie, G. S., Knowles, C. H., Lunniss, P. J., et al.
(2014). Traditional measures of normal anal sphincter function
using high-resolution anorectal manometry (HRAM) in 115 healthy
volunteers. Neurogastroenterol Motil. [0288] Carvello, M.,
Petrelli, A., Vergani, A., Lee, K. M., Tezza, S., Chin, M.,
Orsenigo, E., Staudacher, C., Secchi, A., Dunussi-Joannopoulos, K.,
et al. (2012). Inotuzumab ozogamicin murine analog-mediated B-cell
depletion reduces anti-islet allo- and autoimmune responses.
Diabetes 61, 155-165. [0289] Cox, J., Neuhauser, N., Michalski, A.,
Scheltema, R. A., Olsen, J. V., and Mann, M. (2011). Andromeda: a
peptide search engine integrated into the MaxQuant environment. J
Proteome Res 10, 1794-1805. [0290] D'Addio, F., La Rosa, S.,
Maestroni, A., Jung, P., Orsenigo, E., Ben Nasr, M., Tezza, S.,
Bassi, R., Finzi, G., Marando, A., et al. (2015). Circulating IGF-I
and IGFBP3 Levels Control Human Colonic Stem Cell Function and Are
Disrupted in Diabetic Enteropathy. Cell Stem Cell 17, 486-498.
[0291] D'Addio, F., Valderrama Vasquez, A., Ben Nasr, M., Franek,
E., Zhu, D., Li, L., Ning, G., Snarski, E., and Fiorina, P. (2014).
Autologous nonmyeloablative hematopoietic stem cell transplantation
in new-onset type 1 diabetes: a multicenter analysis. Diabetes 63,
3041-3046. [0292] Di Cairano, E. S., Davalli, A. M., Perego, L.,
Sala, S., Sacchi, V. F., La Rosa, S., Finzi, G., Placidi, C.,
Capella, C., Conti, P., et al. (2011). The glial glutamate
transporter 1 (GLT1) is expressed by pancreatic beta-cells and
prevents glutamate-induced beta-cell death. The Journal of
biological chemistry 286, 14007-14018. [0293] Domenech, A.,
Pasquinelli, G., De Giorgio, R., Gori, A., Bosch, F., Pumarola, M.,
and Jimenez, M. (2011). Morphofunctional changes underlying
intestinal dysmotility in diabetic RIP-I/hIFNbeta transgenic mice.
Int J Exp Pathol 92, 400-412. [0294] Eisenbarth, G. S. (1986). Type
I diabetes mellitus. A chronic autoimmune disease. The New England
journal of medicine 314, 1360-1368. [0295] Faraj, J., Melander, O.,
Sundkvist, G., Olsson, R., Thorsson, O., Ekberg, O., and Ohlsson,
B. (2007). Oesophageal dysmotility, delayed gastric emptying and
gastrointestinal symptoms in patients with diabetes mellitus.
Diabet Med 24, 1235-1239. [0296] Feldman, M., and Schiller, L. R.
(1983). Disorders of gastrointestinal motility associated with
diabetes mellitus. Ann Intern Med 98, 378-384. [0297] Filippi, C.
M., and von Herrath, M. G. (2008). Viral trigger for type 1
diabetes: pros and cons. Diabetes 57, 2863-2871. [0298] Fiorina,
P., Folli, F., Bertuzzi, F., Maffi, P., Finzi, G., Venturini, M.,
Socci, C., Davalli, A., Orsenigo, E., Monti, L., et al. (2003).
Long-term beneficial effect of islet transplantation on diabetic
macro-/microangiopathy in type 1 diabetic kidney-transplanted
patients. Diabetes Care 26, 1129-1136. [0299] Fiorina, P., Folli,
F., D'Angelo, A., Finzi, G., Pellegatta, F., Guzzi, V., Fedeli, C.,
Della Valle, P., Usellini, L., Placidi, C., et al. (2004).
Normalization of multiple hemostatic abnormalities in uremic type 1
diabetic patients after kidney-pancreas transplantation. Diabetes
53, 2291-2300. [0300] Fiorina, P., La Rocca, E., Venturini, M.,
Minicucci, F., Fermo, I., Paroni, R., D'Angelo, A., Sblendido, M.,
Di Carlo, V., Cristallo, M., et al. (2001). Effects of
kidney-pancreas transplantation on atherosclerotic risk factors and
endothelial function in patients with uremia and type 1 diabetes.
Diabetes 50, 496-501. [0301] Fiorina, P., Venturini, M., Folli, F.,
Losio, C., Maffi, P., Placidi, C., La Rosa, S., Orsenigo, E.,
Socci, C., Capella, C., et al. (2005). Natural history of kidney
graft survival, hypertrophy, and vascular function in end-stage
renal disease type 1 diabetic kidney-transplanted patients:
beneficial impact of pancreas and successful islet
cotransplantation. Diabetes Care 28, 1303-1310. [0302] Folli, F.,
Guzzi, V., Perego, L., Coletta, D. K., Finzi, G., Placidi, C., La
Rosa, S., Capella, C., Socci, C., Lauro, D., et al. (2010).
Proteomics reveals novel oxidative and glycolytic mechanisms in
type 1 diabetic patients' skin which are normalized by
kidney-pancreas transplantation. PLoS One 5, e9923. [0303] Forbes,
K., Souquet, B., Garside, R., Aplin, J. D., and Westwood, M.
(2010). Transforming growth factor-{beta} (TGF{beta}) receptors
I/II differentially regulate TGF{beta}1 and IGF-binding protein-3
mitogenic effects in the human placenta. Endocrinology 151,
1723-1731. Giustina, A., Berardelli, R., Gazzaruso, C., and
Mazziotti, G. (2014). Insulin and GH-IGF-I axis: endocrine pacer or
endocrine disruptor? Acta Diabetol. [0304] Gracz, A. D., Fuller, M.
K., Wang, F., Li, L., Stelzner, M., Dunn, J. C., Martin, M. G., and
Magness, S. T. (2013). Brief Report: CD24 and CD44 mark human
intestinal epithelial cell populations with characteristics of
active and facultative stem cells. Stem Cells 31, 2024-2030. [0305]
Hsu, S. M., Raine, L., and Fanger, H. (1981). Use of
avidin-biotin-peroxidase complex (ABC) in immunoperoxidase
techniques: a comparison between ABC and unlabeled antibody (PAP)
procedures. J Histochem Cytochem 29, 577-580. [0306] Hughes, K. R.,
Sablitzky, F., and Mahida, Y. R. (2011). Expression profiling of
Wnt family of genes in normal and inflammatory bowel disease
primary human intestinal myofibroblasts and normal human colonic
crypt epithelial cells. Inflamm Bowel Dis 17, 213-220. [0307] Jung,
P., Sato, T., Merlos-Suarez, A., Barriga, F. M., Iglesias, M.,
Rossell, D., Auer, H., Gallardo, M., Blasco, M. A., Sancho, E., et
al. (2011). Isolation and in vitro expansion of human colonic stem
cells. Nat Med 17, 1225-1227. [0308] Kosinski, C., Li, V. S., Chan,
A. S., Zhang, J., Ho, C., Tsui, W. Y., Chan, T. L., Mifflin, R. C.,
Powell, D. W., Yuen, S. T., et al. (2007). Gene expression patterns
of human colon tops and basal crypts and BMP antagonists as
intestinal stem cell niche factors. Proc Natl Acad Sci USA 104,
15418-15423. [0309] Le Roith, D. (1997). Seminars in medicine of
the Beth Israel Deaconess Medical Center. Insulin-like growth
factors. N Engl J Med 336, 633-640. [0310] Levey, A. S., Bosch, J.
P., Lewis, J. B., Greene, T., Rogers, N., and Roth, D. (1999). A
more accurate method to estimate glomerular filtration rate from
serum creatinine: a new prediction equation. Modification of Diet
in Renal Disease Study Group. Ann Intern Med 130, 461-470. [0311]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
(1951). Protein measurement with the Folin phenol reagent. J Biol
Chem 193, 265-275. [0312] McLean, M. H., Dieguez, D., Jr., Miller,
L. M., and Young, H. A. (2015). Does the microbiota play a role in
the pathogenesis of autoimmune diseases? Gut 64, 332-341. [0313]
Medema, J. P., and Vermeulen, L. (2011). Microenvironmental
regulation of stem cells in intestinal homeostasis and cancer.
Nature 474, 318-326. [0314] Merlos-Suarez, A., Barriga, F. M.,
Jung, P., Iglesias, M., Cespedes, M. V., Rossell, D., Sevillano,
M., Hernando-Momblona, X., da Silva-Diz, V., Munoz, P., et al.
(2011). The intestinal stem cell signature identifies colorectal
cancer stem cells and predicts disease relapse. Cell Stem Cell 8,
511-524. [0315] Munoz, J., Stange, D. E., Schepers, A. G., van de
Wetering, M., Koo, B. K., Itzkovitz, S., Volckmann, R., Kung, K.
S., Koster, J., Radulescu, S., et al. (2012). The Lgr5 intestinal
stem cell signature: robust expression of proposed quiescent `+4`
cell markers. EMBO J 31, 3079-3091. [0316] Muzumdar, R. H., Ma, X.,
Fishman, S., Yang, X., Atzmon, G., Vuguin, P., Einstein, F. H.,
Hwang, D., Cohen, P., and Barzilai, N. (2006). Central and opposing
effects of IGF-I and IGF-binding protein-3 on systemic insulin
action. Diabetes 55, 2788-2796. [0317] Nano, R., Clissi, B., Melzi,
R., Calori, G., Maffi, P., Antonioli, B., Marzorati, S.,
Aldrighetti, L., Freschi, M., Grochowiecki, T., et al. (2005).
Islet isolation for allotransplantation: variables associated with
successful islet yield and graft function. Diabetologia 48,
906-912. [0318] Nathan, D. M. (2015). Diabetes: Advances in
Diagnosis and Treatment. Jama 314, 1052-1062. [0319] Oilinki, T.,
Otonkoski, T., Ilonen, J., Knip, M., and Miettinen, P. J. (2012).
Prevalence and characteristics of diabetes among Somali children
and adolescents living in Helsinki, Finland. Pediatric diabetes 13,
176-180. [0320] Pambianco, G., Costacou, T., Ellis, D., Becker, D.
J., Klein, R., and Orchard, T. J. (2006). The 30-year natural
history of type 1 diabetes complications: the Pittsburgh
Epidemiology of Diabetes Complications Study experience. Diabetes
55, 1463-1469. [0321] Petrelli, A., Carvello, M., Vergani, A., Lee,
K. M., Tezza, S., Du, M., Kleffel, S., Chengwen, L., [0322]
Mfarrej, B. G., Hwu, P., et al. (2011). IL-21 is an antitolerogenic
cytokine of the late-phase alloimmune response. Diabetes 60,
3223-3234. [0323] Pupim, L. B., Heimburger, O., Qureshi, A. R.,
Ikizler, T. A., and Stenvinkel, P. (2005). Accelerated lean body
mass loss in incident chronic dialysis patients with diabetes
mellitus. Kidney Int 68, 2368-2374. [0324] Remes-Troche, J. M.,
De-Ocampo, S., Valestin, J., and Rao, S. S. (2010). Rectoanal
reflexes and sensorimotor response in rectal hyposensitivity. Dis
Colon Rectum 53, 1047-1054. [0325] Sato, T., and Clevers, H.
(2013). Growing self-organizing mini-guts from a single intestinal
stem cell: mechanism and applications. Science 340, 1190-1194.
[0326] Schwarz, P. E., Greaves, C. J., Lindstrom, J., Yates, T.,
and Davies, M. J. (2012). Nonpharmacological interventions for the
prevention of type 2 diabetes mellitus. Nature reviews.
Endocrinology 8, 363-373. [0327] Secchi, A., Caldara, R., La Rocca,
E., Fiorina, P., and Di Carlo, V. (1998). Cardiovascular disease
and neoplasms after pancreas transplantation. Lancet 352, 65;
author reply 66. [0328] Smets, Y. F., Westendorp, R. G., van der
Pijl, J. W., de Charro, F. T., Ringers, J., de Fijter, J. W., and
Lemkes, H. H. (1999). Effect of simultaneous pancreas-kidney
transplantation on mortality of patients with type-1 diabetes
mellitus and end-stage renal failure. Lancet 353, 1915-1919. [0329]
Sridhar, S. S., and Goodwin, P. J. (2009). Insulin-insulin-like
growth factor axis and colon cancer. J Clin Oncol 27, 165-167.
[0330] Stange, D. E., and Clevers, H. (2013). Concise review: the
yin and yang of intestinal (cancer) stem cells and their
progenitors. Stem Cells 31, 2287-2295. [0331] Svedlund, J., Sjodin,
I., and Dotevall, G. (1988). GSRS--a clinical rating scale for
gastrointestinal symptoms in patients with irritable bowel syndrome
and peptic ulcer disease. Dig Dis Sci 33, 129-134. [0332] Talley,
N. J., Young, L., Bytzer, P., Hammer, J., Leemon, M., Jones, M.,
and Horowitz, M. (2001). Impact of chronic gastrointestinal
symptoms in diabetes mellitus on health-related quality of life. Am
J Gastroenterol 96, 71-76.
[0333] van der Flier, L. G., and Clevers, H. (2009). Stem cells,
self-renewal, and differentiation in the intestinal epithelium.
Annual review of physiology 71, 241-260. [0334] Vergani, A.,
D'Addio, F., Jurewicz, M., Petrelli, A., Watanabe, T., Liu, K.,
Law, K., Schuetz, C., Carvello, M., Orsenigo, E., et al. (2010). A
novel clinically relevant strategy to abrogate autoimmunity and
regulate alloimmunity in NOD mice. Diabetes 59, 2253-2264. [0335]
Vergani, A., Fotino, C., D'Addio, F., Tezza, S., Podetta, M.,
Gatti, F., Chin, M., Bassi, R., Molano, R. D., Corradi, D., et al.
(2013). Effect of the purinergic inhibitor oxidized ATP in a model
of islet allograft rejection. Diabetes 62, 1665-1675. [0336]
Williams, A. C., Smartt, H., AM, H. Z., Macfarlane, M., Paraskeva,
C., and Collard, T. J. (2007). Insulin-like growth factor binding
protein 3 (IGFBP-3) potentiates TRAIL-induced apoptosis of human
colorectal carcinoma cells through inhibition of NF-kappaB. Cell
Death Differ 14, 137-145. [0337] Wisniewski, J. R., Zougman, A.,
Nagaraj, N., and Mann, M. (2009). Universal sample preparation
method for proteome analysis. Nat Methods 6, 359-362. [0338] Wu, M.
J., Chang, C. S., Cheng, C. H., Chen, C. H., Lee, W. C., Hsu, Y.
H., Shu, K. H., and Tang, M. J. (2004). Colonic transit time in
long-term dialysis patients. Am J Kidney Dis 44, 322-327. [0339]
Yi, P., Park, J. S., and Melton, D. A. (2014). Perspectives on the
activities of ANGPTL8/betatrophin. Cell 159, 467-468. [0340] Zeki,
S. S., Graham, T. A., and Wright, N. A. (2011). Stem cells and
their implications for colorectal cancer. Nature reviews.
Gastroenterology & hepatology 8, 90-100. [0341] Zhao, J., Yang,
J., and Gregersen, H. (2003). Biomechanical and morphometric
intestinal remodelling during experimental diabetes in rats.
Diabetologia 46, 1688-1697. [0342] Ziegler, A. G., Rewers, M.,
Simell, O., Simell, T., Lempainen, J., Steck, A., Winkler, C.,
Ilonen, J., Veijola, R., Knip, M., et al. (2013). Seroconversion to
multiple islet autoantibodies and risk of progression to diabetes
in children. Jama 309, 2473-2479. [0343] Ziskin, J. L., Dunlap, D.,
Yaylaoglu, M., Fodor, I. K., Forrest, W. F., Patel, R., Ge, N.,
Hutchins, G. G., Pine, J. K., Quirke, P., et al. (2013). In situ
validation of an intestinal stem cell signature in colorectal
cancer. Gut 62, 1012-1023.
Sequence CWU 1
1
41240PRTHomo sapiens 1Met Gly Asn Cys Gln Ala Gly His Asn Leu His
Leu Cys Leu Ala His 1 5 10 15 His Pro Pro Leu Val Cys Ala Thr Leu
Ile Leu Leu Leu Leu Gly Leu 20 25 30 Ser Gly Leu Gly Leu Gly Ser
Phe Leu Leu Thr His Arg Thr Gly Leu 35 40 45 Arg Ser Pro Asp Ile
Pro Gln Asp Trp Val Ser Phe Leu Arg Ser Phe 50 55 60 Gly Gln Leu
Thr Leu Cys Pro Arg Asn Gly Thr Val Thr Gly Lys Trp 65 70 75 80 Arg
Gly Ser His Val Val Gly Leu Leu Thr Thr Leu Asn Phe Gly Asp 85 90
95 Gly Pro Asp Arg Asn Lys Thr Arg Thr Phe Gln Ala Thr Val Leu Gly
100 105 110 Ser Gln Met Gly Leu Lys Gly Ser Ser Ala Gly Gln Leu Val
Leu Ile 115 120 125 Thr Ala Arg Val Thr Thr Glu Arg Thr Ala Gly Thr
Cys Leu Tyr Phe 130 135 140 Ser Ala Val Pro Gly Ile Leu Pro Ser Ser
Gln Pro Pro Ile Ser Cys 145 150 155 160 Ser Glu Glu Gly Ala Gly Asn
Ala Thr Leu Ser Pro Arg Met Gly Glu 165 170 175 Glu Cys Val Ser Val
Trp Ser His Glu Gly Leu Val Leu Thr Lys Leu 180 185 190 Leu Thr Ser
Glu Glu Leu Ala Leu Cys Gly Ser Arg Leu Leu Val Leu 195 200 205 Gly
Ser Phe Leu Leu Leu Phe Cys Gly Leu Leu Cys Cys Val Thr Ala 210 215
220 Met Cys Phe His Pro Arg Arg Glu Ser His Trp Ser Arg Thr Arg Leu
225 230 235 240 2162PRTHomo sapiens 2Thr His Arg Thr Gly Leu Arg
Ser Pro Asp Ile Pro Gln Asp Trp Val 1 5 10 15 Ser Phe Leu Arg Ser
Phe Gly Gln Leu Thr Leu Cys Pro Arg Asn Gly 20 25 30 Thr Val Thr
Gly Lys Trp Arg Gly Ser His Val Val Gly Leu Leu Thr 35 40 45 Thr
Leu Asn Phe Gly Asp Gly Pro Asp Arg Asn Lys Thr Arg Thr Phe 50 55
60 Gln Ala Thr Val Leu Gly Ser Gln Met Gly Leu Lys Gly Ser Ser Ala
65 70 75 80 Gly Gln Leu Val Leu Ile Thr Ala Arg Val Thr Thr Glu Arg
Thr Ala 85 90 95 Gly Thr Cys Leu Tyr Phe Ser Ala Val Pro Gly Ile
Leu Pro Ser Ser 100 105 110 Gln Pro Pro Ile Ser Cys Ser Glu Glu Gly
Ala Gly Asn Ala Thr Leu 115 120 125 Ser Pro Arg Met Gly Glu Glu Cys
Val Ser Val Trp Ser His Glu Gly 130 135 140 Leu Val Leu Thr Lys Leu
Leu Thr Ser Glu Glu Leu Ala Leu Cys Gly 145 150 155 160 Ser Arg
3166PRTHomo sapiens 3Ser Phe Leu Leu Thr His Arg Thr Gly Leu Arg
Ser Pro Asp Ile Pro 1 5 10 15 Gln Asp Trp Val Ser Phe Leu Arg Ser
Phe Gly Gln Leu Thr Leu Cys 20 25 30 Pro Arg Asn Gly Thr Val Thr
Gly Lys Trp Arg Gly Ser His Val Val 35 40 45 Gly Leu Leu Thr Thr
Leu Asn Phe Gly Asp Gly Pro Asp Arg Asn Lys 50 55 60 Thr Arg Thr
Phe Gln Ala Thr Val Leu Gly Ser Gln Met Gly Leu Lys 65 70 75 80 Gly
Ser Ser Ala Gly Gln Leu Val Leu Ile Thr Ala Arg Val Thr Thr 85 90
95 Glu Arg Thr Ala Gly Thr Cys Leu Tyr Phe Ser Ala Val Pro Gly Ile
100 105 110 Leu Pro Ser Ser Gln Pro Pro Ile Ser Cys Ser Glu Glu Gly
Ala Gly 115 120 125 Asn Ala Thr Leu Ser Pro Arg Met Gly Glu Glu Cys
Val Ser Val Trp 130 135 140 Ser His Glu Gly Leu Val Leu Thr Lys Leu
Leu Thr Ser Glu Glu Leu 145 150 155 160 Ala Leu Cys Gly Ser Arg 165
4297PRTHomo sapiens 4Met Gln Arg Ala Arg Pro Thr Leu Trp Ala Ala
Ala Leu Thr Leu Leu 1 5 10 15 Val Leu Leu Arg Gly Pro Pro Val Ala
Arg Ala Gly Ala Ser Ser Ala 20 25 30 Gly Leu Gly Pro Val Val Arg
Cys Glu Pro Cys Asp Ala Arg Ala Leu 35 40 45 Ala Gln Cys Ala Pro
Pro Pro Ala Val Cys Ala Glu Leu Val Arg Glu 50 55 60 Pro Gly Cys
Gly Cys Cys Leu Thr Cys Ala Leu Ser Glu Gly Gln Pro 65 70 75 80 Cys
Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly Leu Arg Cys Gln Pro 85 90
95 Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu Leu Asp Gly Arg Gly
100 105 110 Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu Arg Ala Tyr
Leu Leu 115 120 125 Pro Ala Pro Pro Ala Pro Gly Glu Pro Pro Ala Pro
Gly Asn Ala Ser 130 135 140 Glu Ser Glu Glu Asp Arg Ser Ala Gly Ser
Val Glu Ser Pro Ser Val 145 150 155 160 Ser Ser Thr His Arg Val Ser
Asp Pro Lys Phe His Pro Leu His Ser 165 170 175 Lys Ile Ile Ile Ile
Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr 180 185 190 Lys Val Asp
Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser 195 200 205 Glu
Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu 210 215
220 Asp Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly
225 230 235 240 Val His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys
Lys Lys Gln 245 250 255 Cys Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe
Cys Trp Cys Val Asp 260 265 270 Lys Tyr Gly Gln Pro Leu Pro Gly Tyr
Thr Thr Lys Gly Lys Glu Asp 275 280 285 Val His Cys Tyr Ser Met Gln
Ser Lys 290 295
* * * * *
References