U.S. patent application number 14/159358 was filed with the patent office on 2014-06-19 for cytokine induction of selectin ligands on cells.
The applicant listed for this patent is Robert SACKSTEIN. Invention is credited to Robert SACKSTEIN.
Application Number | 20140170120 14/159358 |
Document ID | / |
Family ID | 38814616 |
Filed Date | 2014-06-19 |
United States Patent
Application |
20140170120 |
Kind Code |
A1 |
SACKSTEIN; Robert |
June 19, 2014 |
CYTOKINE INDUCTION OF SELECTIN LIGANDS ON CELLS
Abstract
Methods and compositions for treating cells with cytokines are
provided herein.
Inventors: |
SACKSTEIN; Robert; (Sudbury,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SACKSTEIN; Robert |
Sudbury |
MA |
US |
|
|
Family ID: |
38814616 |
Appl. No.: |
14/159358 |
Filed: |
January 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13038977 |
Mar 2, 2011 |
8633026 |
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14159358 |
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11779650 |
Jul 18, 2007 |
7998740 |
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13038977 |
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60831525 |
Jul 18, 2006 |
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Current U.S.
Class: |
424/93.7 ;
435/375 |
Current CPC
Class: |
A61P 37/00 20180101;
C12N 5/0634 20130101; A61P 35/00 20180101; A61K 2035/124 20130101;
C07K 2317/76 20130101; A61P 35/02 20180101; A61K 38/193 20130101;
A61K 38/202 20130101; C12N 2501/22 20130101; C12N 5/0669 20130101;
A61P 9/10 20180101; C07K 16/2854 20130101; A61K 38/1709 20130101;
A61P 43/00 20180101; A61K 38/20 20130101; A61P 29/00 20180101; A61K
35/15 20130101 |
Class at
Publication: |
424/93.7 ;
435/375 |
International
Class: |
A61K 35/14 20060101
A61K035/14 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] The work described herein was funded, in part through a
grant from the National Institutes of Health (grant RO1 HL060528).
The United States government has certain rights in the invention.
Claims
1. A method for increasing cell surface expression or activity of
an E-selectin ligand on a myeloid cell comprising, contacting the
myeloid cell with granulocyte colony stimulating factor (G-CSF) and
a sialidase inhibitor, thereby increasing cell surface expression
or activity of an E-selectin ligand on the cell.
2. The method of claim 1, wherein a plurality of myeloid cells are
provided.
3. The method of claim 1, wherein the myeloid cell is
autologous.
4. The method of claim 1, wherein the myeloid cell is
allogeneic.
5. The method of claim 1, wherein the myeloid cell is provided ex
vivo.
6. The method of claim 1, wherein the myeloid cell is contacted
with G-CSF in vitro.
7. The method of claim 1, wherein the E-selectin ligand is a
hematopoietic cell E-/L-selectin Ligand (HCELL) polypeptide.
8. The method of claim 1, wherein said method increases the
expression of a HECA-452-reactive epitope on the cell.
9. The method of claim 1, wherein the cell is treated ex vivo, and
wherein the method further comprises administering the cell to a
subject in need thereof.
10. The method of claim 8, wherein the cell is administered to a
subject as part of treatment with hematopoietic stem cell
transplantation.
11. The method of claim 8, wherein the cell is administered to a
subject who is in need of treatment for tissue injury.
12. The method of claim 1, wherein the sialidase inhibitor is
2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA).
13. The method of claim 1, wherein the cell is administered to a
subject as part of treatment with hematopoietic stem cell
transplantation.
14. The method of claim 1, wherein the cell is administered to a
subject as part of treatment for infection.
15. The method of claim 1, wherein the cell is administered to a
subject who is in need of treatment for tissue injury.
16. The method of claim 1, wherein the cell is a native human
myeloid cell.
17. The method of claim 1, wherein the cell is a native human
immature myeloid cell.
18. The method of claim 1, wherein the cell is a myeloid cell and
wherein the method increases the ability of the cell to migrate to
endothelial beds expressing E-selectin.
Description
RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 11/779,650, filed Jul. 18, 2007, which claims priority to
U.S. Provisional Application No. 60/831,525, filed Jul. 18, 2006,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to methods and compositions for
modulating cell expression of glycoproteins and glycolipids. The
invention also describes reagents and methods of modulating
E-selectin ligand activity, e.g., in cases where cytokine-induced
E-selectin ligand expression are associated with adverse
effects.
BACKGROUND
[0004] The capacity to direct migration of blood-borne cells to a
predetermined location ("homing") has profound implications for a
variety of physiologic and pathologic processes. Recruitment of
circulating cells to a specific anatomic site is initiated by
discrete adhesive interactions between cells in flow and vascular
endothelium at the target tissue(s).
[0005] Selectin-mediated interactions are critical not only for the
rapid and efficient recruitment of leukocytes at a site of injury,
but for steady state, tissue-specific homing as illustrated in: (1)
lymphocyte homing to peripheral lymph nodes, (2) cutaneous tropism
of human skin-homing T-cells and (3) hematopoietic progenitor cell
(HPC) entry into bone marrow.
SUMMARY
[0006] The invention is based, in part, on the discovery of methods
and compositions for modulating the expression or activity of
glycosylation enzymes in a cell. The methods increase the
expression or activity of a glycosylated cell-surface molecule,
such as a glycolipid or glycoprotein (e.g., a glycolipid or
glycoprotein that binds a selectin). The glycosylation enzyme is a
glycosyltransferase such as .alpha.2,3-sialyltransferase
(ST3GalIV), leukocyte .alpha.1,3-fucosyltransferases (FucT-IV,
FucT-VII or FucT-IX), or glycosyltransferase core 2.beta.1-6
N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) or a
glycosidase such sialidase. The methods and compositions described
herein are particularly useful for augmenting selectin ligand or
lewis antigen (e.g. CDS15) expression or activity on various cell
types, and can be applied to enhance the engraftment and/or
tissue-regenerative potential of cells. Accordingly, in one aspect,
the invention features a method for treating a cell by contacting
the cell with one or more cytokines that increase the expression or
activity of a glycosyltransferase polypeptide or glycosidase
polypeptide in a cell. For example the cell is contacted with two,
three, four, five or more cytokines. The method increases
cell-surface expression or activity of a selectin ligand, a lewis
antigen (e.g., lewis x), a VIM-2 epitope or a HECA-452-reactive
epitope on the cell. A selectin ligand is a glycoprotein or a
glycolipid. For example the selectin ligand is an E-selectin
ligand, an L-selectin ligand, a P-selectin ligand. In various
embodiments, the cytokine increases the cell-surface expression or
activity of an E-selectin ligand on the cell. The E-selectin ligand
is, for example, Hematopoietic Cell E-/L-selectin Ligand (HCELL),
or the .about.65 kDa E-selectin described herein. In various
embodiments, the method increases the affinity of the cell for a
selectin.
[0007] The cell can be a hematopoietic cell, such as a
hematopoietic stem cell, e.g., a CD34+ hematopoietic stem cell, a
peripheral blood leukocyte, a lymphocyte, or a myeloid cell, such
as an immature myeloid cell. Other types of cells, including
non-hematopoietic cells, may also be treated according to the
methods. Appropriate non-hematopoietic cells express a receptor for
the cytokine of interest. For example, glial and neuronal cells
express receptors for granulocyte colony stimulating factor
(G-CSF). Neurons are also sensitive to macrophage
colony-stimulating factor (M-CSF) and interleukin-3 (IL-3).
[0008] In various embodiments, the cytokine modulates selectin
ligand expression selectively, on a particular type of cell (e.g.,
the cytokine selectively acts on hematopoietic cells or another
subset of cells). In various embodiments, the cell contacted with
the cytokine is not a T cell.
[0009] In various embodiments, a plurality of cells is provided. A
cell can be provided ex vivo, or in vivo. The cell can also be
contacted with the cytokine in vitro.
[0010] The cytokine is contacted with the cell in a concentration
range of 1-1,000 ng/ml, 1-100 ng/ml, 1-50 ng/ml, 1-25 ng/ml, or
1-10 ng/ml.
[0011] Suitable cytokines include those that modulate the
expression or activity of a selectin ligand and/or enzymes that
modulate the selectin-binding activity of a selectin ligand, such
as carbohydrate modifying enzymes such that expression of
selectin-binding epitopes increases. In one embodiment, the
cytokine is granulocyte colony stimulating factor (G-CSF).
Alternatively, the cytokine is granulocyte macrophage colony
stimulating factor (GM-CSF), macrophage colony stimulating factor
(M-CSF), interleukin-3 (IL-3)/multi colony stimulating factor
(Multi-CSF), transforming growth factor .beta. (TGF.beta.), an
interferon, a chemokine, an interleukin or a tumor necrosis factor.
An interleukin includes, for example, IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,
IL-27, IL-28, and IL-29.
[0012] The cell is treated ex vivo, and the method further includes
administering the cell, or a plurality of cells, to a subject,
e.g., a subject in need of treatment for tissue injury, and/or cell
as part of a hematopoietic stem cell transplantation protocol.
Optionally, the cells which are administered are a selected subset
of cells (e.g., a subset which have been selected based on a
particular phenotype or expression of a cell surface marker, such
as a stem cell marker). The cells may be enriched for those which
express high levels of the selectin ligand. Additionally, the
method includes selecting a subpopulation of cells (e.g., a
subpopulation of leukocytes, or stem cells, such as hematopoietic
stem cells, or stem cells which support regeneration of a desired
tissue) prior to contacting the cells with a cytokine.
[0013] In another aspect, the invention features a kit for
treatment of a cell to increase its engraftment and/or regenerative
potential. The kit includes, for example: a composition comprising
a cytokine (e.g., G-CSF), and instructions for use of the cytokine
to treat a cell under conditions in which the cytokine increases
the cell-surface expression or activity of a selectin ligand
polypeptide on the cell, thereby increasing the engraftment and/or
regenerative potential of the cell.
[0014] In another aspect, the invention features a composition
comprising a glycoprotein isolated from granulocyte colony
stimulating factor-treated peripheral blood leukocytes, wherein the
glycoprotein is approximately 65 kDa, is reactive with monoclonal
antibody HECA-452, and is a ligand for E-selectin. The glycoprotein
may be purified or isolated. In various embodiments, the
glycoprotein includes other features described herein. The
invention also includes derivatives of the 65 kDa glycoprotein
which compete with the natural 65 kDa glycoprotein for binding to
E-selectin.
[0015] In another aspect, the invention features a method for
treating a subject who has received, or is scheduled to receive
granulocyte colony stimulating factor (G-CSF). The method include:
administering to the subject an agent which inhibits a
selectin-mediated (e.g., E-selectin-mediated) interaction with a
selectin ligand. In various embodiments, the method reduces side
effects due to administration of G-CSF, such as enhanced
leukocyte-endothelial interactions that are associated with adverse
inflammatory reactions. The agent is, for example, an antibody or
antigen-binding fragment thereof, a small interfering RNA, or an
antisense oligonucleotide. In various embodiments, the agent
inhibits the expression or activity of a glycosyltransferase and/or
interferes with carbohydrate synthesis. The agent may also be a
soluble carbohydrate or glycosylated polypeptide which directly
inhibits an interaction between a selectin and a selectin ligand,
e.g., by competition for binding to either the selectin or the
selectin ligand. Examples of suitable agents include soluble
mimetics of selectin ligands. The agent can be a soluble form of a
natural selectin, selectin ligand or a derivative thereof which
exhibits enhanced affinity for the selectin ligand or for the
selectin, respectively, or to both.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and are not intended to be
limiting.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 A-G illustrates ML possess enhanced binding to
E-selectin relative to native leukocytes (NL). Human umbilical vein
endothelial cells (HUVEC) were stimulated with TNF-.alpha.for 4-6
hrs. Subsequently, ML or NL were perfused in a parallel plate
apparatus over HUVEC at 1.5 dyne/cm.sup.2. In certain instances,
HUVEC were pretreated with a mAb to E-selectin prior to use in
adhesion assays. (A) Primary tethering and (B) average rolling
velocity of ML or NL on HUVEC was determined. "Stim" indicates
stimulation (+) or no stimulation (-) of HUVEC with TNF-.alpha. for
4-6 hours prior to the assay; .alpha.-E-sel indicates pretreatment
(+) or no pretreatment (-) of HUVEC with a function blocking mAb to
E-selectin, (68-5411); EDTA indicates presence (+) or absence (-)
of 5 mM EDTA in the assay buffer. Values are means.+-.SE of
n.gtoreq.4 different runs. * indicates statistically significant
difference (p<0.05). C-D Are bar graph results of ML or NL
perfused over CHO-E at 1 dyne/cm.sup.2. In certain instances, CHO-E
were pretreated with a function blocking mAb to E-selectin prior to
use in adhesion assays. (C) Primary tethering and (D) average
rolling velocity of ML or NL on CHO-E was determined. .alpha.-E-sel
indicates pretreatment (+) or no pretreatment (-) of CHO-E with a
function blocking mAb to E-selectin, (68-5411); EDTA indicates
presence (+) or absence (-) of 5 mM EDTA in the assay buffer;
Values are means.+-.SE of n.gtoreq.5 different runs. * indicates
statistically significant difference (p<0.05). E-G Show results
of ML and NL that were treated with DiD ex vivo and injected into
the tail vein of mice 5-6 hours after stimulation of one ear
locally with TNF-.alpha.. (E) Represents the average rolling
velocity of ML or NL on inflamed vascular endothelium in
TNF-.alpha.-treated ears of mice. Values represent means.+-.SE of
10-20 leukocytes per mouse with n=3 for each group. (F) Shows
representative images of the adhesive interactions of NL (top) and
ML (bottom) with inflamed vascular endothelium in
TNF-.alpha.-treated ears of two separate mice. Minimal adhesive
interactions were observed in control PBS-treated ear (not shown).
(G) is a bar graph showing adherent leukocytes per field of view.
Values represent means.+-.SE of 15-25 fields of view per mouse with
n=3 for each group. * indicates statistically significant
difference (p<0.05).
[0019] FIGS. 2A-E illustrates how ML express multiple
HECA-452-reactive E-selectin glycoprotein ligands. (A) Shows
HECA-452 blots of cell lysate from (unfractionated) buffy coat of
ML or NL resolved on a reducing 4-20% SDS-PAGE gel. (B) Shows
HECA-452 blots of membrane preparations of mononuclear fraction of
G-CSF mobilized peripheral blood (MPB) leukocytes (20 .mu.g; ML-M),
G-CSF mobilized granulocytes (40 .mu.g; ML-G); native peripheral
blood mononuclear cells (40 .mu.g; PBMC) or native peripheral blood
polymorphonuclear leukocytes (20 .mu.g; PMN) resolved on a reducing
4-20% SDS-PAGE gel. Note the distinct presence of HECA-452-reactive
.about.100 kDa and .about.65 kDa bands in G-CSF mobilized
leukocytes. In all experiments, rat IgM isotype control blots
performed in parallel lacked staining. Results presented are
representative of observations on numerous HECA-452 blots from
numerous clinical samples of G-CSF MPB. (C) Is a bar graph of CHO-E
that were perfused over SDS-PAGE immunoblots of HECA-452 reactive
membrane glycoproteins of ML-M at 0.6 dyne/cm.sup.2 and the number
of interacting cells/mm.sup.2 was tabulated as a function of
molecular weight. The background binding was subtracted and the
results compiled into an adhesion histogram. Results presented are
representative of multiple runs and multiple observations on
numerous HECA-452 blots of membrane preparations of ML-M. (D)
Membrane preparations of ML-M (20 .mu.g) were resolved on a
reducing 4-20% SDS-PAGE gel and immunoblotted with E-selectin-Ig
(E-Ig) chimera in the presence of Ca.sup.2+. The E-Ig chimera
reactive glycoproteins at .about.220 kDa, .about.130 kDa,
.about.100 kDa, and .about.65 kDa corresponded exactly with the
proteins stained by HECA-452. Result shown is representative of
multiple observations on numerous E-Ig blots of various clinical
specimens of ML-M. (E) Shows the results of E-Ig used to
immunoprecipitate E-selectin ligands from membrane preparations of
ML-M, and the resolved immunoprecipitate was blotted with HECA-452.
HECA-452 stained E-Ig immunoprecipitated material at .about.220
kDa, .about.130 kDa, .about.100 kDa and .about.65 kDa bands.
[0020] FIGS. 3A-F illustrates the characterization of PSGL-1, HCELL
and a novel E-selectin ligand on ML. (A) Shows the membrane
preparations of ML-M were resolved on a reducing 4-20% SDS PAGE
gel, and immunoblotted with KPL-1, an antibody to PSGL-1. The bands
at .about.220 kDa and .about.130 kDa corresponded with the
HECA-452-reactive membrane glycoproteins on ML-M. Mouse IgG.sub.1,k
isotype control blots performed in parallel lacked staining. (B)
Shows that KPL-1 was used to immunoprecipitate PSGL-1, and the
resolved immunoprecipitate was blotted with either HECA-452 or
KPL-1. (C) Illustrates the membrane preparations of ML-M were
resolved on a reducing 4-20% SDS PAGE gel, and immunoblotted with
Hermes-1, an antibody to CD44. The band at .about.100 kDa
corresponded with the HECA-452-reactive membrane glycoprotein on
ML-M. Rat IgG isotype control blots performed in parallel lacked
staining. (D) Hermes-1 was used to immunoprecipitate CD44, and the
resolved immunoprecipitate was blotted with either HECA-452 or
another anti-human CD44 mAb, 2C5. (E) Is a graph showing the
results of mAb Dreg-56 used to determine L-selectin expression on
ML and NL using flow cytometry. mIgG.sub.1,k served as isotype
control for Dreg-56. Results shown are typical of multiple clinical
specimens. (F) Shows the results of L-selectin immunoprecipitated
from biotinylated ML-M (MPB) and KG1a cells was resolved on a
reducing SDS-PAGE gel, and immunoblotted with horseradish
peroxidase (HRP) conjugated strepavidin. Note that the .about.80
kDa L-selectin band is present in KG1a cells and absent in
ML-M.
[0021] FIGS. 4A-B illustrates ML possess enhanced levels of
glycosyltransferases ST3GalIV, FucT-IV and FucT-VII. Total RNA from
equal numbers of ML and NL was subjected to RT-PCR followed by PCR
amplification of pairs of cDNAs for ST3GalIV, FucT-IV, FucT-VII and
the housekeeping gene GAPDH. (A) Shows the net intensity of
amplified bands was normalized to the net intensity of respective
GAPDH controls. All values are means.+-.SE of at least 3 different
experiments. (B) Shows that typical blots of PCR amplified products
from NL and ML RNA are presented.
[0022] FIGS. 5A-C shows HCELL and .about.65 kDa glycoprotein are
major E-selectin ligands on ML. (A) Are graphs of PSGL-1, CD44 and
HECA-452 antigen(s) expression determined on untreated (native) and
OSGE-treated ML using flow cytometry. mAbs KPL-1 and F10-44-2 were
used to determine expression of PSGL-1 and CD44, respectively.
mIgG.sub.1,k and mIgM served as isotype controls for KPL-1 and
F10-44-2, respectively. rIgM served as isotype control for
HECA-452. Note that OSGE treatment abrogates surface expression of
PSGL-1 and has minimal effect on expression of CD44 or HECA-452
antigen(s). Results shown are representative of 2 different
experiments. (B) E-Ig blots of cell lysates from equal numbers of
untreated (-) or 30 .mu.g/ml OSGE treated (+) ML resolved on a
reducing 4-20% SDS-PAGE gel. Note that OSGE treatment markedly
abrogates E-selectin binding capacity of PSGL-1 with little to no
effect on E-selectin binding capacity of HCELL and .about.65 kDa
E-selectin ligand. (C) Is a bar graph showing the results of HUVEC
that were stimulated with TNF-.alpha. for 4-6 hrs. Subsequently,
untreated (-) or OSGE (+) treated ML were perfused over HUVEC at
1.5 dyne/cm.sup.2. Primary tethering of untreated or OSGE-treated
ML on HUVEC was determined. Values are means.+-.SE of n.gtoreq.3
different runs.
[0023] FIGS. 6A-E shows in vitro G-CSF treatment of human bone
marrow (BM) cells up-regulates the expression of HCELL and
HECA-452-reactive .about.65 kDa glycoprotein. (A) (left) HECA-452
and (right) E-Ig blots of cell lysates from human BM mononuclear
cells (BM-MNC) or BM CD34+/lineage- cells (Lin-) resolved on a
reducing 4-20% SDS-PAGE gel. (B) (left) HECA-452 and (right) E-Ig
blots of cell lysates from Band 1 (B1), Band 2 (B2) and Band 3 (B3)
ML resolved on a reducing 4-20% SDS-PAGE gel. Note the presence of
HCELL and HECA-452 reactive .about.65 kDa glycoprotein
predominantly in Band 1 and Band 2 cells. (C) (left) HECA-452 and
(right) E-Ig blots of cell lysates from untreated (-) or 72 hr.
G-CSF treated (+) Band 1, Band 2 and Band 3 human BM cells resolved
on a reducing 4-20% SDS-PAGE gel. Note that G-CSF treatment results
in a marked up-regulation of .about.100 kDa HCELL and
HECA-452-reactive .about.65 kDa glycoprotein predominantly in
immature myeloid cells. (D-E) Total RNA from equal numbers of
untreated and G-CSF-treated Band 2 BM cells was subjected to RT-PCR
followed by PCR amplification of pairs of cDNAs for ST3GalIV,
FucT-IV, FucT-VII and the housekeeping gene GAPDH. (D) The net
intensity of amplified bands was normalized to the net intensity of
respective GAPDH controls. All values are means.+-.SE of at least 3
different experiments. (E) Shows typical blots of PCR amplified
products from untreated (U) and G-CSF-treated (G) Band 2 BM cells
RNA are presented.
[0024] FIG. 7 illustrates that there is no distinct difference in
the surface expression of integrin-type homing receptors (e.g.,
LFA-1 (CD11a/CD18; .alpha.L.beta.2) and VLA-4 (CD49d/CD29;
.alpha.4.beta.1)) and chemokine receptor CXCR4 on ML and NL. CD11a,
CD18, CD29, CD49d and CXCR4 expression was determined on ML and NL
using flow cytometry. mAbs 25.3, 7E4, HUTS-21, HP2/1 and 12G5 were
used to determine expression of CD11a, CD18, CD29, CD49d and CXCR4,
respectively. mIgG.sub.1,k served as an isotype control for 25.3,
7E4 and HP2/1 and mIgG.sub.2a served as an isotype control for
HUTS-21 and 12G5. Results shown are representative of 2 separate
experiments.
[0025] FIG. 8 shows HECA-452-reactive glycoproteins of ML are
sensitive to sialidase treatment. Membrane preparations of ML-M (10
.mu.g) were treated with sialidase (+) or buffer treated (-),
resolved on a reducing 4-20% SDS-PAGE gel and immunoblotted with
HECA-452. Absence of staining following sialidase digestion
confirms specificity of HECA-452 staining for sialofucosylated
carbohydrate modifications.
[0026] FIGS. 9A-B illustrate E-Ig-reactive glycoproteins of ML do
not stain in the presence of EDTA or with control human-Ig.
Membrane preparations of ML-M (20 .mu.g) were resolved on a
reducing 4-20% SDS-PAGE gel and immunoblotted with (A)
E-selectin-Ig (E-Ig) chimera in the presence of 10 mM EDTA or (B)
human-Ig. The E-Ig chimera reactive glycoproteins at .about.220
kDa, .about.130 kDa, .about.100 kDa, and .about.65 kDa (FIG. 2d) do
not stain in the presence of EDTA or with control human-Ig. Results
shown are typical of 2 separate experiments.
[0027] FIG. 10 shows HECA-452-reactive .about.65 kDa E-selectin
ligand does not appear to be related to PSGL-1 or CD44. PL2 and 2C5
were used to immunoprecipitate PSGL-1 and CD44, respectively, from
membrane preparations of ML-M. Mouse IgG.sub.1 was used as an
isotype control for immunoprecipitation. The immunoprecipitated
materials were resolved on a reducing 4-20% SDS PAGE gel, and
immunoblotted with HECA-452. Note that PL-2 and 2C5 did not
immunoprecipitate the HECA-452-reactive .about.65 kDa
glycoprotein.
[0028] FIGS. 11A-B illustrates ML possess diminished binding to
P-selectin relative to NL. ML or NL were perfused over CHO-P at 1.0
dyne/cm.sup.2. (A) Illustrates primary tethering and (B) shows
rolling velocity of ML or NL on CHO-P was determined. Values are
means.+-.SE of n.gtoreq.6 different runs. * indicates statistically
significant difference (p<0.05).
[0029] FIGS. 12A-B illustrates G-CSF treatment enhances the
capability of human BM cells to adhere to endothelial E-selectin
under physiologic flow conditions. HUVEC were stimulated with
IL-1.beta. for 4-6 hrs. Subsequently, untreated or 72 hr.
G-CSF-treated Band 2 cells were perfused over HUVEC at 1.5
dyne/cm.sup.2. In certain instances, stimulated HUVEC were
pretreated with a mAb to E-selectin prior to use in adhesion
assays. (A) Illustrates primary tethering and (B) shows rolling
velocity of untreated or G-CSF-treated cells on stimulated HUVEC.
Stim. indicates pretreatment (+) or no pretreatment (-) of HUVEC
with IL-1.beta. for 4-6 hours prior to the assay; .alpha.-E-sel
indicates pretreatment (+) or no pretreatment (-) of HUVEC with a
function blocking mAb to E-selectin, (68-5411); EDTA indicates
presence (+) or absence (-) of 5 mM EDTA in the assay buffer.
Values are means.+-.SE of n.gtoreq.5 different runs. * indicates
statistically significant difference (p<0.05).
[0030] FIG. 13 illustrates that G-CSF treatment increases Fuct-IX
expression in normal progenitors and in mobilized peripheral blood.
Lane 1 are normal cells isolated from bone marrow from healthy
donor. Lane 2 are G-CSF treated normal progenitors isolated from
bone marrow from healthy donor. Lane 3 are untreated cells isolated
from G-CSF mobilized blood. Lane 4 are G-CSF treated cells isolated
from G-CSF mobilized blood.
[0031] FIG. 14 is a bar graph showing that G-CSF treatment of human
myeloid bone marrow cells increases sialidase activity. Sialidase
activity from human myeloid cells (obtained from bone marrow of
normal subjects), before and after treatment with 10 ng/mL of G-CSF
for 24 hours was measured using 4-MU-NANA as substrate, * indicates
significantly different (p.ltoreq.0.05).
[0032] FIG. 15 is a bar graph illustrating that inhibition of
sialidase with DANA blunts G-CSF-induced increase in CD15. Bone
marrow cells were treated with 10 ng/mL of G-CSF for 5 days in the
presence or absence of 100 mM DANA and expression of CD15 was
analyzed by flow cytometry.
[0033] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0034] The invention is based in part on the surprising discovery
that cytokines can induce the expression of glycosylation enzymes
in a cell. Specifically, G-CSF induced the expression of
glycosyltranferases in a cell which resulted in the increased
expression of hematopoietic cell E-/L-selectin ligand (HCELL) and
an approximately 65 kDa E-selectin ligand. Additionally, G-CSF
induced the expression of sialidase which resulted in an increased
expression of CD15.
[0035] Granulocyte colony stimulating factor (G-CSF) is widely used
clinically to augment neutrophil recovery after myelosuppressive
chemo/radiotherapy and for mobilizing bone marrow (BM)
hematopoietic progenitors for use in hematopoietic stem cell
transplantation (HSCT).sup.1. Though generally considered safe,
there are increasing observations that G-CSF administration can
promote leukocyte-endothelial adhesive interactions resulting in
vascular and inflammatory complications.sup.2-13. Indeed, G-CSF
administration has been shown to (i) recruit neutrophils to lung
vasculature resulting in respiratory distress syndrome.sup.4-6,
(ii) stimulate granulocyte adherence to endothelium resulting in
angina pectoris/myocardial infarct.sup.7, (iii) cause neutrophil
infiltration in dermal vessels.sup.8,9 leading to development of
cutaneous leukocytoclastic vasculitis.sup.10, (iv) intensify
arthritic symptoms.sup.11 and (v) precipitate sickle cell
vaso-occlusion.sup.12. Though G-CSF administration may have
favorable effects on myocardial recovery following infarct in
preclinical models.sup.14, a recent report of G-CSF administration
in patients with coronary artery disease revealed a striking
incidence of cardiac ischemic complications.sup.13. A better
understanding of the molecular basis of the enhanced
leukocyte-endothelial interactions accompanying clinical G-CSF
administration could yield strategies to prevent these
complications.
[0036] Despite decades of clinical observations on G-CSF biology,
the effects of G-CSF administration on leukocyte membrane molecules
that bind endothelial counter-receptors under hemodynamic shear
conditions are unknown. In this study, the capacity of leukocytes
mobilized by G-CSF (ML) to bind to inflamed
(TNF-.alpha.-stimulated) endothelium was evaluated. Parallel plate
assays conducted under physiologic flow conditions and intravital
microscopy of a murine inflammation model each showed that,
compared to NL, ML display markedly increased adhesive interactions
with inflamed endothelium, mediated by enhanced E-selectin
receptor/ligand interactions. ML expressed the potent E-selectin
ligand HCELL and another heretofore unrecognized E-selectin
glycoprotein ligand of .about.65 kDa, and possessed enhanced levels
of critical glycosyltransferases (ST3GalIV, FucT-IV, FucT-VII and
FucT-IX) rendering E-selectin ligand activity. Enzymatic removal of
PSGL-1 revealed that these novel ligands are the principal
mediators of the robust ML adhesion to vascular E-selectin.
Treatment of normal human BM cells with clinically-relevant serum
levels of G-CSF in vitro increased the expression of pertinent
glycosyltransferases directly inducing the expression of these two
ligands and resulting in enhanced E-selectin-mediated endothelial
binding. Collectively, these results provide first evidence that
enhanced leukocyte-endothelial interactions following G-CSF
administration is mediated by G-CSF-induced expression of
counter-receptors for vascular E-selectin among circulating myeloid
cells and offer mechanistic insights on the molecular basis of
G-CSF-induced increased E-selectin ligand activity.
[0037] In this study, no distinct change in HECA-452 reactivity of
PSGL-1 on ML compared to that of NL (FIG. 2a) was observed.
Furthermore, OSGE treatment of ML decreased PSGL-1 expression and
function without affecting the overall E-selectin binding capacity
of ML (FIG. 4). Interestingly, ML possessed diminished P-selectin
binding compared to NL (FIG. 11) suggesting that PSGL-1 function is
also altered on ML. Consistent with prior studies.sup.34, we
observed marked decrease in surface L-selectin expression on
circulating leukocytes following G-CSF administration (FIG. 3e),
excluding a role for L-selectin as an E-selectin ligand on ML.
[0038] In contrast to the broad distribution of PSGL-1, HCELL is
characteristically found primarily on normal human BM CD34+
progenitors.sup.28,29. Herein, biochemical studies of ML show that
G-CSF administration results in robust HCELL expression on
circulating (mobilized) myeloid cells, most prominently on immature
myeloid cells (FIG. 6b). The observed augmented HCELL expression is
a direct effect of G-CSF, as exposure to pharmacologically-relevant
G-CSF concentrations in vitro.sup.44,45 results in increases in
glycosyltransferases and the elaboration of HECA-452-reactive
glycosylations rendering the HCELL phenotype on immature human BM
myeloid cells (FIG. 6c). Though low level expression of HCELL on
native circulating human PMNs is observed occasionally, G-CSF had
only a variable effect on inducing HCELL expression among mature
myeloid BM cells. Importantly, HCELL is not expressed on
lymphocytes in BM or in blood, and it is not induced on lymphocytes
by G-CSF treatment.
[0039] In addition to induction of HCELL, G-CSF administration in
vivo and in vitro also induces the expression of a
HECA-452-reactive .about.65 kDa glycoprotein. The results of
blot-rolling assays, Western blot staining with E-Ig, and
immunoprecipitation with E-Ig of membrane preparations of ML show
that the .about.65 kDa glycoprotein is a high affinity E-selectin
ligand. The G-CSF-induced .about.65 kDa E-selectin ligand does not
appear to be a glycoform of other previously described E-selectin
ligands (PSGL-1, CD44, and L-selectin (FIG. 3)), and thus
represents a novel E-selectin ligand. Importantly, this .about.65
kDa glycoprotein is found predominantly among cells within the
mononuclear fraction of ML (ML-M cells) (FIG. 6b). Although the
various leukocyte subset(s) that express this .about.65 kDa
glycoprotein are currently unknown and warrant further
investigation, its expression is conspicuously absent from
lymphocytes (both T- and B-cells) within the ML-M fraction.
[0040] From a clinical perspective, defining the molecular
mechanism(s) of G-CSF-induced vascular and inflammatory
complications is of paramount importance as healthy individuals are
increasingly being exposed to this agent to serve as donors for
hematopoietic stem cell therapy (HSCT). It is plausible that G-CSF
may preferentially mobilize subset(s) of myeloid cells which
express high affinity E-selectin ligands. Consistent with this
notion are clinical observations that G-CSF-associated adverse
events occur in parallel to increases in leukocyte
numbers.sup.4,9,12. However, not all donors with high leukocyte
counts will exhibit complications, suggesting that some individuals
may be particularly susceptible to G-CSF-induced vascular and
inflammatory problems. To some extent, this may reflect
variabilities in the capacity of G-CSF to induce E-selectin ligand
expression on circulating myeloid cells and/or responsiveness of
the endothelial cells of G-CSF recipients to the induction of
E-selectin expression. However, in multiple clinical ML
collections, it was observed that G-CSF uniformly increased
E-selectin ligand activity of circulating leukocytes and
upregulated the expression of the E-selectin ligands HCELL and the
HECA-452-reactive .about.65 kDa glycoprotein. The cumulative effect
of these additional, G-CSF-induced E-selectin ligands to that of
natively expressed PSGL-1 on immature (and mature) myeloid cells
may prime these circulating cells to adhere to inflamed/ischemic
endothelium, consistent with emerging clinical experiences/reports
raising warnings for the use of G-CSF in individuals with known or
suspected inflammatory or cardiovascular diseases.
[0041] Under physiologic blood flow conditions, leukocytes
initially make contact on the vessel surface by engagement of
counter-receptors for relevant endothelial molecules that mediate
shear-resistant interactions.sup.15,16. One of the principal
effectors of these interactions is E-selectin, which is an
inducible endothelial molecule expressed at sites of
inflammation.sup.16,17 that binds sialofucosylated carbohydrate
ligands expressed on leukocytes.sup.18. An expanding body of
evidence causally links upregulated E-selectin expression to
vascular complications of G-CSF administration.sup.19-22. Notably,
the receptor for G-CSF is expressed on endothelium.sup.23 and G-CSF
directly induces E-selectin expression on endothelial cells in
culture.sup.24. However, there is little information on whether
G-CSF administration modifies E-selectin ligand expression on
mobilized, circulating leukocytes.
[0042] These findings provide new perspectives on selectin ligands
and on the biology of G-CSF, and indicate that induction of potent
counter-receptors for E-selectin is contributory to the vascular
and inflammatory complications observed with the use of this
agent.
[0043] Methods of Increasing Glycosylation Enzyme Expression or
Activity
[0044] Glycosylation enzyme expression or activity in a cell is
increased by contacting a cell with a cytokine. Glycosylation
enzymes include for example glycosytransferases or glycosidases.
Glycosyltransferase catalyze the transfer of glycosyl groups to an
acceptor and are responsible for the formation of glycosidic bonds.
In contrast, glycosidases catalyze hydrolysis of the glycosidic
linkage and are responsible for the trimming of glycans during
carbohydrate synthesis. Glycosyltransferases and glycosidases form
the major catalytic machinery for the synthesis and breakage of
glycosidic bonds involved in carbohydrate synthesis.
[0045] Glycosyltransferase includes for example, core 1
glycosyltransferase (e.g., .beta.-3-galactosyltransferase); core 2
glycosyltransferase (e.g., N-acetylglucosaminyltransferases such as
.beta.-(1,6) N-acetylglucosaminyltransferase, GnTI, GnTII, GnTIII,
GlcNAcT1; sialyltransferases (e.g., .alpha.-sialyltransferases,
such as .alpha.-2,3 sialyltransferases (ST3GalIV) .alpha.-2,6
sialyltransferases, and .beta.-sialyltransferases);
fucosyltransferases (e.g., .alpha.-fucosyltransferases, such as
.alpha.-1,3 fucosyltransferases (FucT IV, FucT VII, FucT IX) and
.beta.-fucosyltransferases; galactosyltransferases; (e.g.,
.alpha.-galactosyltransferases, such as .alpha.1,3
galactosyltransferases and .beta.-galactosyltransferases);
mannosyltransferases (e.g., .alpha.-mannosyltransferases and
.beta.-mannosyltransferases), or N-acetylgalactosaminyltransferases
(e.g., .alpha.-(1,3) N-acetylgalactosaminyltransferase and
.beta.-(1,4) N-acetylgalactosaminyltransferases).
[0046] Glycosidases include for example glucosidases, mannosidases,
fucosidases, sialidases, galactosidases, xylanases, lactases,
amylases, chitinases, sucrases, maltases, neuraminidases,
invertases, hyaluronidase or lysozymes.
[0047] Cytokines include for example, granulocyte colony
stimulating factor (G-CSF), granulocyte macrophage colony
stimulating factor (GM-CSF), macrophage colony stimulating factor
(M-CSF), interleukin-3 (IL-3)/multi colony stimulating factor
(Multi-CSF), transforming growth factor .beta. (TGF.beta.), an
interferon, a chemokine, a tumor necrosis factor or an interleukin,
such as interleukin-1 (IL-1) IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-14, IL-16, IL-17,
IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,
IL-27, IL-28, and IL-29.
[0048] Cells to be treated include any cell capable of expressing a
glycoprotein or a glycolipid. The cell is a hematopoietic cell,
such as a hematopoietic stem cell, e.g., a CD34+ hematopoietic stem
cell, a bone marrow cell, a neutrophil, a peripheral blood
leukocyte, a lymphocyte, or a myeloid cell, such as an immature
myeloid cell. Alternatively, the cell is a non-hematopoietic cells
such as a liver cell, a lung cell, a pancreatic cell, a cardiac
cell, a gastric cell or a kidney cell. The cell is contacted in
vitro, ex vivo or in vivo.
[0049] Alternatively, the cell is contacted with a cytokine in an
amount sufficient to increase expression or activity of a
glycoprotein or glycolipid. The glycoprotein or glycolipid is
cytosolic or cell membrane protein or lipid. By increased
expression or activity of a glycoprotein or a glycolipid it is
meant that the cell expresses a greater amount of the glycoprotein
or a glycolipid as compared to a cell that has not been contacted
with the cytokine or that the affinity of the cell for the ligand
of the glycoprotein or a glycolipid is increased. For example, the
treated cell has an increase affinity for a selectin. Affinity is
measured by methods known in the art.
[0050] For example, cell surface expression of a selectin ligand,
or a lewis antigen is increased following treatment of the cell
with the cytokine. Selectins include E-selectin, L-selectin or
P-selectin. The selectin ligands include, for example, MAdCAM1,
CD334, PSGL-1, CD24, ESL-1 or the VIM-2 epitope. Preferably, the
selectin ligand is, a hematopoietic cell E-/L-selectin ligand
(HCELL) or a 65 kDa E-selectin ligand described herein. Lewis
antigens include Le.sup.a epitope, the Le.sup.b epitope, Le.sup.x
or the Le.sup.y epitope. The Lewis antigen is sialyated.
Preferably, cell surface expression of a Le.sup.x epitope or the
siayl Le.sup.x epitope is increased following cytokine treatment of
the cell. Exemplary, Le.sup.x antigens include CD15 and CD15s.
[0051] The invention also provides methods to treat or alleviate
the symptoms of a variety of disorders. For example, cells produced
by the methods of the invention can be administered to a subject to
treat a tissue injury or as part of a stem cell transplant
protocol. The cells treated according to the method of the
invention have an increased regenerative/engraftment potential and
are useful for a variety of therapeutic methods including, tissue
repair, tissue regeneration, and tissue engineering. By increased
regenerative/engraftment potential it is meant that the cell has a
greater survival rate after transplantation as compared to an
untreated cell.
[0052] For example, the cells treated according to the methods of
the invention are useful in bone regeneration, cardiac
regeneration, vascular regeneration, neural regeneration and the
treatment of ischemic disorders. Ischemic conditions include, but
are not limited to, limb ischemia, congestive heart failure,
cardiac ischemia, kidney ischemia, ESRD, stroke, and ischemia of
the eye. The cells are administered to mammalian subjects, to
effect tissue repair or regeneration. The cells are administered
allogeneically or autogeneically.
[0053] The cell can be of mesodermal, ectodermal or endoderamal
origin. Preferably, the cell is a stem cell. More preferably the
cell is of mesodermal origin. For example, the cell is a
hematopoietic progenitor cell.
[0054] Included in the invention is a method of increasing the
engraftment potential of a cell by providing a cell and contacting
said cell with one or more cytokines that increases cell-surface
expression or activity of a selectin ligand, e.g. HCELL on the
cell. The invention further provides a method of increasing levels
of engrafted stem cells in a subject, e.g., human, by administering
to the subject a cytokine that increases cell-surface or expression
of a selectin ligand on one or more stem cells in the subject. The
cytokine can be administered in vivo, ex vivo or in vitro.
[0055] The subject is preferably a mammal. The mammal can be, e.g.,
a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
Additionally, the subject suffers from or is at risk of developing
a hematopoietic disorder, e.g, leukemia, cancer, or a tissue
injury. A mammal suffering from or at risk of developing a
hematopoietic disorder, e.g, leukemia, cancer, or tissue injury can
be identified by the detection of a known risk factor, e.g.,
gender, age, prior history of smoking, genetic or familial
predisposition, attributed to the particular disorder.
Alternatively, a mammal suffering from or at risk of developing a
hematopoietic disorder, e.g, leukemia, or tissue injury can be
identified by methods known in the art to diagnosis a particular
disorder.
[0056] Pharmaceutical Administration and Dosage Forms
[0057] The described cells can be administered as a
pharmaceutically or physiologically acceptable preparation or
composition containing a physiologically acceptable carrier,
excipient, or diluent, and administered to the tissues of the
recipient organism of interest, including humans and non-human
animals. Cell-containing compositions can be prepared by
resuspending the cells in a suitable liquid or solution such as
sterile physiological saline or other physiologically acceptable
injectable aqueous liquids. The amounts of the components to be
used in such compositions can be routinely determined by those
having skill in the art.
[0058] The cells or compositions thereof can be administered by
placement of the cell suspensions onto absorbent or adherent
material, i.e., a collagen sponge matrix, and insertion of the
cell-containing material into or onto the site of interest.
Alternatively, the cells can be administered by parenteral routes
of injection, including subcutaneous, intravenous, intramuscular,
and intrasternal. Other modes of administration include, but are
not limited to, intranasal, intrathecal, intracutaneous,
percutaneous, enteral, and sublingual. In one embodiment of the
present invention, administration of the cells can be mediated by
endoscopic surgery.
[0059] For injectable administration, the composition is in sterile
solution or suspension or can be resuspended in pharmaceutically-
and physiologically-acceptable aqueous or oleaginous vehicles,
which may contain preservatives, stabilizers, and material for
rendering the solution or suspension isotonic with body fluids
(i.e. blood) of the recipient. Non-limiting examples of excipients
suitable for use include water, phosphate buffered saline, pH 7.4,
0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute
ethanol, and the like, and mixtures thereof. Illustrative
stabilizers are polyethylene glycol, proteins, saccharides, amino
acids, inorganic acids, and organic acids, which may be used either
on their own or as admixtures. The amounts or quantities, as well
as the routes of administration used, are determined on an
individual basis, and correspond to the amounts used in similar
types of applications or indications known to those of skill in the
art.
[0060] Consistent with the present invention, the cell can be
administered to body tissues, including liver, pancreas, lung,
salivary gland, blood vessel, bone, skin, cartilage, tendon,
ligament, brain, hair, kidney, muscle, cardiac muscle, nerve,
skeletal muscle, joints, and limb.
[0061] The number of cells in a cell suspension and the mode of
administration may vary depending on the site and condition being
treated. As non-limiting examples, in accordance with the present
invention, about 35-300.times.10.sup.6 cells are injected to effect
tissue repair. Consistent with the Examples disclosed herein, a
skilled practitioner can modulate the amounts and methods of
cell-based treatments according to requirements, limitations,
and/or optimizations determined for each case.
[0062] The preferred suspension solution is Multiple Electrolyte
Injection Type 1 (USP/EP). Each 100 mL of Multiple Electrolyte
Injection Type 1 contains 234 mg of Sodium Chloride, USP (NaCl);
128 mg of Potassium Acetate, USP (C.sub.2H.sub.3KO.sub.2); and 32
mg of Magnesium Acetate Tetrahydrate
(Mg(C.sub.2H.sub.3O.sub.2).sub.2.4H.sub.2O). It contains no
antimicrobial agents. The pH is adjusted with hydrochloric acid.
The pH is 5.5 (4.0 to 8.0). The Multiple Electrolyte Injection Type
1 is preferably supplemented with 0.5% human serum albumin
(USP/EP). Preferably, the cell pharmaceutical composition is stored
at 0-12.degree. C., unfrozen.
[0063] Indications and Modes of Delivery for Cells
[0064] Cells may be manufactured and processed for delivery to
patients using the described methods where the final formulation is
the cells with all culture components substantially removed to the
levels deemed safe by the FDA. It is critical for the cells to have
a final viability greater than 70%, however the higher the
viability of the final cell suspension the more potent and
efficacious the final cell dose will be, and the less cellular
debris (cell membrane, organelles and free nucleic acid from dead
cells), so processes that enhance cell viability while maintaining
the substantially low culture and harvest components, while
maintaining closed aseptic processing systems are highly
desirable.
[0065] Limb Ischemia
[0066] It has been demonstrated that bone marrow-derived cells are
used for vascular regeneration in patients with critical limb
ischemia, peripheral vascular disease, or Burger's syndrome. The
cells delivered to patients with ischemic limbs, and have been
shown to enhance vascular regeneration. Cells are delivered to
patients by creating a cell suspension and removing the cells from
the supplied bag or vial in which they are delivered. A syringe is
used to remove the cell suspension, and then smaller 0.25 ml to 1
ml individual injection volumes are loaded from the main syringe
using a syringe adaptor, and then several individual injection
volumes are delivered via intramuscular injection to the site of
limb ischemia and where vascular formation is required. The cells
may be delivered through a wide range of needle sizes, from large
16 gauge needles to very small 30 gauge needles, as well as very
long 28 gauge catheters for minimally invasive procedures.
Alternatively, the cells may also be delivered intravascularly and
allowed to home to the site of ischemia to drive local tissue
regeneration.
[0067] Cardiac Regeneration
[0068] There are a variety of modes of delivery for driving cardiac
tissue regeneration. The cells are delivered intra-vascularly and
allowed to home to the site of regeneration. Alternatively, the
cells are also be delivered directly into the cardiac muscle,
either epicardially or endocardially, as well as transvascularly.
The cells may be delivered during an open-chest procedure, or via
minimally invasive procedures such as with delivery via a catheter.
The cells are delivered to these patients by creating a cell
suspension and removing the cells from the supplied bag or vial in
which they are delivered. A syringe is used to remove the cell
suspension, and then smaller 0.25 ml to 1 ml individual injection
volumes are loaded from the main syringe using a syringe adaptor,
and then several individual injection volume are delivered via
intramuscular injection to the site of cardiac ischemia and where
vascular formation is required. The cells may be delivered through
a wide range of needle sizes, from large 16 gauge needles to very
small 30 gauge needles, as well as very long 28 gauge catheters for
minimally invasive procedures.
[0069] Spinal Cord Regeneration
[0070] There are a variety of ways that cells are used for
regeneration after spinal cord injury (SCI). Cells may be injected
directly into the site of SCI, seeded onto a matrix (chosen from
the list below for bone regeneration) and seeded into re-sected
spinal cord or placed at the site such that the cells may migrate
to the injury site. Alternatively, the cells are delivered
intravascularly and allowed to home to the site of injury to drive
local tissue regeneration.
[0071] There are a variety of other applications where the cells
may be delivered locally to the tissue via direct injection,
seeding onto a matrix for localized delivery, or delivered via the
vascular system allowing for cells to home to the site of injury or
disease. These diseases are limb ischemia, congestive heart
failure, cardiac ischemia, kidney ischemia, end stage renal
disease, stroke, and ischemia of the eye.
[0072] Orthopedic Indications for Bone Regenerations
[0073] Cells have been used successfully in bone regeneration
applications in humans. Optionally, cells are mixed with 3D
matrices to enhance delivery and localization at the site where
bone regeneration is required. The three-dimensional matrices come
in a range of physical and chemical forms, and viscous or gelled
binding materials may also be added to aid handling and delivery
properties.
[0074] Three dimensional matrices include for example,
demineralized bone particles, mineralized bone particles, synthetic
ceramics of the calcium phosphate family such as alpha tri-calcium
phosphates (TCP), beta TCP, hydroxyappatites, and complex mixtures
of these materials. Other matrices include, for example,
collagen-based sponges, polysaccharide-based materials such as
hyaluronan and alginates, synthetic biodegradable polymeric
materials such as poly-lactides, poly-glycolides, poly-fumarates,
poly-ethylene glycol, co-polymers of these as well as other
materials known in the art.
[0075] Any of the matrices used with cells may be processed into
different physical forms that are common in the art for tissue
regeneration applications. These physical forms are open and closed
pore foams and sponges, fiber-based woven or non-woven meshes, or
small particles ranging from nano-particles to micron-sized
particles (1 micrometer-1000 micrometers) and macro-particles in
the millimeter size scale. The small particles also often have an
open porosity, with nanopores aiding in nutrient and metabolite
transport and micropores providing pores large enough to facilitate
cell seeding and tissue integration.
[0076] When the matrices used for cell delivery are small particles
delivered to wound sites, at times viscous materials or gels are
used to bind the particles that aid in materials handling and
delivery, as well as helping to keep the particles and the cells
localized at the site after placement. Viscous binding materials
include for example, hyaluronan, alginates, collagens, poly
ethylene glycols, poly fumarates, blood clots and fibrin-based
clots, as well as mixtures of these materials, either in the form
of viscous fluids to soft or hard hydrogels. Other viscous
materials and hydrogels are known in the art
[0077] In various embodiments, cells are delivered with TCP,
demineralized bone, and mineralized bone particles in sizes ranging
from 200 micrometers to 5 millimeters, depending on the specific
application. Optionally, these materials are bound with
fibrin-based clots made from autologous freshly prepared plasma
from the patient. Other fibrin clots or different hydrogels, or
matrix materials common may also be used.
[0078] Generally, cells are mixed with the matrices just prior to
surgery when used for bone regeneration. For long-bone
regeneration, typically the area of bone non-union is opened by the
surgeon, and the necrotic bone is removed. The non-unioned bone or
area where bone is needed may or may not be de-corticated by the
surgeon to allow bleeding at the site, at which point the
cell-matrix mixture is placed by the surgeon between the bones
where regeneration will occur. This mixture of the cells and matrix
drive tissue regeneration with the physical matrix guiding the
location of bone regeneration and the cells providing the tissue
repair stimulus for driving angiogenesis, would healing, and bone
regeneration. The remaining cell/matrix mixture is optionally
placed around the fracture line after any orthopedic hardware has
been placed such as plates, rods, screws or nails.
Methods of Reducing Inflammation
[0079] Inflammation is inhibited (e.g., reduced) by administering
to tissue a selectin inhibitor. Tissues to be treated include any
tissue subject to inflammation such as a gastrointestinal tissue,
e.g., intestinal tissue, a cardiac tissue, a muscle tissue, an
epithelial tissue, an endothelium tissue, a vascular tissue, a
pulmonary tissue, a dermal tissue, or a hepatic tissue. For
example, the tissue is an epithelial tissue such as an intestinal
epithelial tissue, pulmonary epithelial tissue, dermal tissue
(i.e., skin), or liver epithelial tissue.
[0080] Inhibition of inflammation is characterized by a reduction
of redness, pain and swelling of the treated tissue compared to a
tissue that has not been contacted with a selectin inhibitor.
Tissues are directly contacted with an inhibitor. Alternatively,
the inhibitor is administered systemically. Selectin inhibitors are
administered in an amount sufficient to decrease (e.g., inhibit)
leukocyte-endothelial interaction. The selectin inhibitor is
administered to a subject prior to, during or after receiving G-CSF
therapy. An inflammatory response is evaluated by morphologically
by observing tissue damage, localized redness, and swelling of the
affected area. Alternatively, an inflammatory response is evaluated
by measuring c-reactive protein, or IL-1 in the tissue or in the
serum or plasma. Efficaciousness of treatment is determined in
association with any known method for diagnosing or treating the
particular inflammatory disorder. Alleviation of one or more
symptoms of the inflammatory disorder indicates that the compound
confers a clinical benefit.
[0081] The methods described herein lead to a reduction in the
severity or the alleviation of one or more symptoms of an
inflammatory disorder. The inflammatory disorder is acute or
chronic. For example, the methods described herein reduce the
severity of vascular and inflammatory complications associated with
G-CSF therapy. Complications associated with G-CSF therapy include,
for example, respiratory distress syndrome, angina pectoris,
myocardial infarct, cutaneous leukocytoclastic vasculitis,
arthritis, precipitate sickle cell vaso-occlusion, and cardiac
ischemia. Disorders are diagnosed and or monitored, typically by a
physician using standard methodologies.
[0082] The subject is preferably a mammal. The mammal can be, e.g.,
a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
The subject suffers from a disorder in which G-CSF therapy is
indicated. For example, the subject is receiving a hematopoietic
stem cell transplant.
[0083] A selectin inhibitor is a compound meant a compound that
inhibits or reduces selectin-ligand interaction. Selectin
inhibitors are known in the art such as those described in U.S.
Pat. No. 5,728,685 (the contents of which are incorporated herein
by reference) or are identified using methods described herein. The
selectin inhibitor is, for example, a small molecule, and antisense
nucleic acid, a short-interfering RNA, or a ribozyme.
EXAMPLES
Example 1
General Methods
[0084] Materials:
[0085] G-CSF mobilized peripheral blood (MPB) was obtained from
pheresis products of donors for clinical HSCT at Brigham and
Women's Hospital/Dana Farber Cancer Institute (Boston, Mass.).
G-CSF MPB leukocytes (ML) were isolated from buffy coat of dextran
sedimentated whole blood, followed by hypotonic lysis to remove
contaminating red cells. Mononuclear fraction of these cells (ML-M)
was isolated by Ficoll-Hypaque (1.077 g/ml; Sigma Aldrich) density
gradient centrifugation. Native (unmobilized) peripheral blood was
obtained from consenting healthy volunteers, and NL (buffy coat)
were isolated using dextran sedimentation as for ML. Peripheral
blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque
density gradient centrifugation. Polymorphonuclear leukocytes (PMN)
were isolated by dextran sedimentation followed by collecting the
cell pellet after Ficoll-Hypaque density gradient centrifugation.
Contaminated red cells were removed by hypotonic lysis.
[0086] Normal human bone marrow (BM) cells were isolated from human
BM harvest material (Massachusetts General Hospital, Boston,
Mass.). Red cells were separated using dextran sedimentation
method. The leukocyte-rich supernatant was subjected to a two-step
discontinuous Percoll (Amersham Pharmacia Biotech; Piscataway,
N.J.) density gradient centrifugation (1.065 g/ml and 1.080 g/ml;
1000 g for 20 min at 4.degree. C.). This resulted in separation of
BM cells into three different "bands" according to myeloid cell
maturity.sup.50. The cells with least density found at the upper
band ("Band 1") contained early immature cells (myeloblasts and
promyelocytes), "Band 2" contained late immature cells (primarily
myelocytes and metamyelocytes), and "Band 3" contained the most
mature leukocytes (predominantly band and segmented neutrophils) as
well as some contaminating red cells, which were subsequently
removed by hypotonic lysis. The cells in different bands were
collected, washed and used for further studies. In some instances,
BM mononuclear cells (BM-MNC) were isolated by Ficoll-Hypaque
density gradient centrifugation. CD34+/lineage- subpopulation was
isolated from BM-MNC using a negative cell selection StemSep.TM.
human progenitor enrichment cocktail (Stem Cell Technologies Inc.);
CD34+ cells were then further isolated by positive selection using
anti-CD34 immunomagnetic beads (Miltenyi Biotech.), routinely
resulting in populations of >98% CD34+ cells.
[0087] All human samples were obtained and used in accordance with
the procedures approved by the Human Experimentation and Ethics
Committees of Partners Cancer Care Institutions (Massachusetts
General Hospital, Brigham and Women's Hospital and Dana Farber
Cancer Institute (Boston, Mass.)).
[0088] Antibodies and E-Selectin Chimera:
[0089] The following antibodies were from BD Pharmingen (San Diego,
Calif.): function blocking murine anti-human E-selectin (68-5411;
IgG.sub.1), rat anti-human CLA (HECA-452; IgM), murine anti-human
PSGL-1 (KPL-1; IgG.sub.1), purified and fluorescein isothiocynate
(FITC)-conjugated murine anti-human L-selectin (DREG-56;
IgG.sub.1), murine anti-human CXCR4 (12G5; IgG.sub.2a), murine
anti-human CD29 (HUTS-21; IgG.sub.2a), mouse IgG.sub.1, .kappa.
isotype, mouse IgG.sub.2a isotype, rat IgG isotype, rat IgM
isotype, FITC-conjugated goat anti-mouse Ig and FITC-conjugated
goat anti-rat IgM. Rat anti-human CD44 (Hermes-1; IgG.sub.2a) was a
gift of Dr. Brenda Sandmaier (Fred Hutchinson Cancer Research
Center; Seattle, Wash.). Murine anti-human CD44 (F10-44-2; mIgM),
phycoerythrin (PE)-conjugated strepavidin, alkaline phosphatase
(AP)-conjugated anti-rat IgM, anti-rat IgG, anti-mouse Ig, and
anti-human Ig were from Southern Biotechnology Associates
(Birmingham, Ala.). Recombinant murine E-selectin/human Ig chimera
(E-Ig) and murine anti-human CD44 (2C5; IgG.sub.2a) were from
R&D Systems (Minneapolis, Minn.). Murine anti-human PSGL-1
(PL-2; IgG.sub.1), murine anti-human CD11a (25.3, IgG.sub.1),
function blocking murine anti-human CD18 (7E4, IgG.sub.1), murine
anti-human CD49d (HP2/1, IgG.sub.1), purified and PE-conjugated
murine anti-human CD34 (QBEND10; IgG.sub.1) and PE-conjugated mouse
IgG.sub.1, .kappa. isotype were from Coulter-Immunotech (Miami,
Fla.). Function blocking rat anti-murine E-selectin (9A9;
IgG.sub.1) was a kind gift of Drs. Barry Wolitzky (CHIRON BioPharma
Research; Emeryville, Calif.) and Klaus Ley (University of
Virginia; Charlottesville, Va.).sup.26,27.
[0090] Cell Culture and Treatment of HUVEC:
[0091] HUVEC were obtained from the tissue culture core facility at
Brigham and Women's Hospital's Pathology Department and were
cultured in M199 supplemented with 15% FBS, 5 units/ml heparin, 50
.mu.g/ml endothelial growth factor, 100 units/ml penicillin and 100
.mu.g/ml streptomycin. For adhesion assays, the HUVEC were cultured
at the center of 100-mm tissue culture dishes (BD Falcon; Franklin
Lakes, N.J.) coated with 10 .mu.g/ml human plasma fibronectin
(Sigma). All experiments were performed with confluent HUVEC
monolayers. To stimulate expression of endothelial adhesion
molecules including E-selectin, HUVEC were pre-treated with 20
ng/ml of recombinant human TNF-.alpha. (endotoxin<0.1 ng/.mu.g
TNF-.alpha.; Research Diagnostics, Inc; Concord, Mass.) or 2 ng/ml
of recombinant human IL-1.beta. (endotoxin<0.1 ng/.mu.g
IL-1.beta.; Research Diagnostics, Inc.) for 4-6 hrs prior to use in
the adhesion studies.
[0092] Human hematopoietic KG1a cell line (ATCC; Manassas, Va.) was
cultured as described previously.sup.28,39. Chinese hamster ovary
cells (CHO) stably transfected with full-length cDNA encoding human
E-selectin (CHO-E cells) or human P-selectin (CHO-P cells) and
mock-transfected CHO cells (CHO-mock).sup.29 were cultured in MEM
supplemented with 10% FBS, 1% sodium pyruvate, 1% non-essential
amino acids, 100 units/ml penicillin and 100 .mu.g/ml
streptomycin.
[0093] Parallel Plate Flow Chamber Adhesion Assays:
[0094] A tissue culture dish containing confluent HUVEC monolayer
or CHO cells was loaded into a parallel plate flow chamber
(Glycotech; Gaithersburg, Md.). The flow chamber was mounted on an
inverted microscope connected to a videocamera, VCR, and monitor.
The field of view was standardized to the mid-point of the flow
chamber. After a brief rinse with HBSS/10 mM HEPES/2 mM CaCl.sub.2
(assay buffer), ML or NL (1.times.10.sup.6 cells/run in assay
buffer) were drawn over the HUVEC monolayer or CHO cells at a shear
stress of 1.5 dyne/cm.sup.2 or 1 dyne/cm.sup.2 respectively. In
certain experiments, TNF-.alpha.-stimulated HUVEC or CHO-E cells
were treated with anti-human E-selectin mAb, 68-5411. The mAb
treated HUVEC or CHO-E cells were then incubated at 37.degree. C.
for 15 min prior to use in adhesion assays. In other experiments,
ML were treated with anti-human L-selectin mAb, Dreg-56 or
anti-human CD18 (.beta.2 integrin) mAb, 7E4 or anti-human CD29
(.beta.1 integrin) mAb, HUTS-21 or mouse IgG.sub.1,k at 4.degree.
C. for 15 min prior to use in adhesion assays. Primary
tethering.sup.49 was determined by quantifying the number of ML or
NL that attached (i.e., interacted) from the free stream directly
onto HUVEC monolayer or CHO cells during the first 2 minutes of
flow. Secondary attachments (i.e., flowing leukocytes interacting
with an already adherent leukocyte on HUVEC monolayer or CHO cells)
or leukocytes that rolled into the field of observation from the
upstream region were not counted. Average rolling velocity, a
quantitative measure of selectin binding strength, was computed
(using Scion Image) as the displacement by the centroid of the cell
divided by the time interval of observation, 5 sec.
[0095] In Vivo Imaging of Cellular Trafficking:
[0096] All studies were performed in accordance with NIH guidelines
for the care and use of animals and under approval of the
Institutional Animal Care and Use Committees of Partners Affiliated
Institutions and the Harvard Medical School. Groups of C57BL/6 mice
received intradermal injection of 200 ng/ml of TNF-.alpha. in a
volume of 10 .mu.l into the right ear. As a control, the left ear
pinnae received intradermal injection of PBS. Six hours later, an
intravenous catheter was inserted into the tail vein of
anesthetized mice. The anesthetized mice were placed in a heated
tube on the stage of a video rate scanning laser confocal
microscope platform.sup.25. To image the vasculature, the ears of
mice were placed on a coverslip and high-resolution images with
cellular details was obtained through the intact mouse skin at
depths of up to 250 .mu.m from the surface using a Olympus
60.times.1.2NA water immersion objective lens. For cell tracking,
animals received 5.times.10.sup.6 of DiD labeled (Molecular Probes)
ML or NL suspended in 200 .mu.l sterile saline (tail-vein
injection), while on the stage, and their interaction with ear
vasculature was viewed using in vivo confocal microscopy. DiD was
excited with a 656 nm diode laser and detected with a
photomultiplier tube through a 695+/-27.5 nm bandpass filter (Omega
Optical). The system operates at a user-selectable frame rate from
15-30 fps. The images are simultaneously recorded by a digital
video recorder (Canon) and captured by a Macintosh computer
equipped with a Scion LG-3 board for frame averaging.sup.25. In
some experiments, mice were treated with 70 .mu.g of mAb 9A9 for 1
hr. prior to injection of leukocytes. Average rolling velocity was
computed (using ImageJ) as the displacement by the centroid of the
cell divided by the time interval of observation.
[0097] OSGE Treatment:
[0098] ML (10.times.10.sup.6 cells/ml) were treated at 37.degree.
C. for 1 hr with 30 .mu.g/ml OSGE (Cedarlane Laboratories, Ontario,
Canada). Following the incubation, the cells were washed and
divided proportionately for use in flow cytometry, Western blot
analysis, and parallel plate flow chamber adhesion assays.
[0099] G-CSF Treatment:
[0100] For in vitro G-CSF treatment, isolated subsets of BM cells
(1.times.10.sup.6 cells/ml) were cultured at 37.degree. C. for 72
hr in the presence of recombinant human G-CSF (10 ng/ml in RPMI,
10% FBS; Amgen, Thousand Oaks, Calif.)); PBS diluent was used for
control (untreated) cells. Note that the in vitro dose of G-CSF
utilized is well within the expected levels in human
serum/extracellular fluids (after a single subcutaneous dose of 5
or 10 .mu.g/kg, peak serum levels range from .about.15.1 to
.about.100.5 ng/ml).sup.44,45. In all experiments, L-selectin
expression was determined by flow cytometry on untreated and
G-CSF-treated cells to test the efficacy of G-CSF treatment. At the
end of the culture period, equal numbers of untreated and G-CSF
treated cells were divided proportionately for use in flow
cytometry, Western blot analysis, RT-PCR and parallel plate flow
chamber adhesion assays. For Western blot analysis, cells were
lysed in 2% NP-40 in Buffer A (consisting of 150 mM NaCl, 50 mM
Tris-HCl pH 7.4, 1 mM EDTA, 0.02% sodium azide, 20 mg/ml PMSF and 1
complete protease inhibitor cocktail tablet/100 ml buffer). The
lysate was used immediately or stored at -20.degree. C. for later
use.
[0101] Membrane Preparations, SDS Separation and Western Blots:
[0102] Membrane proteins of purified ML-M, ML-G, PMN, or PBMC were
isolated as described previously.sup.28,39. Membrane protein
suspensions were aliquoted and stored at -20.degree. C. For
SDS-PAGE and Western blotting, membrane preparations were diluted
in reducing sample buffer, boiled and then separated on 4-20% or
7.5% Criterion Tris-HCl SDS-PAGE gels (Bio-Rad Laboratories).
Resolved membrane proteins were transferred to Sequi-blot
polyvinylidene diflouride (PVDF) membrane (Bio-Rad Laboratories)
and blocked with heat inactivated FBS. Blots were incubated with
primary antibodies or E-Ig (each at 1 .mu.g/ml). Appropriate
isotype control immunoblots (each at 1 .mu.g/ml) were performed in
parallel to evaluate nonspecific binding to protein bands. After
extensive washing with TBS/0.1% Tween 20, blots were incubated with
appropriate alkaline phosphatase (AP)-conjugated secondary
antibodies (1:1000). Western Blue AP substrate (Promega, Madison,
Wis.) was used to develop the blots.
[0103] Immunoprecipitation Studies:
[0104] Membrane proteins of ML-M were incubated with
immunoprecipitating antibodies or E-Ig, or with appropriate isotype
controls and then incubated with Protein G-agarose.
Immunoprecipitates were washed extensively using Buffer A
containing 2% NP-40, 1% SDS. E-Ig precipitated material was washed
extensively with Buffer A without EDTA containing 2% NP-40 and 2 mM
CaCl.sub.2. All immunoprecipitates were diluted in reducing sample
buffer, boiled, then subjected to SDS-PAGE, transferred to PVDF
membrane, and immunostained with HECA-452 or appropriate mAbs.
[0105] In some experiments, the surface proteins on ML-M and KG1a
cells were biotinylated using EZ-Link.RTM. NHS-PEO.sub.4-Biotin
(Pierce Biotechnology, Inc.; Rockford, Ill.). An aliquot of cells
(1.times.10.sup.6 cells) was removed and the efficiency of
biotinylation determined using flow cytometry. The remaining cells
were solubilized in Buffer A containing 2% NP-40. L-selectin was
immunoprecipitated using polyclonal rabbit anti-human L-selectin
antiserum (prepared by Covance, Princeton, N.J.). Control
immunoprecipitation was performed in parallel using rabbit
pre-immune serum (Covance). Immunoprecipitated proteins were
diluted in reducing sample buffer, boiled, then subjected to
SDS-PAGE, transferred to PVDF membrane and immunostained with
horseradish peroxidase (HRP) conjugated strepavidin (1:500; Dako
Cytomation; Carpinteria, Calif.). Vector Nova Red HRP substrate
(Vector Laboratories; Burlingame, Calif.) was used to develop the
blots.
[0106] Blot Rolling Assays:
[0107] The blot rolling assay has been described previously.sup.29.
CHO-E were washed in PBS, and resuspended to 2.times.10.sup.6
cells/ml in assay buffer/10% glycerol. Western blots of ML-M
membrane preparations stained with HECA-452 were rendered
translucent by immersion in assay buffer/10% glycerol. These blots
were then placed in the parallel plate flow chamber, and CHO-E were
perfused into the chamber at a shear stress of 0.6 dyne/cm.sup.2;
an adjustment in the volumetric flow rate was made to account for
the increase in viscosity due to the presence of 10% glycerol in
the assay buffer. Molecular weight standards (SeeBlue.RTM. Plus2
prestained molecular weight standard; Invitrogen Corporation;
Carlsbad, Calif.) were co-electrophoresed on adjacent lanes and
served as a guide to aid placement of the flow chamber over the
stained bands of interest. The number of tethering and rolling
CHO-E was tabulated as function of the molecular weight region and
compiled into an adhesion histogram. Negative controls were
prepared by adding 10 mM EDTA to the assay buffer to chelate
Ca.sup.2+ required for binding or treating CHO-E with anti
E-selectin mAb, 68-5411, at 4.degree. C. for 15 min. prior to use
in adhesion assays.
[0108] Flow Cytometry:
[0109] Aliquots of .about.2-5.times.10.sup.5 cells were washed with
PBS, 2% FBS and incubated with primary mAbs. Subsequently, the
cells were washed and incubated with species and isotype matched
FITC- or PE-labeled polyclonal antibodies. Following this
incubation, the cells were washed and FITC or PE fluorescence of
cells was determined using a Cytomics FC 500 MPL flow cytometer
(Beckman Coulter Inc., Fullerton, Calif.).
[0110] RT-PCR:
[0111] Total cellular RNA was isolated from equal numbers of NL and
ML or untreated or G-CSF-treated human BM cells using Trizol.RTM.
LS reagent (Life Technologies, Inc.) according to the
manufacturer's protocol. The isolated RNA was quantified by
spectrophotometric absorbance readings at 260 nm. Equal amounts of
RNA were then taken and used as templates for RT-PCR with Titan.TM.
One Tube RT-PCR System (Roche Molecular Biochemicals) and the
following primers: ST3Gal IV, sense CTC TCC GAT ATC TGT TTT ATT TTC
CCA TCC CAG AGA GAA GAA GGA G (SEQ ID NO:1) and antisense GAT TAA
GGT ACC AGG TCA GAA GGA GGT GAG GTT CTT (SEQ ID NO:2); FucT-VII,
sense CCC ACC GTG GCC CAG TAC TAC CGC TTC T (SEQ ID NO:3) and
antisense CTG ACC TCT GTG CCC AGC CTC CCG T (SEQ ID NO:4); FucT-IV,
sense CGG GTG TGC CAG GCT GTA CAG AGG (SEQ ID NO:5) and antisense
TCG GGA ACA GTT GTG TAT GAG ATT (SEQ ID NO:6); GAPDH sense GAA GGT
GAA GGT CGG AGT C (SEQ ID NO:8) and antisense GAA GAT GGT GAT GGG
ATT TC (SEQ ID NO:9).
[0112] A total of 30 cycles were found to be below the plateau
phase of amplification for all primers giving an accurate
reflection of the relative concentration of mRNA. Optical PCR
conditions were 94.degree. C. for 2 min, 60.degree. C. for 45 sec.,
and 72.degree. C. for 1 min on a PTC-200 Peltier Thermal cycler (MJ
Research). Amplified bands were visualized after 1% agarose (Sigma
Aldrich) gel electrophoresis of the PCR products. Analysis of
digital images of amplified bands was done using Kodak software.
Mean intensities were determined of fixed size regions set over
each band. The background intensity for each lane was subtracted
from mean intensity in the same lane to arrive at net intensity.
The net intensity of the specific band was then normalized to the
net intensity of GAPDH control.
[0113] Statistics:
[0114] When comparing two means, statistical analyses were done by
unpaired Student's t-test of the means. P values<0.05 were
considered statistically significant. Unless stated otherwise, all
error bars represent standard error of mean.
Example 2
ML Possess Enhanced Binding to E-Selectin Relative to NL
[0115] We analyzed adhesive interactions of ML and NL on
TNF-.alpha.-stimulated human umbilical vein endothelial cells
(HUVEC) in a parallel plate flow chamber assay. Under hemodynamic
flow conditions (1.5 dyne/cm.sup.2), ML displayed markedly enhanced
E-selectin-mediated and Ca.sup.2+-dependent primary tethering on
stimulated HUVEC compared to NL (FIG. 1a). Moreover, ML rolled
distinctly slower than NL on stimulated HUVEC (FIG. 1b). Because
activated integrins support deceleration of cells in flow, we
measured the surface expression of activation-dependent epitopes of
integrins LFA-1 (CD11a/CD18;.alpha..sub.L.beta..sub.2) and VLA-4
(CD49d/CD29;.alpha..sub.4.beta..sub.1), and of chemokine-receptor
CXCR4 on ML and NL. Flow cytometry revealed no difference in the
expression of these molecules (FIG. 7a) suggesting that the marked
decrease in rolling velocity of ML was primarily due to their
increased capacity to engage endothelial E-selectin. To further
characterize the enhanced E-selectin binding capacity of ML, we
examined the adhesion of ML and NL on Chinese hamster ovary cells
transfected with human E-selectin (CHO-E). ML displayed
significantly enhanced E-selectin-mediated and Ca.sup.2+-dependent
primary tethering on CHO-E (FIG. 1c). Furthermore, the rolling
velocity of ML on CHO-E was significantly lower than that of NL
(FIG. 1d).
[0116] To assess whether the observed enhanced E-selectin binding
of ML in vitro could have a meaningful physiologic effect in vivo,
we utilized TNF-.alpha.-induced murine ear inflammation model and
employed dynamic real-time intravital confocal microscopy.sup.25 to
visualize the adhesive interactions of ML and NL with inflamed ear
vasculature. Compared to NL, ML displayed significantly slower
rolling (FIG. 1e) and significantly enhanced adhesion (FIGS. 1f and
1g) to vascular endothelium within the TNF-.alpha.-treated ear. A
function blocking mAb to murine E-selectin, 9A9.sup.26,27,
prominently increased the rolling velocity and diminished leukocyte
adhesion to inflamed endothelium, highlighting a critical role for
vascular E-selectin/leukocyte E-selectin ligand interactions in
mediating this enhanced adhesion. Collectively, these data
demonstrate that ML possess heightened adhesive interactions with
endothelium mediated by enhanced binding to E-selectin.
Example 3
ML Express Hcell and a Novel HECA-452-Reactive .about.65 kDa
E-Selectin Glycoprotein Ligand
[0117] To identify the E-selectin ligand(s) expressed by ML, we
performed Western blot analysis using mAb HECA-452 as a probe. This
mAb HECA-452 recognizes sialofucosylated oligosaccharides,
prototypically sialyl lewis-X (sLe.sup.x), that serve as selectin
binding determinants and HECA-452 reactivity of glycoproteins
correlates with E-selectin ligand activity.sup.28,29. Western-blot
analysis of unfractionated ML lysates revealed several
sialidase-sensitive HECA-452-reactive bands (.about.220 kDa,
.about.130 kDa, .about.100 kDa, and .about.65 kDa) (FIG. 2a and
FIG. 8). Consistent with results of prior studies.sup.30,
unfractionated NL lysates revealed two prominent HECA-452-reactive
bands (.about.220 kDa and .about.130 kDa) (FIG. 2a). Notably,
comparison of ML to NL showed no significant difference in HECA-452
staining of bands at .about.220 kDa and .about.130 kDa; however, ML
lysates showed distinct and prominent HECA-452 staining at bands of
.about.100 kDa and .about.65 kDa (FIG. 2a). Western blot analysis
of membrane proteins of mononuclear (ML-M) and polymorphonuclear
(ML-G) fractions of ML each showed the marked expression of
HECA-452-reactive species of .about.100 kDa and .about.65 kDa (FIG.
2b).
[0118] To determine whether the HECA-452-reactive membrane
glycoproteins from ML represented E-selectin ligands, we utilized
the blot-rolling assay.sup.29,31. For these experiments, we could
not assess binding interactions over the entire lane due to the
length restriction of the flow chamber; because the differences in
HECA-reactivity in proteins from ML and NL clustered within
mobilities encompassing 30 kDa-170 kDa, we set the field view over
this range. Among ML lysates, E-selectin ligand activity was
reproducibly observed on HECA-452 stained bands at .about.130 kDa,
.about.100 kDa and .about.65 kDa (FIG. 2c). Notably, the number of
interacting CHO-E was much greater on the .about.100 kDa and
.about.65 kDa bands compared to the .about.130 kDa band (FIG. 2c),
suggesting that these glycoproteins were major E-selectin ligands.
Specificity for E-selectin binding was verified by significant
diminution of CHO-E binding by addition of EDTA to cell suspension
or by incubating cells with function blocking mAb to E-selectin
(not shown). Moreover, no interactions were observed when
mock-transfected CHO cells were perfused over HECA-452 blots of ML
(not shown).
[0119] In a complementary approach, we probed the expression of
E-selectin ligands using murine E-selectin-human Ig chimera (E-Ig)
in ligand blots. E-Ig, in the presence of Ca.sup.2+, stained ML-M
membrane proteins at .about.220 kDa, .about.130 kDa, .about.100
kDa, and .about.65 kDa (FIG. 2d) corresponding with the
HECA-452-reactive membrane glycoproteins, whereas these bands did
not stain in the presence of EDTA or with control human-Ig (FIG.
9). HECA-452 blots of membrane proteins of ML-M immunoprecipitated
using E-Ig also revealed staining at .about.220 kDa, .about.130
kDa, .about.100 kDa and .about.65 kDa (FIG. 2e). Control
immunoprecipitates using human-Ig or E-Ig in the presence of EDTA
showed absence of HECA-452-reactive proteins (FIG. 2e, and not
shown). Collectively, these results show that the observed
glycoproteins migrating at .about.220 kDa, .about.130 kDa,
.about.100 kDa and .about.65 kDa represent the E-selectin ligands
of ML.
[0120] The electrophoretic mobilities of several bands bearing
E-selectin ligand activity coincided with that of two previously
characterized human E-selectin ligands, i.e., PSGL-1 (mw 220-240
kDa (dimer) and 120-140 kDa (monomer)) and the Hcell glycoform of
CD44 (mw .about.100 kDa).sup.28,31. Thus, we sought to determine
whether these bands represented these molecules. KPL-1
(anti-PSGL-1) blots of ML-M membrane proteins showed bands of
.about.220 kDa and .about.130 kDa under reducing conditions (FIG.
3a). Subsequently, blots of immunoprecipitated PSGL-1 were stained
with either HECA-452 or KPL-1. As shown in FIG. 3b, ML express both
the monomer and dimer forms of PSGL-1, which represent the
.about.130 kDa and .about.220 kDa HECA-452-reactive glycoproteins.
Blots of ML-M membrane preparations stained with Hermes-1
(anti-CD44) showed a band of .about.100 kDa under reducing
conditions (FIG. 3c). Subsequently, blots of
Hermes-1-immunoprecipitated CD44 were stained with either HECA-452
or 2C5, another anti-human CD44 antibody. As shown in FIG. 3d, ML
express Hcell, evident as a HECA-452-reactive glycoform of
CD44.sup.28.
[0121] The HECA-452-reactive .about.65 kDa glycoprotein was not
immunoprecipitated by mAbs KPL-1 or Hermes-1 (FIGS. 3b and 3d).
Other mAbs to PSGL-1 (e.g., PL-2) and CD44 (e.g., 2C5) also did not
immunoprecipitate the .about.65 kDa protein (FIG. 10), suggesting
that this protein is not related to either PSGL-1 or CD44. Human
neutrophil L-selectin (mw .about.75-90 kDa) has been reported to be
an E-selectin ligand.sup.32. Since the HECA-452-reactive .about.65
kDa glycoprotein resolved in Western blots in the molecular weight
range of L-selectin.sup.33, we investigated whether this structure
was L-selectin. Flow cytometry revealed that ML express little
L-selectin (FIG. 3e). This observation is consistent with a
previous study demonstrating that G-CSF treatment of leukocytes
down-regulates L-selectin expression.sup.34. In agreement with the
flow cytometry results, we were unable to detect L-selectin in
immunoprecipitates of ML-M (FIG. 3f). Collectively, these data
demonstrate that PSGL-1 and Hcell serve as E-selectin ligands on ML
and that the HECA-452-reactive .about.65 kDa protein is not a
glycoform of PSGL-1, CD44 or L-selectin.
Example 4
ML Possess Enhanced Levels of ST3GalIV, FucT-IV and FucT-VII
[0122] The capacity of E-selectin to recognize its relevant
glycoprotein leukocyte ligand(s) is dependent on carbohydrate
decoration of the core protein.sup.18,28,35. Based on prior
observations that glycosyltransferases are regulated by
cytokines.sup.36, we sought to investigate whether G-CSF affects
expression of relevant glycosyltransferases that create pertinent
sialofucosylations. The carbohydrate modifications rendering the
expression of E-selectin binding determinants are critically
mediated by specific glycosyltransferases:
.alpha.2,3-sialyltransferase (ST3GalIV) and leukocyte
.alpha.1,3-fucosyltransferases (FucT-IV and FucT-VII).sup.37,38.
RT-PCR analysis of the expression of ST3GalIV, FucT-IV and FucT-VII
revealed that the transcripts for each of these
glycosyltransferases were increased in ML relative to NL (FIG.
4).
Example 5
Hcell and .about.65 kDa Glycoprotein are Major E-Selectin Ligands
on ML
[0123] The E-/L-selectin ligand activity of Hcell is resistant to
O-sialoglycoprotein endopeptidase (OSGE) treatment.sup.28,31,39,
whereas OSGE treatment abrogates PSGL-1 binding to all three
selectins.sup.40,41. Accordingly, to determine the contribution of
PSGL-1 to the observed enhanced E-selectin ligand activity of ML,
we performed OSGE digestion and measured residual E-selectin
binding activity. OSGE digestion of ML abrogated surface expression
of PSGL-1 and had minimal effect on CD44 and HECA-452 antigen
levels (FIG. 5a). E-Ig blots of cell lysates of ML showed distinct
reduction of E-selectin binding by PSGL-1 on OSGE-treated cells
(absent binding at dimer and marked diminution at monomer), while
E-selectin binding determinants of Hcell and the .about.65 kDa
glycoprotein were intact (FIG. 5b). Despite significant decreases
in PSGL-1 expression and function, OSGE treatment had no effect on
primary tethering of ML on TNF-.alpha.-stimulated HUVEC (FIG. 5c).
Combined, these data demonstrate that Hcell and the .about.65 kDa
glycoprotein are major E-selectin ligands on ML. Interestingly,
compared to NL, ML displayed markedly diminished
P-selectin-mediated and Ca.sup.2+-dependent primary tethering on
Chinese hamster ovary cells transfected with human P-selectin
(CHO-P) (FIG. 11a). Furthermore, ML rolled significantly faster
than NL on CHO-P (FIG. 11b). Given that PSGL-1 is the predominant
ligand for P-selectin, the decreased PSGL-1 function on ML,
combined with the findings that G-CSF treatment down-regulates
PSGL-1 expression in humans.sup.42, indicates that Hcell and
.about.65 kDa glycoprotein contribute to the augmented E-selectin
binding of ML.
Example 6
In Vitro G-CSF Treatment of Human BM Cells Up-Regulates the
Expression of ST3GalIV, FucT-IV and FucT-VII with Associated
Increases in Expression of Hcell and HECA-452-Reactive .about.65
kDa Glycoprotein and E-Selectin Binding
[0124] While PSGL-1 is broadly expressed on myeloid, lymphoid and
dendritic lineage cells.sup.30,43 and also on CD34+ hematopoietic
progenitor cells (FIG. 6a), Hcell is typically expressed only on
CD34+ progenitors in human BM (FIG. 6a and.sup.28). Separation of
ML into early immature (Band 1), late immature (Band 2) and mature
(Band 3) myeloid fractions by Percoll gradient showed that
predominantly Band 1 and Band 2 cells possessed Hcell and also
expressed the HECA-452-reactive .about.65 kDa glycoprotein (FIG.
6b). The presence of these structures in association with G-CSF
administration prompted us to determine whether G-CSF directly
induces their expression. Freshly isolated BM cells were separated
into early immature (Band 1), late immature (Band 2) and mature
(Band 3) myeloid fractions. The different subsets of cells were
then cultured at 37.degree. C. for 72 hr in the presence of 10
ng/ml G-CSF. Importantly, the in vitro dose of G-CSF utilized is
well within the expected levels in human serum/extracellular fluids
(after a single subcutaneous dose of 5 or 10 .mu.g/kg, peak serum
levels range from .about.15.1 to .about.100.5 ng/ml).sup.44,45. The
expression of Hcell and .about.65 kDa glycoprotein were analyzed by
reactivity to HECA-452 and E-Ig using Western blot analysis. In all
experiments, efficacy of G-CSF treatment was confirmed by observing
the down-regulation of L-selectin expression on G-CSF treated cells
relative to untreated cells (not shown). As shown in FIG. 6c, G-CSF
treatment consistently resulted in up-regulation in expression of
Hcell and the HECA-452-reactive .about.65 kDa glycoprotein
predominantly on immature Band 1 and Band 2 cells. In association
with G-CSF-induced increases in HCELL and HECA-452-reactive
.about.65 kDa protein, in vitro treatment of immature human BM
cells (Band 2) with G-CSF resulted in increases in expression
levels of ST3GalIV, FucT-IV and FucT-VII (FIGS. 6d and 6e) together
with enhanced E-selectin binding (FIG. 12).
Example 7
In Vitro G-CSF Treatment of Human BM Cells and Blood Cells
Up-Regulates the Expression of Fucosyltransferase IX (FuT-IX) with
Associated Increases in Expression of CD15
[0125] Freshly isolated bone marrow and peripheral blood were t
cultured at 37.degree. C. for 72 hr in the presence of 10 ng/ml
G-CSF. Treatment increases Fuct-IX expression in normal progenitors
and in mobilized peripheral blood. (FIG. 13) Fuct-IX has been found
to regulate CD15 expression (which is the non-sialylated core of
sLex) and is expressed on the surface of neutrophils. CD15 plays a
role in innate immunity in priming dendritic cells. Moreover, FTIX
fucosylates internal lactosamine units yielding the VIM-2 epitope.
While, the VIM-2 is not sLex, but it consists of a terminal sialic
acid with an "internal fucosylation" that can function as an
E-selectin ligand
Example 8
In Vitro G-CSF Treatment of Human BM Cells and Leukocytes Cells
Up-Regulates the Expression of Sialidase with Associated Increases
in Expression of CD15
[0126] Freshly isolated BM cells were separated into early immature
(Band 1), late immature (Band 2) and mature (Band 3) myeloid
fractions. Early immature bone marrow cells, late mature bone
marrow cells and leukocytes were then cultured at 37.degree. C. for
72 hr in the presence of 10 ng/ml G-CSF. Treatment of bone marrow
and leukocytes resulted in an increase expression of sialidase.
Treatment of early immature none marrow did not has an effect on
the expression of CD15. However treatment of the late mature bone
marrow and leukocytes resulted in an increased expression of CD15.
Furthermore, this increase of CD15 could be prevented by the
inhibition of sialidase with
2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA). These results
indicate that CD15 expression was a direct result of the increase
of sialidase expression resulting from G-CSF treatment.
REFERENCES
[0127] 1. Elfenbein, G. J. & Sackstein, R. Primed marrow for
autologous and allogeneic transplantation: a review comparing
primed marrow to mobilized blood and steady-state marrow. Exp
Hematol 32, 327-39 (2004). [0128] 2. Lindemann, A. & Rumberger,
B. Vascular complications in patients treated with granulocyte
colony-stimulating factor (G-CSF). Eur J Cancer 29A, 2338-9 (1993).
[0129] 3. Hill, J. M. & Bartunek, J. The end of granulocyte
colony-stimulating factor in acute myocardial infarction? Reaping
the benefits beyond cytokine mobilization. Circulation 113, 1926-8
(2006). [0130] 4. Azoulay, E., Attalah, H., Harf, A., Schlemmer, B.
& Delclaux, C. Granulocyte colony-stimulating factor or
neutrophil-induced pulmonary toxicity: myth or reality? Systematic
review of clinical case reports and experimental data. Chest 120,
1695-701 (2001). [0131] 5. Azoulay, E. et al. Exacerbation by
granulocyte colony-stimulating factor of prior acute lung injury:
implication of neutrophils. Crit. Care Med 30, 2115-22 (2002).
[0132] 6. Arimura, K. et al. Acute lung Injury in a healthy donor
during mobilization of peripheral blood stem cells using
granulocyte-colony stimulating factor alone. Haematologica 90,
ECR10 (2005). [0133] 7. Fukumoto, Y. et al. Angina pectoris
occurring during granulocyte colony-stimulating factor-combined
preparatory regimen for autologous peripheral blood stem cell
transplantation in a patient with acute myelogenous leukaemia. Br J
Haematol 97, 666-8 (1997). [0134] 8. Mossner, R., Beckmann, I.,
Hallermann, C., Neumann, C. & Reich, K. Granulocyte
colony-stimulating-factor-induced psoriasiform dermatitis resembles
psoriasis with regard to abnormal cytokine expression and epidermal
activation. Exp Dermatol 13, 340-6 (2004). [0135] 9. Dereure, O.,
Hillaire-Buys, D. & Guilhou, J. J. Neutrophil-dependent
cutaneous side-effects of leucocyte colony-stimulating factors:
manifestations of a neutrophil recovery syndrome? Br J Dermatol
150, 1228-30 (2004). [0136] 10. Jain, K. K. Cutaneous vasculitis
associated with granulocyte colony-stimulating factor. J Am Acad
Dermatol 31, 213-5 (1994). [0137] 11. Stricker, R. B. &
Goldberg, B. G-CSF and exacerbation of rheumatoid arthritis. Am J
Med 100, 665-6 (1996). [0138] 12. Adler, B. K. et al. Fatal sickle
cell crisis after granulocyte colony-stimulating factor
administration. Blood 97, 3313-4 (2001). [0139] 13. Hill, J. M. et
al. Outcomes and risks of granulocyte colony-stimulating factor in
patients with coronary artery disease. J Am Coll Cardiol 46, 1643-8
(2005). [0140] 14. Harada, M. et al. G-CSF prevents cardiac
remodeling after myocardial infarction by activating the Jak-Stat
pathway in cardiomyocytes. Nat Med 11, 305-11 (2005). [0141] 15.
Butcher, E. C. Leukocyte-endothelial cell recognition: three (or
more) steps to specificity and diversity. Cell 67, 1033-6 (1991).
[0142] 16. Sackstein, R. The lymphocyte homing receptors:
gatekeepers of the multistep paradigm. Curr Opin Hematol 12, 444-50
(2005). [0143] 17. van der Wal, A. C., Das, P. K., Tigges, A. J.
& Becker, A. E. Adhesion molecules on the endothelium and
mononuclear cells in human atherosclerotic lesions. Am J Pathol
141, 1427-33 (1992). [0144] 18. Kansas, G. S. Selectins and their
ligands: current concepts and controversies. Blood 88, 3259-87
(1996). [0145] 19. Albert, R. K. Mechanisms of the adult
respiratory distress syndrome: selectins. Thorax 50 Suppl 1, S49-52
(1995). [0146] 20. Kriegsmann, J. et al. Expression of E-selectin
messenger RNA and protein in rheumatoid arthritis. Arthritis Rheum
38, 750-4 (1995). [0147] 21. Groves, R. W., Allen, M. H., Barker,
J. N., Haskard, D. O. & MacDonald, D. M. Endothelial leucocyte
adhesion molecule-1 (ELAM-1) expression in cutaneous inflammation.
Br J Dermatol 124, 117-23 (1991). [0148] 22. Glass, L. F.,
Fotopoulos, T. & Messina, J. L. A generalized cutaneous
reaction induced by granulocyte colony-stimulating factor. J Am
Acad Dermatol 34, 455-9 (1996). [0149] 23. Bussolino, F. et al.
Granulocyte- and granulocyte-macrophage-colony stimulating factors
induce human endothelial cells to migrate and proliferate. Nature
337, 471-3 (1989). [0150] 24. Fuste, B. et al. Granulocyte
colony-stimulating factor increases expression of adhesion
receptors on endothelial cells through activation of p38 MAPK.
Haematologica 89, 578-85 (2004). [0151] 25. Sipkins, D. A. et al.
In vivo imaging of specialized bone marrow endothelial microdomains
for tumour engraftment. Nature 435, 969-73 (2005). [0152] 26.
Smith, M. L., Olson, T. S. & Ley, K. CXCR2- and
E-selectin-induced neutrophil arrest during inflammation in vivo. J
Exp Med 200, 935-9 (2004). [0153] 27. Kunkel, E. J. & Ley, K.
Distinct phenotype of E-selectin-deficient mice. E-selectin is
required for slow leukocyte rolling in vivo. Circ Res 79, 1196-204
(1996). [0154] 28. Dimitroff, C. J., Lee, J. Y., Rafii, S.,
Fuhlbrigge, R. C. & Sackstein, R. CD44 is a major E-selectin
ligand on human hematopoietic progenitor cells. J Cell Biol 153,
1277-86 (2001). [0155] 29. Fuhlbrigge, R. C., King, S. L.,
Dimitroff, C. J., Kupper, T. S. & Sackstein, R. Direct
real-time observation of E- and P-selectin-mediated rolling on
cutaneous lymphocyte-associated antigen immobilized on Western
blots. J Immunol 168, 5645-51 (2002). [0156] 30. Kieffer, J. D. et
al. Neutrophils, monocytes, and dendritic cells express the same
specialized form of PSGL-1 as do skin-homing memory T cells:
cutaneous lymphocyte antigen. Biochem Biophys Res Commun 285,
577-87 (2001). [0157] 31. Dimitroff, C. J., Lee, J. Y., Fuhlbrigge,
R. C. & Sackstein, R. A distinct glycoform of CD44 is an
L-selectin ligand on human hematopoietic cells. Proc Natl Acad Sci
USA 97, 13841-6 (2000). [0158] 32. Zollner, O. et al. L-selectin
from human, but not from mouse neutrophils binds directly to
E-selectin. J Cell Biol 136, 707-16 (1997). [0159] 33.
Schleiffenbaum, B., Spertini, O. & Tedder, T. F. Soluble
L-selectin is present in human plasma at high levels and retains
functional activity. J Cell Biol 119, 229-38 (1992). [0160] 34.
Ohsaka, A. et al. Granulocyte colony-stimulating factor
down-regulates the surface expression of the human leucocyte
adhesion molecule-1 on human neutrophils in vitro and in vivo. Br J
Haematol 84, 574-80 (1993). [0161] 35. Fuhlbrigge, R. C., Kieffer,
J. D., Armerding, D. & Kupper, T. S. Cutaneous lymphocyte
antigen is a specialized form of PSGL-1 expressed on skin-homing T
cells. Nature 389, 978-81 (1997). [0162] 36. Wagers, A. J., Waters,
C. M., Stoolman, L. M. & Kansas, G. S. Interleukin 12 and
interleukin 4 control T cell adhesion to endothelial selectins
through opposite effects on alpha 1,3-fucosyltransferase VII gene
expression. J Exp Med 188, 2225-31 (1998). [0163] 37. Ellies, L. G.
et al. Sialyltransferase specificity in selectin ligand formation.
Blood 100, 3618-25 (2002). [0164] 38. Wagers, A. J., Stoolman, L.
M., Kannagi, R., Craig, R. & Kansas, G. S. Expression of
leukocyte fucosyltransferases regulates binding to E-selectin:
relationship to previously implicated carbohydrate epitopes. J
Immunol 159, 1917-29 (1997). [0165] 39. Dimitroff, C. J., Lee, J.
Y., Schor, K. S., Sandmaier, B. M. & Sackstein, R. Differential
L-selectin binding activities of human hematopoietic cell
L-selectin ligands, Hcell and PSGL-1. J Biol Chem 276, 47623-31
(2001). [0166] 40. Goetz, D. J. et al. Isolated P-selectin
glycoprotein ligand-1 dynamic adhesion to P- and E-selectin. J Cell
Biol 137, 509-19 (1997). [0167] 41. Spertini, O., Cordey, A. S.,
Monai, N., Giuffre, L. & Schapira, M. P-selectin glycoprotein
ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and
CD34+ hematopoietic progenitor cells. J Cell Biol 135, 523-31
(1996). [0168] 42. Jilma, B. et al. Rapid down modulation of
P-selectin glycoprotein ligand-1 (PSGL-1, CD162) by G-CSF in
humans. Transfusion 42, 328-33 (2002). [0169] 43. Laszik, Z. et al.
P-selectin glycoprotein ligand-1 is broadly expressed in cells of
myeloid, lymphoid, and dendritic lineage and in some
nonhematopoietic cells. Blood 88, 3010-21 (1996). [0170] 44. van
Der Auwera, P. et al. Pharmacodynamics and pharmacokinetics of
single doses of subcutaneous pegylated human G-CSF mutant (Ro
25-8315) in healthy volunteers: comparison with single and multiple
daily doses of filgrastim. Am J Hematol 66, 245-51 (2001). [0171]
45. Faulkner, L. B. et al. G-CSF serum pharmacokinetics during
peripheral blood progenitor cell mobilization: neutrophil
count-adjusted dosage might potentially improve mobilization and be
more cost-effective. Bone Marrow Transplant 21, 1091-5 (1998).
[0172] 46. Hakans son, L. et al. Effects of in vivo administration
of G-CSF on neutrophil and eosinophil adhesion. Br J Haematol 98,
603-11 (1997). [0173] 47. Xia, L. et al. P-selectin glycoprotein
ligand-1-deficient mice have impaired leukocyte tethering to
E-selectin under flow. J Clin Invest 109, 939-50 (2002). [0174] 48.
Yang, J. et al. Targeted gene disruption demonstrates that
P-selectin glycoprotein ligand 1 (PSGL-1) is required for
P-selectin-mediated but not E-selectin-mediated neutrophil rolling
and migration. J Exp Med 190, 1769-82 (1999). [0175] 49. Zou, X. et
al. PSGL-1 Derived from Human Neutrophils is a High Efficiency
Ligand for Endothelial Expressed E-selectin under Flow. Am J
Physiol Cell Physiol (2005). [0176] 50. Cowland, J. B. &
Borregaard, N. Isolation of neutrophil precursors from bone marrow
for biochemical and transcriptional analysis. J Immunol Methods
232, 191-200 (1999).
OTHER EMBODIMENTS
[0177] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
8146DNAArtificial SequenceChemically Synthesized 1ctctccgata
tctgttttat tttcccatcc cagagagaag aaggag 46236DNAArtificial
SequenceChemically Synthesized 2gattaaggta ccaggtcaga aggaggtgag
gttctt 36328DNAArtificial SequenceChemically Synthesized
3cccaccgtgg cccagtacta ccgcttct 28425DNAArtificial
SequenceChemically Synthesized 4ctgacctctg tgcccagcct cccgt
25524DNAArtificial SequenceChemically Synthesized 5cgggtgtgcc
aggctgtaca gagg 24624DNAArtificial SequenceChemically Synthesized
6tcgggaacag ttgtgtatga gatt 24719DNAArtificial SequenceChemically
Synthesized 7gaaggtgaag gtcggagtc 19819DNAArtificial
SequenceChemically Synthesized 8aagatggtga tgggatttc 19
* * * * *