1. suffer from an autoimmune disease
2. read and discuss patent no. with me and others
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| United States Patent |
8,697,077 |
|
Faustman
|
April 15, 2014
|
Methods and compositions for treating autoimmune diseases
Abstract
The invention features methods for increasing or maintaining the number
of functional cells of a predetermined type, for example, insulin
producing cells of the pancreas, blood cells, spleen cells, brain cells,
heart cells, vascular tissue cells, cells of the bile duct, or skin
cells, in a mammal (e.g., a human patient) that has injured or damaged
cells of the predetermined type.
| Inventors: |
Faustman; Denise L. (Boston, MA) |
| Applicant: |
| Name | City | State | Country | Type |
Faustman; Denise L. |
Boston |
MA |
US | |
|
|---|
| Assignee: |
The General Hospital Corporation
(Boston,
MA)
|
| Family ID:
|
30002891
|
|---|
| Appl. No.:
|
13/462,160 |
|---|
| Filed:
|
May 2, 2012 |
|---|
Prior Publication Data
|
|
|
|---|
|
| Document Identifier | Publication Date |
|---|
| | US 20120213731 A1 | Aug 23, 2012 |
|
|
Related U.S. Patent Documents
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|---|
|
| Application Number | Filing Date | Patent Number | Issue Date |
|---|
| | 12632452 | Dec 7, 2009 | 8173129 |
|
| | 10358664 | Feb 5, 2003 | 7628988 |
|
| | 60392687 | Jun 27, 2002 |
|
|
|
|
| Current U.S. Class: | 424/139.1; 424/142.1; 424/154.1; 424/577 |
| Current CPC Class: |
A61K 35/26 (20130101);
A61K 39/04 (20130101); A61K 35/39 (20130101); A61K 35/44 (20130101);
A61K 38/1866 (20130101); A61K 38/191 (20130101); A61K 35/28 (20130101);
A61K 35/30 (20130101); A61K 38/1774 (20130101); A61K 35/28 (20130101);
A61K 35/44 (20130101); A61K 38/191 (20130101); A61K 38/1866 (20130101);
A61K 35/39 (20130101); A61K 35/26 (20130101); A61K 38/1774 (20130101);
A61K 2300/00 (20130101); A61K 2300/00 (20130101); A61K
2300/00 (20130101); A61K 2300/00 (20130101); A61K 2300/00 (20130101);
A61K 2300/00 (20130101); A61K 2300/00 (20130101) |
| Current International Class: |
A61K 35/26 (20060101); A61K 35/28 (20060101); A61K 39/40 (20060101); A61K 39/395 (20060101) |
References Cited [Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
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|
|---|
|
| WO-92/04033 |
|
Mar 1992 |
|
WO |
|
| WO-95/24914 |
|
Sep 1995 |
|
WO |
|
| WO-95/25533 |
|
Sep 1995 |
|
WO |
|
| WO-97/08328 |
|
Mar 1997 |
|
WO |
|
| WO-97/21802 |
|
Jun 1997 |
|
WO |
|
| WO-00/53209 |
|
Sep 2000 |
|
WO |
|
| WO-02/26819 |
|
Apr 2002 |
|
WO |
|
| WO-2004/003164 |
|
Jan 2004 |
|
WO |
|
|
Other References
Kaufman et al., J of Immunol. 1997, V.158, pp. 2435-2442. cited by examiner
.
Feldman et al., Transplant. Proc. 1998, 30, 4126-4127. cited by examiner
.
Mestas et al J. of Immunology, 2004, 172, pp. 2731-238. cited by examiner
.
Corbett et al. (Proc Natl Acad Sci U S A. Mar. 1, 1993 ;90(5): 1731-5. cited by examiner
.
Couzin Science, 2006, V.311, p. 1694. cited by examiner
.
Fischer et al., "A TNF receptor 2 selective agonist rescues human
neurons from oxidative stress-induced cell death," PloS One.
6(11):e27621(1-11) (2011). cited by applicant
.
Loetscher et al., "Human tumor necrosis factor .alpha. (TNF
.alpha.) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF
receptors," J Biol Chem. 268(35):26350-26357 (1993). cited by applicant
.
Van Zee et al., "A human tumor necrosis factor (TNF) alpha mutant
that binds exclusively to the p55 TNF receptor produces toxicity in the
baboon," J Exp Med. 179(4):1185-1191 (1994). cited by applicant
.
Welborn et al., "A human tumor necrosis factor p75 receptor agonist
stimulates in vitro T cell proliferation but does not produce
inflammation or shock in the baboon," J Exp Med. 184(1):165-171 (1996).
cited by applicant
.
Creasey et al., "Biological effects of recombinant human tumor
necrosis factor and its novel muteins on tumor and normal cell lines,"
Cancer Res. 47(1):145-9 (1987). cited by applicant
.
Tavernier et al., "Analysis of the structure-function relationship
of tumour necrosis factor. Human/mouse chimeric TNF proteins: general
properties and epitope analysis," J Mol Biol. 21 1(2):493-501 (1990).
cited by applicant
.
Al-Awquati et al., "Stem cells in the kidney," Kidney Int. 61:387-395 (2002). cited by applicant
.
Aldrich et al., "Positive selection of self- and alloreactive CD8+ T
cells in Tap-1 mutant mice," Proc Natl Acad Sci USA. 91:6525-6528
(1994). cited by applicant
.
Alison et al., "Hepatocytes from non-hepatic adult stem cells," Nature. 406:257 (2000). cited by applicant
.
Allen et al., "Effect of Bacillus calmette-guerin vaccination on
new-onset Type 1 Diabetes," Diabetes Care. 22(1):1703-1707 (1999). cited
by applicant
.
Altomonte et al., "Serum levels of interleukin-1b, tumour necrosis
factor-a and interleukin-2 in rheumatoid arthritis. Correlation with
disease activity," Clin Rheumatol. 11(2):202-205 (1992). cited by
applicant
.
Anderson et al., "The NOD mouse: A model of immune dysregulation," Annu Rev Immunol. 23:447-485 (2005). cited by applicant
.
Anderson et al., "Can stem cells cross lineage boundaries?," Nat Med. 7:393-395 (2001). cited by applicant
.
Anderson et al., "Studies on the cytophilic properties of human
.beta.2 microglobulin," J Immunol. 114:997-1000 (1975). cited by
applicant
.
Aristarkhov et al., " E2-C, a cyclin-selective ubiquitin carrier
protein required for the destruction of mitotic cyclins," Proc Natl Acad
Sci USA. 93:4294-4299 (1996). cited by applicant
.
Aranda et al., "Analysis of intestinal lymphocytes in mouse colitis
mediated by transfer of CD4+, CD45RBhigh T Cells to SCID recipients," J
Immunol. 158(7):3464-3473 (1997). cited by applicant
.
Ashton-Rickardt et al., "Peptide contributes to the specificity of
positive selection of CD8+ T Cells in the thymus," Cell. 73:1041-1049
(1993). cited by applicant
.
Ashton-Rickardt et al., "Evidence for a differential avidity model
of T Cell selection in the thymus," Cell . 76:651-663 (1994). cited by
applicant
.
Atkinson et al., "The NOD mouse model of Type 1 Diabetes: As good as it gets?," Nat Med. 5(6):601-604 (1999). cited by applicant
.
Baeuerle et al, "NF-kB: Ten years after," Cell. 87:13-20 (1996). cited by applicant
.
Baeza et al., "Pancreatic regenerating gene overexpression in the
nonobese diabetic mouse during active diabetogensis," Diabetes.
45(1):67-70 (1996). cited by applicant
.
Baeza et al., "Reg protein: a potential beta-cell-specific growth
factor?," Diabetes Metab. 22:229-234 (1996). cited by applicant
.
Baeza et al., "Specific reg II gene overexpression in the non-obese
diabetic mouse pancreas during active diabetogenesis," FEBS Letters.
416:364-368 (1997). cited by applicant
.
Baik et al., "BCG vaccine prevents insulitis in low dose
streptozotocin-induced diabetic mice," Diabetes Res. and Clin. Pract.
46: 91-97, 1999. cited by applicant
.
Baldwin, "The NF-.sub.kB and I.sub.kB proteins: new discoveries and
insights," Ann. Rev. Immunol. 14:649-683 (1996). cited by applicant
.
Ban et al., "Selective death of autoreactive T Cells in human
diabetes by TNF or TNF receptor 2 agonism," Proc. Natl. Acad. Sci. USA
105: 13644-13649, 2008. cited by applicant
.
Barres, "A new role for glia: generation of neurons!," Cell 97: 667-670, 1999. cited by applicant
.
Baxter et al., "Mycobacteria precipitate an SLE-like syndrome in
diabetes-prone NOD mice," Immunology. 83:227-231 (1994). cited by
applicant
.
Beg et al., "An essential role for NF-KB in preventing
TNF-.alpha.-induced cell death," Science. 274:782-784 (1996). cited by
applicant
.
Bernabeu et al., ".beta.2 -microglobulin from serum associates with
MHC class I antigens on the surface of cultured cells," Nature.
308:642-645 (1984). cited by applicant
.
Bendelac et al., "Syngeneic transfer of autoimmune diabetes from
diabetic NOD mice to healthy neonates," J. Exp. Med. 166:823-832 (1987).
cited by applicant
.
Bill et al, "Use of soluble MHC class II/peptide multimers to
detect antigen-specific T cells in human disease," Arthritis Res.
4:261-265 (2002). cited by applicant
.
Bjornson et al., "Turning brain into blood: A hematopoietic fate
adopted by adult neural stem cells in vitro," Science. 283:534-537
(1999). cited by applicant
.
Boches et al, "Role for the adenosine triphosphate-dependent
proteolytic pathway in reticulocyte maturation," Science. 215:978-980
(1982). cited by applicant
.
Bras et al., "Diabetes-prone NOD mice are resistant to
Mycobacterium avium and the infection prevents autoimmune disease,"
Immunology. 89:20-25 (1996). cited by applicant
.
Brayer et al., "Alleles from chromosomes 1 and 3 of NOD mice
combine to influence Sjogren's syndrome-like autoimmune exocrinopathy, "
J. Rheumatol. 27(8):1896-1904 (2000). cited by applicant
.
Brazelton et al., "From marrow to brain: expression of neuronal
phenotypes in adult mice," Science. 290:1775-1779 (2000). cited by
applicant
.
Brod et al., "Ingested interferon .alpha. suppresses Type I
diabetes in non-obese diabetic mice," Diabetologia. 41:1227-1232 (1998).
cited by applicant
.
Brodbeck et al., "Genetic determination of nephrogenesis: the
pax/eya/six gene network," Pediatr. Nephrol. 19:249-255 (2004).
(Abstract Only). cited by applicant
.
Bunting et al., "Enforced P-glycoprotein pump function in murine
bone marrow cells results in expansion of side population stem cells in
vitro and repopulating cells in vivo," Blood. 96(3):902-909 (2000).
cited by applicant
.
Cavallo et al., "BCG vaccine with and without nicotinamide in
recent onset IDDM: a multicenter randomized trial," Autoimmunity
24(Suppl.1):A063 (1996). cited by applicant
.
Caetano et al., "Effect of methotrexate (MTX) on NAD(P)+
dehydrogenases of HeLa cells: malic enzyme, 2- oxoglutarate and
isocitrate dehydrogenases," Cell Biochem Funct. 15(4):259-264 (1997).
cited by applicant
.
Chatenoud et al., "CD3 antibody-induced dominant self tolerance in
overtly diabetic NOD mice," J Immunol. 158(6):2947-2954 (1997). cited by
applicant
.
Choi et al., "Prevention of Encephalomyocarditis virus-induced
diabetes by live recombinant Mycobacterium bovis Bacillus
Calmette-Guerin in susceptible mice," Diabetes. 49:1459-1467 (2000).
cited by applicant
.
Cole et al., "Two ParaHox genes, SpLox and SpCdx, interact to
partition the posterior endoderm in the formation of a functional gut,"
Development. 136:541-549 (2009). cited by applicant
.
Colucci et al., "Programmed cell death in the pathogenesis of
murine IDDM: resistance to apoptosis induced in lymphocytes by
cyclophosphamide," J Autoimmunity 9:271-276 (1996). cited by applicant
.
Corbett et al., "Nitric oxide mediates cytokine-induced inhibition
of insulin secretion by human islets of langerhans" Proc Natl Acad Sci
USA. 90(5):1731-1735 (1993). cited by applicant
.
Coux et al., "Enzymes catalyzing ubiquitination and proteolytic
processing of the p105 precursor of nuclear factor .sub.KB1," J Biol
Chem. 273(15):8820-8828 (1998). cited by applicant
.
Couzin, "Diabetes studies conflict on power of spleen cells," Science. 311:1694 (2006). cited by applicant
.
Darzynkiewicz et al., "Use of flow and laser scanning cytometry to
study mechanisms regulating cell cycle and controlling cell death,"
Clinics in Laboratory Medicine. 21:857-873 (2001). cited by applicant
.
Dear et al., "The Hox11 gene is essential for cell survival during
spleen development," Development. 121:2909-2915 (1995). cited by
applicant
.
Declaration of Dr. Denise Faustman from U.S. Appl. No. 10/358,644, filed on May 15, 2009. cited by applicant
.
Dieguez-Acuna et al., "Characterization of mouse spleen cells by
subtractive proteomics," Mol Cell Proteomics. 4(10):1459-1470 (2005).
cited by applicant
.
Dieguez-Acuna et al., "Proteomics identifies multipotent and low
oncogenic risk stem cells of the spleen," Int J Biochem Cell Biol.
42:1651-1660 (2009). cited by applicant
.
Dilts et al., "Autoimmune diabetes: The involvement of benign and
malignant autoimmunity," J Autoimmun. 12:229-232 (1999). cited by
applicant
.
Dinarello, "Interleukin-1, Interleukin-1 receptors and
Interleukin-1 receptor antagonist," Intern Rev Immunol. 16:457-499
(1998). cited by applicant
.
Driscoll et al., "The proteasome (multicatalytic protease) is a
component of the 1500-kDa proteolytic complex which degrades
ubiquitin-conjugated proteins," J Biol Chem. 265:4789-4792 (1990). cited
by applicant
.
Durand et al., "Mesenchymal lineage potentials of
Aorta-Gonad-Mesonephros stromal clones," Heamatologica. 91:1172-1179
(2006). cited by applicant
.
Eglitis et al., "Hematopoietic cells differentiate into both
microglia and macroglia in the brains of adult mice," Proc Natl Acad Sci
USA. 94:4080-4085 (1997). cited by applicant
.
Elliott et al., "Effect of Bacille Calmette-Guerin vaccination on
C-Peptide secretion in children newly diagnosed with IDDM," Diabetes
Care. 21(10):1691-1693 (1998). cited by applicant
.
Eytan et al., "ATP-dependent incorporation of 20S protease into the
26S complex that degrades proteins conjugated to ubiquitin," Proc Natl
Acad Sci USA. 86:7751-7755 (1989). cited by applicant
.
Fan et al., "Generation of p50 subunit of NF-KB by processing of
p105 through an ATP-dependent pathway," Nature. 354:395-398 (1991).
cited by applicant
.
Faustman et al., "Abnormal T-lymphocyte subsets in Type I Diabetes," Diabetes. 38:1462- 1468 (1989). cited by applicant
.
Faustman et al., "Linkage of faulty major histocompatibility
complex class I to autoimmune diabetes," Science. 254:1756-1761 (1991).
cited by applicant
.
Faustman et al., "Murine pancreatic .beta.-Cells express H-2K and
H-2D but not la Antigens," J Exp Med. 151:1563-1568 (1980). cited by
applicant
.
Faustman et al., "Prevention of xenograft rejection by masking
donor HLA class I antigens," Science. 252:1700-1702 (1991). cited by
applicant
.
Faustman et al., "T-lymphocyte changes linked to autoantibodies.
Association of insulin autoantibodies with CD4+CD45R+ lymphocyte
subpopulation in prediabetic subjects," Diabetes. 40:590-597 (1991).
cited by applicant
.
Feldman et al., "Anti-TNF.alpha. therapy is useful in rheumatoid
arthritis and Crohn's disease: Analysis of the mechanism of action
predicts utility in other diseases," Transplant Proc. 30(8):4126-4127
(1998). cited by applicant
.
Ferrando et al., "Adult T-Cell ALL patients whose lymphoblasts
express the HOX11 oncogene have an excellent prognosis when treated with
chemotherapy and are not candidates for allogeneic bone marrow
transplantation in first remission," Blood.
11:154A (2002). cited by applicant
.
Fischer et al., "An improved flow cytometric assay for the
determination of cytotoxic T lymphocyte activity," J Immunol Methods.
259:159-169 (2002). cited by applicant
.
Foulis, "C.L. Oakley lecture (1987). The pathogenesis of beta cell
destruction in Type I (insulin-dependent) diabetes mellitus," J Pathol.
152(3):141-148 (1987). cited by applicant
.
Fu et al., "Antigen processing and autoimmunity: Evaluation of mRNA
abundance and function of HLA-Linked genes," Ann NY Acad Sci.
842:138-155 (1998). cited by applicant
.
Fu et al., "Defective major histocompatibility complex class I
expression on lymphoid cells in autoimmunity," J Clin Invest.
91:2301-2307 (1993). cited by applicant
.
Fukada et al., "Two signals are necessary for cell proliferation
induced by a cytokine receptor gp130: Involvement of STAT3 in
anti-apoptosis," Immunity. 5:449-460 (1996). cited by applicant
.
Gage, "Mammalian neural stem cells," Science. 287:1433-1438 (2000). cited by applicant
.
Gage et al., "Multipotent progenitor cells in the adult dentate gyrus," J Neurobiol. 36:249-266 (1998). cited by applicant
.
Ganoth et al., "A multicomponent system that degrades proteins
conjugated to ubiquitin. Resolution of factors and evidence for
ATP-dependent complex formation," J Biol Chem. 263(25):12412-12419
(1988). cited by applicant
.
Gaur et al., "Induction of islet allotolerance in nonhuman primates," Ann NY Acad Sci. 958:199-203 (2002). cited by applicant
.
Gazda et al., "Diabetes results from a late change in the
autoimmune response of NOD mice," J Autoimmun. 10:261-270 (1997). cited
by applicant
.
Gazda et al., "Regulation of autoimmune diabetes: characteristics
of non-islet-antigen specific therapies," Immunol Cell Biol. 74: 401-407
(1996). cited by applicant
.
Genestier et al., "Immunosuppressive properties of methotrexate:
Apoptosis and clonal deletion of activated peripheral T Cells," J Clin
Invest. 102(2):322-328 (1998). cited by applicant
.
Gerich et al., "Advances in diabetes for the millenium:
Understanding insulin resistance," MedGenMed. 6(Suppl): 11:1-9 (2004).
cited by applicant
.
Ghosh et al., "Activation in vitro of NF-.sub.kB by phosphorylation
of its inhibitor I.sub.kB," Nature. 344:678-682 (1990). cited by
applicant
.
Glas et al., "The CD8+ T Cell repertoire in
.beta.2-microglobulin-deficient mice is biased towards reactivity
against self-major histocompatibility class I," J Exp Med.
179(2):661-672 (1994). cited by applicant
.
Goldberg, "Functions of the proteasome: The lysis at the end of the tunnel," Science. 268:522-523 (1995). cited by applicant
.
Goldberg, "The mechanism and functions of ATP-dependent proteases
in bacterial and animal cells," Eur J Biochem. 203:9-23 (1992). cited by
applicant
.
Gottlieb et al., "Cell acidification in apoptosis: Granulocyte
colony-stimulating factor delays programmed cell death in neutrophils by
up-regulating the vacuolar H.sup.+-ATPase," Proc Natl Acad Sci USA.
92:5965-5968 (1995). cited by applicant
.
Graves et al., "Lack of association between early childhood
immunizations and .beta.-Cell autoimmunity," Diabetes Care 22:1694-1967
(1999). cited by applicant
.
Grewal et al., "Local expression of transgene encoded TNF.alpha. in
islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by
preventing the development of auto-reactive islet-specific T Cells," J
Exp Med. 184:1963-1974 (1996). cited by
applicant
.
Grilli et al., "Neuroprotection by aspirin and sodium salicylate
through blockade of NF-kB activation," Science. 274:1383-1385 (1996).
cited by applicant
.
Gronostajski et al., "The ATP dependence of the degradation of
short- and long-lived proteins in growing fibroblasts," J Biol Chem.
260(6):3344-3349 (1985). cited by applicant
.
Gueckel et al., "Mutations in the yeast proteasome B-Type subunit
Pre3 uncover position-dependent effects on proteasomal peptidase
activity and in vivo function," J Biol Chem. 273(31): 19443-19452
(1998). cited by applicant
.
Gupta, "Molecular steps of tumor necrosis factor receptor-mediated
apoptosis," Curr Mol Med. 1(3):317-324 (2001). cited by applicant
.
Haas et al., "Pathways of ubiquitin conjugation," FASEB J. 11:1257-1268 (1997). cited by applicant
.
Hao et al., "Effect of mycophenolate mofetil on islet allografting
to chemically induced or spontaneously diabetic animals," Transplant
Proc. 24(6): 2843-2844 (1992). cited by applicant
.
Harada et al., "Prevention of overt diabetes and insulitis in NOD
mice by a single BCG vaccination," Diabetes Res Clin Pract. 8:85-89
(1990). cited by applicant
.
Hartwell et al., "Aberrant cytokine regulation in macrophages from
young autoimmune-prone mice: Evidence that the intrinsic defect in MRL
macrophage IL-1 expression is transcriptionally controlled," Mol
Immunol. 32(10):743-751 (1995). cited by
applicant
.
Hayashi et al., "Essential role of human leukocyte antigen-encoded
proteasome subunits in NF-kB activation and prevention of tumor necrosis
Factor-.alpha.-induced apoptosis," J Biol Chem. 275(7):5238-5247
(2000). cited by applicant
.
Hayashi et al., "NOD mice are defective in proteasome production
and activation of NF-KB," Mol Cell Biol. 19(12):8646-8659 (1999). cited
by applicant
.
Hershko et al., "The ubiquitin system for protein degradation," Annu Rev Biochem. 61: 761- 807 (1992). cited by applicant
.
Hostikka et al., "The mouse hoxc11 gene: genomic structure and
expression pattern," Mech Dev. 70:133-145 (1998). (Abstract Only). cited
by applicant
.
Hester et al., "Studies on the cytophilic properties of human
.beta.2-microglobulin. II. The role of histocompatibility antigens,"
Scand J Immunol. 9(2):125-134 (1979). cited by applicant
.
Horsfall et al., "Characterization and specificity of B-cell
responses in lupus induced by Mycobacterium bovis in NOD/Lt mice,"
Immunology 95:8-17 (1998). cited by applicant
.
Horwitz et al., "Recombinant baccillus calmette-guerin (BCG)
vaccines expressing the Mycobacterium Tuberculosis 30-kDa major
secretory protein induce greater protective immunity against
Tuberculosis than conventional BCG vaccines in a highly
susceptible animal model," Proc Natl Acad Sci USA. 97:13853-13858
(2000). cited by applicant
.
Hsu et al., "TRADD-TRAF2 and TRADD-FADD interactions define two
distinct TNF receptor 1 signal transduction pathways," Cell. 84:299-308
(1996). cited by applicant
.
Humphreys-Behr et al., "New concepts for the development of
autoimmune exocrinopathy derived from studies with the NOD mouse model,"
Arch Oral Biol. 44:S21-S25 (1999). (Abstract Only). cited by applicant
.
Hyafil et al., "Dissociation and exchange of the
.sub..beta.2-micoglobulin subunit of HLA-A and HLA-B antigens," Proc
Natl Acad Sci USA. 76(11):5834-5838 (1979). cited by applicant
.
Hymowitz et al., "Toward small-molecule agonists of TNF receptors," Nat Chem Biol. 1(7):353-354 (2005). cited by applicant
.
Jackson et al., "Hematopoietic potential of stem cells isolated
from murine skeletal muscle," Proc Natl Acad Sci USA. 96(25):14482-14486
(1999). cited by applicant
.
Jacob et al., "Monoclonal anti-tumor necrosis factor antibody
renders non-obese diabetic mice hypersensitive to irradiation and
enhances insulitis development," Int Immunol. 4(5):611-614 (1992). cited
by applicant
.
Jacob et al., "Prevention of diabetes in nonobese diabetic mice by
tumor necrosis factor (TNF): Similarities between TNF-.alpha. and
interleukin 1," Proc Natl Acad Sci USA. 87:968-972 (1990). cited by
applicant
.
Jacob et al., "Tumour necrosis factor-.alpha. in murine autoimmune
`lupus` nephritis," Nature. 331:356-358 (1988). cited by applicant
.
Jakubowski et al., "Phase I trial of intramuscularly administered
tumor necrosis factor in patients with advanced cancer," J Clin Oncol.
7(3):298-303 (1989). cited by applicant
.
Jiang et al., "Pluripotency of mesenchymal stem cells derived from adult marrow," Nature. 418:41-49 (2002). cited by applicant
.
Johansson et al' "Identification of a neural stem cell in the adult
mammalian central nervous system," Cell. 96:25-34 (1999). cited by
applicant
.
Juang et al., "Beneficial influence of glycemic control upon the
growth and function of transplanted islets," Diabetes 43:1334-1339
(1994). cited by applicant
.
Kaijzel et al., "Functional analysis of a human tumor necrosis
factor .alpha. (TNF-.alpha.) promoter polymorphism related to joint
damage in rheumatoid arthritis," Mol Med. 4:724-733 (1998). cited by
applicant
.
Kanzler et al, "Hox11 acts cell autonomously in spleen development
and its absence results in altered cell fate of mesenchymal spleen
precursors," Devel Biol. 234:231-243 (2001). cited by applicant
.
Kaufman et al., "Patterns of hemopoietic reconstitution in nonobese
diabetic mice: dichotomy of allogeneic resistance versus competitive
advantage of disease-resistant marrow," J Immunol. 158(5):2435-2442
(1997). cited by applicant
.
Kawaski et al., "Prevention of type 1 diabetes: from the view point
of .beta. cell damage," Diabetes Res Clin Pract. 66:S27-S32 (2004).
cited by applicant
.
Kieran et al., "The DNA binding subunit of NF-KB is identical to
factor KBF1 and homologous to the rel oncogene product," Cell
62:1007-1018 (1990). cited by applicant
.
Klingensmith et al., "Vaccination with BCG at diagnosis does not
alter the course of IDDM," abstract presented at the 31st research
symposium on prevention of type I diabetes in general population.
American Diabetes Association, Estes Park,
Colorado, 27-29 (1996). cited by applicant
.
Koarada et al., "B Cells lacking RP105, A novel B cell antigen, in
systemic lupus erythematosus," Arthritis & Rheumatism
42(12):2593-2600 (1999). cited by applicant
.
Kodama et al., "Islet regeneration during the reversal of
autoimmune diabetes in NOD mice," Science. 302:1223-1227 (2003). cited
by applicant
.
Kodama et al., "Regenerative medicine: A radical reappraisal of the
spleen," Trends Mol Med. 11(6):271-276 (2005). cited by applicant
.
Kodama et al., "The therapeutic potential of tumor necrosis factor
for autoimmune disease: A mechanically based hypothesis," Cell Mol Life
Sci. 62:1850-1862 (2005). cited by applicant
.
Kopp and Ghosh, "Inhibition of NF-KB by sodium salicylate and aspirin," Science. 265:956-959 (1994). cited by applicant
.
Kouskoff et al., "Organ-specific disease provoked by systemic
autoimmunity," Cell. 87:811-822 (1996) (Abstract Only). cited by
applicant
.
Koyama et al., "Hox11 genes establish synovial joint organization
and phylogenetic characteristics in developing mouse zeugopod skeletal
elements," Development. 137: 3795, 2010 (Abstract Only). cited by
applicant
.
Krause et al., "Multi-organ, multi-lineage engraftment by a single
bone marrow-derived stem cell," Cell. 105:369-377 (2001). cited by
applicant
.
Kuehnle and Goodell, "The therapeutic potential of stem cells from adults," BMJ. 325:372-376 (2002). cited by applicant
.
Kwon et al., "Evidence for involvement of the proteasome complex
(26S) and NFKB in IL-1.beta.-induced nitric oxide and prostaglandin
production by rat islets and RINm5F Cells" Diabetes. 47:583-591 (1998).
cited by applicant
.
Kwon et al., "Interleukin-1.beta. -induced nitric oxide synthase
expression by rat pancreatic .beta.-cells: Evidence for the involvement
of nuclear factor .sub.kB in the signaling mechanism," Endocrinology.
136(11):4790-4795 (1995). cited by
applicant
.
Laakko et al., "Versatility of merocyanine 540 for the flow
cytometric detection of apoptosis in human and murine cells," J Immunol
Methods. 261:129-139 (2002). cited by applicant
.
Lahav-Baratz et al., "Reversible phosphorylation controls the
activity of cyclosome-associated cyclin-ubiquitin ligase," Proc Natl
Acad Sci USA 92:9303-9307 (1995). cited by applicant
.
Lakey et al., "BCG immunotherapy prevents recurrence of diabetes in
islet grafts transplanted into spontaneously diabetic NOD mice,"
Transplantation. 57(8):1213-1217 (1994). cited by applicant
.
Lammert et al., "Induction of pancreatic differentiation by signals
from blood vessels," Science. 294:564-567 (2001). cited by applicant
.
Lanza et al., "Transplantation of encapsulated canine islets into
spontaneously diabetic BB/Wor rats without immunosuppression,"
Endocrinology. 131:637-642 (1992). cited by applicant
.
Lapchak et al., "Tumor necrosis factor production is deficient in
diabetes-prone BB rats and can be corrected by complete Freund's
adjuvant: A possible immunoregulatory role of tumor necrosis factor in
the prevention of diabetes," Clin Immunol
Immunopathol. 65(2):129-134 (1992). cited by applicant
.
Lawrence et al., "Differential hepatocyte toxicity of recombinant
Apo2L/TRAIL versions," Nat Med. 7(4):383-385 (2001). cited by applicant
.
Lewis et al., "Integrins regulate the apoptotic response to DNA
damage through modulation of p53" Proc Natl Acad Sci USA.
99(6):3627-3632 (2002). cited by applicant
.
Li and Faustman, "Use of donor .beta.2-microglobulin-deficient
transgenic mouse liver cells for isografts, allografts, and xenografts,"
Transplantation. 55(4):940-946 (1993). cited by applicant
.
Li et al., "Abnormal class I assembly and peptide presentation in
the nonobese diabetic mouse," Proc Natl Acad Sci USA. 91:11128-11132
(1994). cited by applicant
.
Li et al., "Reduced expression of peptide-loaded HLA class I
molecules on multiple sclerosis lymphocytes," Ann Neurol. 38:147-154
(1995). cited by applicant
.
Ljunggren et al., "MHC class I expression and CD8+ T cell
development in TAP1/.beta.2-microglobulin double mutant mice," Int
Immunol. 7(6):975-984 (1995). cited by applicant
.
Macchi et al., "Impaired apoptosis in mitogen-stimulated
lymphocytes of patients with multiple sclerosis," NeuroReport.
10:399-402 (1999). cited by applicant
.
Mak et al., "Signaling for survival and apoptosis in the immune
system," Arthritis Res. 4(Suppl 3):S243-S252 (2002). cited by applicant
.
Markiewicz et al., "Long-term T cell memory requires the surface
expression of self-peptide/major histocompatibility complex molecules,"
Proc Natl Sci USA. 95:3065-3070 (1998). cited by applicant
.
Markmann et al., "Indefinite survival of MHC class I-deficient
murine pancreatic islet allografts," Transplantation. 54:1085-1089
(1992). cited by applicant
.
Marriott, "TNF-.alpha. antagonists: Monoclonal antibodies, soluble
receptors, thalidomide and other novel approaches," Expert Opin Investig
Drugs. 6(8):1105-1108 (1997). cited by applicant
.
Matsumoto et al., "Liver organogenesis promoted by endothelial
cells prior to vascular function," Science. 294:559-563 (2001). cited by
applicant
.
Mayer-Proschel et al., "Isolation of lineage-restricted neuronal
precursors from multipotent neuroepithelial stem cells," Neuron.
19:773-785 (1997). cited by applicant
.
McGuire et al., "An enzyme related to the high molecular weight
multicatalytic proteinases, macropain, participates in a
ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble
extracts of BHK 21/C13 fibroblasts," Biochim Biophys Acta.
967:195-203 (1988). cited by applicant
.
McInerney et al., "Prevention of insulitis and diabetes onset by
treatment with complete Freund's adjuvant in NOD mice," Diabetes.
40:715-725 (1991). cited by applicant
.
McKay et al., "Mammalian deconstruction for stem cell reconstruction," Nat Med. 6:747-748 (2000). cited by applicant
.
Mercurio et al., "p105 and p98 precursor proteins play an active
role in NF-Kappa B-mediated signal transduction," Genes Dev. 7:705-718
(1993). cited by applicant
.
Mestas et al., "Of mice and not Men: Differences between mouse and
human immunology," J Immunol. 172:2731-2738 (2004). cited by applicant
.
Mezey et al., "Turning blood into brain: Cells bearing neuronal
antigens generated in vivo from bone marrow," Science. 290: 1779-1782
(2000). cited by applicant
.
Miller et al., "Both the Lyt-2+ and L3T4+ T cell subsets are
required for the transfer of diabetes in nonobese diabetic mice," J
Immunol. 140:52-58 (1988). cited by applicant
.
Mittleman et al., "A phase I pharmacokinetic study of recombinant
human tumor necrosis factor administered by a 5-day continuous
infusion," Invest New Drugs. 10(3):183-190 (1992). cited by applicant
.
Miyazaki et al., "Predominance of T lymphocytes in pancreatic
islets and spleen of pre-diabetic non-obese diabetic (NOD) mice: A
longitudinal study," Clin Exp Immunol. 60:622-630 (1985). cited by
applicant
.
Morrison, "Stem cell potential: Can anything make anything?" Curr Biol. 11:R7-R9 (2001). cited by applicant
.
Nomikos et al., "Combined treatment with nicotinamide and
desferrioxamine prevents islet allograft destruction in NOD mice,"
Diabetes. 35:1302-1304 (1986). cited by applicant
.
Murthi et al., "Novel homeobox genes are differentially expressed
in placental microvascular endothelial cells compared with macrovascular
cells," Placenta. 29:624-630 (2008). (Abstract Only). cited by
applicant
.
Offield et al., "PDX-1 is required for pancreatic outgrowth and
differentiation of the rostral duodenum," Development. 122:983-995
(1996). cited by applicant
.
Ono et al., "IDDM in BB rats. Enhanced MHC Class I heavy-chain gene
expression in pancreatic islets," Diabetes. 37:1411-1418 (1988). cited
by applicant
.
Orlowski, "The multicatalytic proteinase complex, a major
extralysosomal proteolytic system," Biochemistry. 29(45):10289-10297
(1990). cited by applicant
.
Osorio et al., "Beta-2 microglobulin gene disruption prolongs
murine islet allograft survival in NOD mice," Transplant Proc. 26(2):752
(1994). cited by applicant
.
Palombella et al., "The ubiquitin-proteasome pathway is required
for processing the NF-.sub.KB1 precursor protein and the activation of
NF- .sub.KB," Cell. 78:773-785 (1994). cited by applicant
.
Pestano et al., "Inactivation of misselected CD8 T cells by CD8
gene methylation and cell death," Science. 284:1187-1191 (1999). cited
by applicant
.
Petersen et al., "Bone marrow as a potential source of hepatic oval cells," Science. 284:1168-1170 (1999). cited by applicant
.
Pozzilli, "BCG vaccine in insulin-dependent diabetes mellitus," Lancet. 349:1520-1521 (1997). cited by applicant
.
Paolillo et al., "The effect of bacille calmette-guerin on the
evolution of new enhancing lesions to hypointense T1 lesions in
relapsing remitting MS," J Neurol. 250:247-248 (2003). cited by
applicant
.
Pontesilli et al., "Circulating lymphocyte populations and
autoantibodies in non-obese diabetic (NOD) mice: A longitudinal study,"
Clin Exp Immunol. 70:84-93 (1987). cited by applicant
.
Prieto et al., "Apoptotic rate: A new indicator for the
quantification of the incidence of apoptosis in cell cultures,"
Cytometry. 48:185-193 (2002). cited by applicant
.
Qin et al., "Complete Freud's adjuvant-induced T cells prevent the
development and adoptive transfer of diabetes in nonobese diabetic
mice," J Immunol. 150:2072-2080 (1993). cited by applicant
.
Qin et al., "BCG vaccination prevents insulin-dependent diabetes
mellitus (IDDM) in NOD mice after disease acceleration with
cyclophosphamide," J Autoimmun.10:271-278 (1997). cited by applicant
.
Quintana et al., "Experimental autoimmune myasthenia gravis in
naive non-obese diabetic (NOD/LtJ) mice: Susceptibility associated with
natural IgG antibodies to the acetylcholine receptor," Intl Immunol.
15:11-16 (2003). cited by applicant
.
Raab et al., "In vitro evaluation of methotrexate and azathioprine
for antipsoriatic activity," Arch Derm Res. 253:77-84 (1975). cited by
applicant
.
Rabinovitch et al., "TNF-.alpha. down-regulates type 1 cytokines
and prolongs survival of syngeneic islet grafts in nonobese diabetic
mice," J Immunol. 159:6298-6303 (1997). cited by applicant
.
Rabinovitch et al., "Tumor necrosis factor mediates the protective
effect of freund's adjuvant against autoimmune diabetes in BB rats," J
Autoimmun. 8:357-366 (1995). cited by applicant
.
Rajagopalan et al., "Pathogenic anti-DNA autoantibody-inducing T
helper cell lines from patients with active lupus nephritis: Isolation
of CD4-8- T helper cell lines that express the .gamma..delta. T-cell
antigen receptor," Proc Natl Acad Sci USA.
87:7020-7024 (1990). cited by applicant
.
Raju et al., "Characterization and developmental expression of
Tlx-1, the murine homolog of HOX11," Mech Dev. 44:51-64 (1993). cited by
applicant
.
Ramiya et al., "Reversal of insulin-dependent diabetes using islets
generated in vitro from pancreatic stem cells," Nat Med. 6(3):278-282
(2000). cited by applicant
.
Rath el al., "TNF-induced signaling in apoptosis," J Clin Immuno1.19(6):350-364 (1999). cited by applicant
.
Rechsteiner, "Ubiquitin-mediated pathways for intracellular proteolysis," Annu Rev Cell Biol. 3:1-30 (1987). cited by applicant
.
Rietze et al., "Purification of a pluripotent neural stem cell from
the adult mouse brain," Nature. 412:736-739 (2001). cited by applicant
.
Ristori et al., "Use of Bacille Calmette-Guerin (BCG) in multiple sclerosis," Neurology. 53:1588-1589 (1999). cited by applicant
.
Roberts et al., "Hox11 controls the genesis of the spleen," Nature. 368:747-749 (1994). cited by applicant
.
Robertson et al., "Preservation of insulin mRNA levels and insulin
secretion in HIT cells by avoidance of chronic exposure to high glucose
concentrations," J Clin Invest. 90:320-325 (1992). cited by applicant
.
Roberts et al., "Developmental expression of Hox11 and
specification of splenic cell fate," Am J Pathol. 146:1089-1101 (1995).
cited by applicant
.
Robinson et al., "Elevated levels of cysteine protease activity in
salive and salivary glands of the non obese diabetic (NOD) mouse model
for Sjogren Syndrome," Proc Natl Acad Sci USA. 94:5767-5771 (1997).
cited by applicant
.
Robinson et al., "A novel NOD-derived murine model of primary
Sjogren's Syndrome," Arth Rheum. 41:150-156 (1998). cited by applicant
.
Rolfe et al., "The ubiquitin-mediated proteolytic pathway as a therapeutic area," J Mol Med. 75:5-17 (1997). cited by applicant
.
Rosenthal, "Prometheus's vulture and the stem-cell promise," n. Engl J Med. 349:67-274 (2003). cited by applicant
.
Ryu et al., "Reversal of established autoimmune diabetes by
restoration of endogenous .beta. cell function," J Clin Invest.
108:63-72 (2001). cited by applicant
.
Sadelan et al., "Prevention of type I diabetes in NOD mice by
adjuvant immunotherapy," Diabetes. 39:583-589 (1990). cited by applicant
.
Sarin et al., "Cytotoxic effect of TNF and lymphotoxin on T lymphoblasts," J Immunol. 151:3716-3718 (1995). cited by applicant
.
Satoh et al., "Inhibition of type I diabetes in BB rats with
recombinant human tumor necrosis factor-.alpha. ," J Immunol.
145(5):1395-1399 (1990). cited by applicant
.
Satoh et al., "Recombinant human tumor necrosis factor .alpha.
suppresses autoimmune diabetes in non obese diabetic mice," J Clin
Invest. 84:1345-1348 (1989). cited by applicant
.
Schatz et al., "Defective inducer T-cell function before the onset
of insulin-dependent diabetes mellitus," J Autoimmun. 4:125-136 (1991).
cited by applicant
.
Schmidt et al., "Interspecies exchange of .beta.2-microglobulin and
associated MHC and differentiation antigens," Immunogenetics.
13(6):483-49 (1981). cited by applicant
.
Schuppan, "Current concepts of Celiac Disease pathogenesis," Gastroenterology. 119:234- 242 (2000). cited by applicant
.
Sears et al., "NF-kB p105 processing via the ubiquitin-proteasome
pathway," J Biol Chem. 273:1409-1419 (1998). cited by applicant
.
Serrano et al., "Non-HLA associations with autoimmune diseases," Autoimmun Rev. 5:209- 214 (2006). cited by applicant
.
Serup, "Panning for pancreatic stem cells," Nat Genet. 25:134-135 (2000). cited by applicant
.
Serup et al., "Islet and stem cell transplantation for treating diabetes," BMJ. 322:29-32 (2001). cited by applicant
.
Serreze et al., "Th1 to Th2 cytokine shifts in nonobese diabetic
mice: Sometimes an outcome, rather than the cause, of diabetes
resistance elicited by immunostimulation," J Immunol. 166:1352-1359
(2001). cited by applicant
.
Shehadeh et al., "Effect of adjuvant therapy on development of
diabetes in mouse and man," Lancet. 343:706-707 (1994). cited by
applicant
.
Shihabuddin et al., "Adult spinal cord stem cells generate neurons
after transplantation in the adult dentate gyrus," J Neurosci.
20:8727-8735 (2000). cited by applicant
.
Shohami et al., "Dual role of tumor necrosis factor alpha in brain
injury," Cytokine Growth Factor Rev. 10:119-130 (1999). cited by
applicant
.
Singh et al., "Can progression of IDDM be prevented in newly
diagnosed patients by BCG immunotherapy?" Diabetes Metab Rev. 13:320-321
(1997). cited by applicant
.
Silva et al., "Prevention of autoimmune diabetes through
immunostimulation with Q fever complement-fixing antigen," Ann NY Acad
Sci. 1005:423-430 (2003). cited by applicant
.
Slack, "Stem cells in epithelial tissues," Science. 287:1431-1433 (2000). cited by applicant
.
Song et al., "Tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle
progression," J Exp Med. 191(7):1095-1103 (2000). cited by applicant
.
Speiser et al., "Loss of ATP-dependent proteolysis with maturation
of reticulocytes and erythrocytes," J Biol Chem. 257(23):14122-14127
(1982). cited by applicant
.
Sreenan et al., "Increased .beta.-Cell proliferation and reduced
mass before diabetes onset in the nonobese diabetic mouse," Diabetes.
48:989-996 (1999). cited by applicant
.
Stephens et al., "Protection of NIT-1 pancreatic .beta.-cells from
immune attack by inhibition of NF-kB," J Autoimmun. 10:293-298 (1997).
cited by applicant
.
Storms et al., "Hoechst dye efflux reveals a novel CD7+CD34-
lymphoid progenitor in human umbilical cord blood," Blood. 96:2125-2133
(2000). cited by applicant
.
Sun et al., "MHC class I multimers," Arthritis Res. 9:265-269 (2001). cited by applicant
.
Szdoray et al., "Programmed cell death in rheumatoid arthritis
peripheral blood T-cell subpopulations determined by laser scanning
cytometry," Lab Invest. 83(12):1839-1848 (2003). cited by applicant
.
Shehadeh et al., "Repeated BCG vaccination is more effective than a
single dose in preventing diabetes in non-obese diabetic (NOD) mice,"
Isr J Med Sci. 33(11):711-715 (1997). cited by applicant
.
Swirski et al., "Identification of splenic reservoir monocytes and
their deployment to inflammatory sites," Science. 325:612-616 (2009).
cited by applicant
.
Tamura et al., "In vivo differentiation of stem cells in the
aorta-gonad-mesonephros region of mouse embryo and adult bone marrow,"
Exp Hematol. 30:957-966 (2002) (Abstract Only). cited by applicant
.
Tartaglia et al, "The two different receptors for tumor necrosis
factor mediate distinct cellular responses," Proc Natl Acad Sci USA.
88:9292-9296 (1991). cited by applicant
.
Terada et al., "Bone marrow cells adopt the phenotype of other
cells by spontaneous cell fusion," Nature. 416:542-545 (2002). cited by
applicant
.
The Merck Manual of Diagnosis and Therapy, Beers and Berkow,
Published by Merck Research Laboratories, 17th Ed., 165-171, 1999. cited
by applicant
.
Toma et al., "Isolation of multipotent adult stem cells from the
dermis of mammalian skin," Nat Cell Bio. 3:778-784 (2001). cited by
applicant
.
Totpal et al., "TNF and its receptor antibody agonist differ in
mediation of cellular responses," J Immunol. 153:2248-2257 (1994). cited
by applicant
.
Townsley et al., "Dominant-negative cyclin-selective ubiquitin
carrier protein E2-C/UbcH10 blocks cells in metaphase," Proc Natl Acad
Sci USA. 94:2362-2367 (1997). cited by applicant
.
Trowsdale et al., "Sequences encoded in the class II region of the
MHC related to the `ABC` superfamily of transporters," Nature.
348:741-744 (1990). cited by applicant
.
Ulaeto et al., "A T-cell dormant state in the autoimmune process of
nonobese diabetic mice treated with complete Freund's adjuvant," Proc
Natl Acad Sci USA. 89:3927-3931 (1992). cited by applicant
.
Tran et al., "Reversal of Sjogren's-like Syndrome in non-obese
diabetic mice," Ann Rheum Dis. 66:812-814 (2007). cited by applicant
.
Van der Kooy et al., "Why stem cells?," Science. 287:1439-1441 (2000). cited by applicant
.
Van Nocker et al., "The multiubiquitin-chain-binding protein Mcb1
is a component of the 26S proteasome in Saccharomyces cerevisiae and
plays a nonessential, substrate-specific role in protein turnover," Mol
Cell Biol. 16(11):6020-6028 (1996). cited
by applicant
.
Van Noort et al., "Cell biology of autoimmune diseases," Int Rev Cytol. 178:127-204 (1998). cited by applicant
.
Vidal-Puig et al., "Tolerance to peripheral tissue Is transient and
maintained by tissue-specific class I expression," Transplant Proc.
26:3314-3316 (1994). cited by applicant
.
Vogel et al., "Studies cast doubt on plasticity of adult cells," Science. 295:1989-1991 (2002). cited by applicant
.
Von Herrath et al., "In vivo treatment with a MHC class
I-restricted blocking peptide can prevent virus-induced autoimmune
diabetes," J Immunol. 161:5087-5096 (1998). cited by applicant
.
Wang et al., "Prevention of recurrence of IDDM in
islet-transplanted diabetic NOD mice by adjuvant immunotherapy,"
Diabetes. 41:114-117 (1992). cited by applicant
.
Watt et al., "Out of eden: stem cells and their niches," Science. 287:1427-1430 (2000). cited by applicant
.
Waxman et al., "Demonstration of two distinct high molecular weight
proteases in rabbit reticulocytes, one of which degrades ubiquitin
conjugates," J Biol Chem. 262(6):2451-2457 (1987). cited by applicant
.
Weissman, "Translating stem and progenitor cell biology to the
clinic: barriers and opportunities," Science. 287:1442-1446 (2000).
cited by applicant
.
Weringer et al., "Identification of T cell subsets and Class I and
Class II antigen expression in islet grafts and pancreatic islets of
diabetic BioBreeding/Worcester rats," Am J Pathol. 132(1):292-303
(1988). cited by applicant
.
Wellik et al., "Hox11 paralogous genes are essential for
metanephric kidney induction," Genes Dev. 16:1423-1432 (2002). cited by
applicant
.
Wellik, "The role of Hox11 paralogous genes in prostate development," Grant Detail. (Abstract only) (2009). cited by applicant
.
Wicker et al., "Transfer of autoimmune diabetes mellitus with
splenocytes from nonobese diabetic (NOD) mice," Diabetes. 35:855-860
(1986). cited by applicant
.
Willis et al., "Type 1 Diabetes in insulin-treated adult-onset
diabetic subjects," Diabetes Res Clin Pract. 42:49-53 (1998). cited by
applicant
.
Winston, "Embryonic stem cell research: the case for . . . ," Nat Med. 7:396-397 (2001). cited by applicant
.
Wong et al., "Identification of an MHC class I-restricted
autoantigen in Type I Diabetes by screening an organ-specific cDNA
library," Nat Med. 5:1026-1031 (1999). cited by applicant
.
Xu et al., "MHC/peptide tetramer-based studies of T cell function," J Immunol Methods. 268:21-28 (2002). cited by applicant
.
Yagi et al., "Possible mechanism of the preventive effect of BCG
against diabetes mellitus in NOD Mouse. I. Generation of suppressor
macrophages in spleen cells of BCG-vaccinated mice," Cell Immunol.
138:130-141 (1991). cited by applicant
.
Yagi et al., "Possible mechanism of the preventive effect of BCG
against diabetes mellitus in NOD Mouse. II. Suppression of pathogenesis
by macrophage transfer from BCG-vaccinated mice," Cell Immunol.
138:142-149 (1991). cited by applicant
.
Yan et al., "Reduced expression of Tap1 and Lmp2 antigen-processing
genes in the nonobese diabetic (NOD) mouse due to a mutation in their
shared bidirectional promoter," J Immunol. 159:3068-3080 (1997). cited
by applicant
.
Yang et al., "Effect of tumor necrosis factor .alpha. on
insulin-dependent diabetes mellitus in NOD Mice. I. The early
development of autoimmunity and the diabetogenic process," J Exp Med.
180:995-1004 (1994). cited by applicant
.
Ying et al., "Changing potency by spontaneous fusion," Nature. 416:545-548 (2002). cited by applicant
.
Zoller et al., "Apoptosis resistance in peripheral blood
lymphocytes of alopecia areata patients," J Autoimmun. 23(3):241-256
(2004). cited by applicant
.
Zulewski et al., "Multipotential nestin-positive stem cells
isolated from adult pancreatic islets differentiate ex vivo into
pancreatic endocrine, exocrine, and hepatic phenotypes," Diabetes.
50:521-533 (2001). cited by applicant
.
International Search Report for International Application No. PCT/US2004/037998, mailed Feb. 28, 2008. cited by applicant
.
Written Opinion for International Application No. PCT/US2004/037998, mailed Feb. 28, 2008. cited by applicant
.
Supplementary Partial European Search Report for European
Application No. 0481753.4 (PCT/US2004/037998), dated Oct. 19, 2009.
cited by applicant. |
Primary Examiner: Belyavskyi; Michail
Attorney, Agent or Firm: Clark & Elbing LLP
Armstrong; Todd
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No.
12/632,452 (now U.S. Pat. No. 8,173,129), filed Dec. 7, 2009, which is a
divisional of, and claims priority from U.S. application Ser. No.
10/358,664 (now U.S. Pat. No. 7,628,988), filed Feb. 5, 2003, which
claims the benefit of the filing date of U.S. Provisional Application No.
60/392,687, filed Jun. 27, 2002, each of which is hereby incorporated by
reference in its entirety.
Claims
What is claimed is:
1. A method for treating or stabilizing an autoimmune disorder
selected from the group consisting of type I diabetes, celiac
sprue-dermatitis, Crohn's disease, Graves'
disease, hypothyroidism, lupus, multiple sclerosis, psoriasis,
rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, and ulcerative
colitis in a mammal comprising administering to said mammal a
composition comprising a tumor necrosis factor (TNF)-alpha
receptor agonist that specifically binds or activates TNF-alpha receptor
II but not TNF-alpha receptor I.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said TNF-alpha receptor
agonist treats or stabilizes said autoimmune disorder by selectively
killing blood cells with increased sensitivity to cell death, and
wherein killing of said blood cells treats or
stabilizes said disorder.
4. The method of claim 1, wherein said TNF-alpha receptor
agonist treats or stabilizes said autoimmune disorder by promoting
cellular regeneration, wherein said regeneration treats or stabilizes
said disorder.
5. The method of claim 2, wherein said disorder is type I diabetes.
6. The method of claim 2, wherein said disorder is celiac sprue-dermatitis.
7. The method of claim 2, wherein said disorder is Crohn's disease.
8. The method of claim 2, wherein said disorder is Graves' disease.
9. The method of claim 2, wherein said disorder is hypothyroidism.
10. The method of claim 2, wherein said disorder is lupus.
11. The method of claim 2, wherein said disorder is multiple sclerosis.
12. The method of claim 2, wherein said disorder is psoriasis.
13. The method of claim 2, wherein said disorder is rheumatoid arthritis.
14. The method of claim 2, wherein said disorder is sarcoidosis.
15. The method of claim 2, wherein said disorder is Sjogren's syndrome.
16. The method of claim 2, wherein said disorder is ulcerative colitis.
17. The method of claim 3, wherein said blood cells are T-cells, B-cells, or macrophages.
18. The method of claim 1, wherein said mammal already has symptoms of said disorder.
19. The method of claim 1, wherein said TNF-alpha receptor
agonist is formulated for intramuscular, intravenous, intraperitoneal,
intravesicular, intraarticular, intralesional, or subcutaneous
administration.
20. The method of claim 1, wherein said TNF-alpha receptor agonist is administered in a single dose.
21. The method of claim 1, wherein said TNF-alpha receptor agonist is administered in multiple doses.
22. The method of claim 4, wherein said cellular regeneration
comprises regeneration of an organ or tissue that is injured, damaged,
or deficient in said mammal, wherein said organ or tissue is, or is part
of, bladder, brain, nervous system
tissue, blood vessels, skin, eye structures, gut, bone, muscle,
ligament, cartilage, esophagus, heart, pancreas, intestines,
gallbladder, bile duct, kidney, liver, lung, fallopian tubes, ovaries,
prostate, spinal cord, spleen, stomach, testes, thymus,
thyroid, trachea, ureter, urethra, uterus, or fat.
Description
BACKGROUND OF THE INVENTION
The invention relates to repairing and regenerating damaged
tissue in a human. Such damage may result from an existing autoimmune
disease, or may be the result of a non-autoimmune insult. We have
previously shown that eliminating autoimmune
cells and re-educating the immune system are important components of an
effective treatment of an autoimmune disease (described in U.S. patent
application Ser. Nos. 09/521,064, 09/768,769, and Ryu et al., Journal
of Clinical Investigations, "Reversal
of Established Autoimmune Diabetes by Restoration of Endogenous Beta
Cell Function," 108:31-33, 2001), which are hereby incorporated by
reference). While an autoimmune disease may be successfully treated,
the individual may nonetheless have significant
tissue damage as a result of the prior autoimmune attack.
Many tissues have an innate ability to repair themselves once
the damage causing insult is eliminated, but this ability to repair
damage decreases in correlation with the duration of the insult. For
example, the regenerative capacity of
endogenous pancreatic islets is virtually eliminated in long-term Type I
diabetics, i.e., patients who have had the disease for more than 15
years. In cases where the endogenous tissue has lost its regenerative
capacity, the damage may be repaired by
providing exogenous tissue to the individual, for example, a transplant.
A promising treatment for diabetes, islet transplantation, has been
the subject of human clinical trials for over ten years. While there
have been many successes with islet
transplantation in animals, these have occurred where the animals are
diabetic due to chemical treatment, rather than natural disease. The
only substantiated peer reviewed studies using non-barrier and non-toxic
methods and showing success with islet
transplants in naturally diabetic mice use isogeneic (self) islets. The
isogeneic islets were transplanted into non-obese diabetic (NOD) mice
with active diabetes, which were pre-treated with TNF-alpha (tumor
necrosis factor-alpha); BCG (Bacillus
Clamette-Guerin, an attenuated strain of mycobacterium bovis); or CFA
(Complete Freund's Adjuvant), which is an inducer of TNF-alpha
(Rabinovitch et al., J. Immunol. 159:6298-6303, 1997). This approach
is not clinically applicable primarily because
syngeneic islets are not available. Furthermore, existing cell
replacement strategies have not prevented end-stage diseases or
permanently reversed insulitis. In the allograft setting of islet
transplantation, grafts are eventually rejected, even with
immunosuppression. Furthermore, diabetic host treatments such as body
irradiation and bone marrow transplantation are unacceptably toxic,
rendering the short-term alternative of insulin therapy more attractive.
Recently, islet transplantation has achieved limited success in
clinical trials, such as that observed for allogenic transplants
combined with multi-drug immunosuppression therapy, with type 1 diabetic
patients having a sustained return to
normoglycemia over a 6 month period. These results have been obtained
with continuous, and sometimes toxic, drug therapy, often in the setting
of a simultaneous life-saving renal transplant. However, these
moderately successful islet transplants show
failures after about one year, speculated to be due in part to the drug
therapy itself inducing insulin resistance. The earlier failure of
islet transplants in type 1 diabetics, compared to non-diabetic patients
receiving islet transplants (such as in
cancer patients who have had their pancreas removed), raises the concern
that immunosuppressive therapy shows greater efficacy for graft
rejection over autoimmunity prevention. Lending credence to these
concerns is the observation of the inefficiency of
immunosuppression therapy for the prevention of graft rejection of
allogenic or xenogeneic islet transplants in animal studies using
non-obese diabetic (NOD) mice.
We previously described a transplantation method to introduce
allogeneic and xenogeneic tissues into non-immunosuppressed hosts in
which the cells are modified such that the donor antigens are disguised
from the host's immune system (U.S. Pat.
No. 5,283,058, which is hereby incorporated by reference). Generally,
masked islets or transgenic islets with ablated MHC class I molecules
are only partially protected from recurrent autoimmunity in NOD mice
(Markmann et al., Transplantation
54:1085-1089, 1992). A need exists for methods of regenerating damaged
tissue that are not only applicable to tissue damage that results from
autoimmune attack, but also to non-autoimmune induced damage.
SUMMARY OF THE INVENTION
The invention features methods for organ regeneration in a
mammal (e.g., a human patient). Accordingly, in a first aspect, the
invention features a method for increasing or maintaining the number of
functional cells of a predetermined type in a
mammal (i) who has injured or damaged cells of the predetermined type or
who has a deficiency of cells of the predetermined type (e.g., a mammal
with a lower than normal number of these cells or a mammal lacking
these cells) and (ii) who does not have an
autoimmune disease. This method involves administering to the mammal a
composition that induces lymphopenia and that increases the number of
cells of the predetermined cell type in the animal. In desirable
embodiments, the composition activates a
receptor on the surface of cells of the predetermined cell type or on
the surface of precursor cells that differentiate into cells of the
predetermined cell type in the mammal. Desirably, the method also
includes administering cells of the predetermined
cell type to the mammal. In desirable embodiments, the method includes
administering cells that recapitulate a developmental sequence (e.g.,
endoderm with mesoderm; endoderm with ectoderm; or ectoderm with
mesoderm to promote the regeneration of the
tissue of interest). In some embodiments, the method also includes
administering precursor cells that differentiate into cells of the
predetermined cell type to the mammal. In particular embodiments, the
method also includes inducing damage to the
cells of a predetermined type in the mammal or inducing damage in a site
of the mammal in which cells of the predetermined type are desirable.
For example, damage can be induced in cells of the predetermined type or
cells of another type within 10
inches, 5 inches, 1 inch, 10 cm, 5 cm, 1 cm, 10 mm, or 1 mm of the
location in which cells of the predetermined type are desirable.
In another aspect, the invention features a method for treating
or stabilizing an established autoimmune disease in a mammal. This
method involves (a) administering to the mammal a first composition that
selectively kills a predetermined
subpopulation of blood cells, in an amount sufficient to selectively
kill at least 10%, preferably at least 75%, of the subpopulation of
blood cells in the mammal, (b) repeating step (a) one or more times, and
(c) optionally monitoring the glucose level
in the mammal two or more times. Desirably, the method also includes
administering to the mammal a second composition that selectively kills a
predetermined subpopulation of blood cells, in an amount sufficient to
selectively kill at least 10%,
preferably at least 75%, of the subpopulation of blood cells in the
mammal. In some embodiments, the method includes determining whether
the mammal has a subpopulation of blood cells with higher than normal
sensitivity to the first composition prior to
step (a).
In a related aspect, the invention features another method for
treating or stabilizing an established autoimmune disease in a mammal.
This method involves (a) administering to the mammal a first composition
that selectively kills a
predetermined subpopulation of blood cells, in an amount sufficient to
selectively kill at least 10%, preferably at least 75%, of the
subpopulation of blood cells in the mammal, (b) repeating step (a) one
or more times, and (c) optionally maintaining the
blood glucose level in the mammal within a normal range. Desirably, the
method also includes administering to the mammal a second composition
that selectively kills a predetermined subpopulation of blood cells, in
an amount sufficient to selectively
kill at least 10%, preferably at least 75%, of the subpopulation of
blood cells in the mammal. In some embodiments, the method includes
determining whether the mammal has a subpopulation of blood cells with
higher than normal sensitivity to the first
composition prior to step (a).
In another aspect, the invention features a method for
treating, stabilizing, or preventing an autoimmune disease (e.g., an
established autoimmune disease) and/or increasing or maintaining the
number of functional cells of a predetermined type
in a mammal. This method involves (a) administering to the mammal a
composition that selectively kills a predetermined subpopulation of
blood cells, in an amount sufficient to selectively kill at least 10%,
preferably at least 75%, of the subpopulation
of blood cells in the mammal, and (b) prior to, after, or concurrently
with step (a), administering to the mammal cells that have the potential
to differentiate into the predetermined type or that are of the
predetermined cell type. In one embodiment,
the method includes determining, prior to step (a), whether the blood of
the mammal contains a subpopulation of blood cells with higher than
normal sensitivity to the composition to be administered; if this is the
case, the decision to employ the
composition is reinforced.
In another aspect, the invention features a method for
treating, stabilizing, or preventing an autoimmune disease (e.g., an
established autoimmune disease) and/or increasing or maintaining the
number of functional cells of a predetermined type
in a mammal. This method involves (a) administering to the mammal a
first composition that selectively kills a pre-determined subpopulation
of stimulated blood cells, in an amount sufficient to selectively kill
at least 10%, preferably at least 75%, of
the subpopulation of stimulated blood cells in the mammal, (b) prior to,
after, or concurrently with step (a), administering to the mammal a
second composition that selectively kills a predetermined subpopulation
of unstimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of unstimulated blood cells in the mammal, and (c)
prior to, after, or concurrently with steps (a) or (b), administering to
the mammal cells that have the
potential to differentiate into the predetermined type or that are of
the predetermined cell type. In one embodiment, prior to step (a), the
method includes determining whether the blood of the mammal contains a
subpopulation of stimulated blood cells
with increased sensitivity to the first composition; if this is the
case, the decision to employ the first composition is reinforced. In
another embodiment, prior to step (b), the method includes determining
whether the blood of the mammal contains a
subpopulation of unstimulated blood cells with increased sensitivity to
the second composition; if this is the case, the decision to employ the
second composition is reinforced.
In another aspect, the invention features a method for
treating, stabilizing, or preventing an autoimmune disease (e.g., an
established autoimmune disease). This method involves administering to
the mammal a composition that selectively kills a
pre-determined subpopulation of unstimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of unstimulated blood cells in the mammal. In one
embodiment, the method includes
determining whether the blood of the mammal contains a subpopulation of
unstimulated blood cells with increased sensitivity to the composition
to be administered; if this is the case, the decision to employ the
composition is reinforced.
In another aspect, the invention features a method for
treating, stabilizing, or preventing an autoimmune disease (e.g., an
established autoimmune disease). This method involves administering to
the mammal a composition that selectively kills a
pre-determined subpopulation of stimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of stimulated blood cells in the mammal. In one
embodiment, the method includes determining
whether the blood of the mammal contains a subpopulation of stimulated
blood cells with increased sensitivity to the composition to be
administered; if this is the case, the decision to employ the
composition is reinforced.
In another aspect, the invention features a method for
treating, stabilizing, or preventing an autoimmune disease (e.g., an
established autoimmune disease). This method involves administering to
the mammal a composition that selectively kills a
pre-determined subpopulation of blood cells, in an amount sufficient to
selectively kill at least 10%, preferably at least 75%, of the
subpopulation of blood cells in the mammal. In one embodiment, the
method includes determining whether the blood of
the mammal contains a subpopulation of blood cells with increased
sensitivity to the composition to be administered; if this is the case,
the decision to employ the composition is reinforced.
In another aspect, the invention features a method of
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves (a) administering
to the mammal a composition that selectively kills a
predetermined subpopulation of stimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of stimulated blood cells in the mammal, and (b)
prior to, after, or concurrently with step
(a), administering to the mammal cells that have the potential to
differentiate into the predetermined type or that are of the
predetermined cell type. In one embodiment, prior to step (a), the
method includes determining whether the blood of the mammal
contains a subpopulation of stimulated blood cells with increased
sensitivity to the composition to be administered; if this is the case,
the decision to employ the composition is reinforced. In some
embodiments, the mammal has an injured or diseased
organ that has increased normal functional activity after administration
of the composition.
In another aspect, the invention features a method of
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves (a) administering
to the mammal a composition that selectively kills a
predetermined subpopulation of stimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of stimulated blood cells in the mammal, and (b)
prior to, after, or concurrently with step
(a), administering to the mammal one or more precursor cells that
differentiate into cells of the predetermined type in vivo. Desirably,
the precursor cells are stem cells and step (a) is performed prior to
step (b). In other embodiments, non-islet
cells are administered for the treatment or prevention of diabetes. In
certain embodiments, the method also involves regulating blood sugar
levels in diabetic patients using, e.g., a glucose clamp or administered
insulin. Desirably, a composition that
kills unstimulated blood cells is administered to the patient in an
amount sufficient to selectively kill a subpopulation of unstimulated
blood cells in the patient.
In another aspect, the invention features a method for
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves (a) administering
to the mammal one or more cells of blood origin or
endothelial origin, and (b) prior to, after, or concurrently with step
(a), administering to the mammal a composition that selectively kills a
predetermined subpopulation of stimulated blood cells, in an amount
sufficient to selectively kill at least
10%, preferably at least 75%, of the subpopulation of stimulated blood
cells in the mammal. In some embodiments, the patient has an autoimmune
disease (e.g., diabetes) or an increased risk for an autoimmune
disease. Desirably, a composition that kills
unstimulated blood cells is administered to the patient in an amount
sufficient to selectively kill a subpopulation of unstimulated blood
cells in the patient.
In another aspect, the invention features a method for
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves (a) administering
to the mammal one or more cells of blood origin or of
endothelial, mesoderm or ectodermic origin, and (b) prior to, after, or
concurrently with step (a), administering to the mammal a composition
that promotes or recapitulates the embryonic program of cellular
differentiation in the host. In desirable
embodiments, the method includes administering to the mammal a
proteasome activity-promoting substance, such as gamma interferon. In
some embodiments, the method includes administering to the mammal an
agent that increases Flk or Flt expression or
function. Examples include TNF-, IL-1, HAT, or NF-B induction, or cAMP
inhibition, using agents known to achieve these functions. Other
examples include stimulation of AP-2, EGF-1, Sp1, AP-1, NFkB, GATA
stimulation with the induction of PECAM-1,
activator protein-2, CT-rich Sp1 binging activity, PDGF-A, PDGF-B,
monocyte chemoattractant protein-1, TF, Ets1, SCL/Tal-1, FGF, HATs
P/CAF, CBP/p300 and HIF-2alpha (HRF, EPAS, HLF). These functions may
also be achieved by TGF-beta inhibition, TGF-beta
receptor blockade, or inhibition of CREB (camp response element binding
protein). In certain embodiments, the method includes administering to
the mammal an agent that increases VEGF, VEGF1, VEGF2, VEGF1R, or VEGF2R
expression or function, such as a
VEGF polypeptide or a nucleic acid molecule encoding a VEGF polypeptide
or substance that activates the promoter of a VEGF protein receptor.
VEGF polypeptides include full-length VEGF proteins, as well as
biologically active VEFG fragments. These
agents are in some cases preferred for mesoderm/endodermal activation
for differentiation. For mural differentiation (cells usually of neural
crest or pericardial origins), host treatment with PDGF or PDGF-BB can
desirably be included in the method.
For BV endothelium differentiation or regrowth of tissue, treatment of
the host with an FGF and/or IGF-1 can be desirable. Furthermore, for
promotion of regeneration can in some instances be accomplished using
just one agent, or with two or more agents,
administered with or without pluripotent cells.
In another aspect, the invention features a method of
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves administering to
the mammal a composition that selectively kills a
predetermined subpopulation of stimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of stimulated blood cells in the mammal. In one
embodiment the method includes determining
whether the blood of the mammal contains a subpopulation of stimulated
blood cells with higher than normal sensitivity to the composition to be
administered; if this is the case, the decision to employ the
composition is reinforced. In some embodiments,
the mammal has an injured or diseased organ that increased normal
functional activity after administration of the composition.
In another aspect the invention features a method of increasing
or maintaining the number of functional cells of a predetermined type
in a mammal with an autoimmune disease or an increased risk for an
autoimmune disease. This method involves
administering to the mammal one or more precursor cells that
differentiate into one or more cells of the predetermined type in vivo
or that promote proliferation of endogenous cells of the predetermined
type in vivo. The differentiated cell(s) will
eventually present MHC class I and peptide, and the MHC class I has at
least one allele that matches an MHC class I allele expressed by the
mammal.
In another aspect, the invention features a method of
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves (a) administering
to the mammal one or more cells of the predetermined
type, and (b) prior to, after, or concurrently with step (a)
administering to the mammal a composition that kills unstimulated blood
cells, in an amount sufficient to selectively kill at least 10%,
preferably at least 75%, of the subpopulation of
unstimulated blood cells in the mammal. In one embodiment, prior to
step (a), the method includes determining whether the blood of the
mammal contains a subpopulation of unstimulated blood cells with higher
than normal sensitivity to the composition to
be administered. In some embodiments, the mammal has an injured or
diseased organ that has increased normal functional activity after
administration of the composition.
In another aspect, the invention features a method of
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves administering to
the mammal a composition that selectively kills a
predetermined subpopulation of stimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of stimulated blood cells in the mammal. In one
embodiment the method includes determining
whether the mammal has a subpopulation of stimulated blood cells with
higher than normal sensitivity to the composition to be administered.
In some embodiments, the mammal has an injured or diseased organ that
has increased normal functional activity
after administration of the composition.
In yet another aspect, the invention features a method of
increasing or maintaining the number of functional cells of a
predetermined type in a mammal. This method involves (a) administering
to the mammal one or more precursor cells that
differentiate into one or more cells of the predetermined type in vivo,
and (b) prior to, after, or concurrently with step (a), administering to
the mammal a composition that selectively kills a predetermined
subpopulation of unstimulated blood cells, in
an amount sufficient to selectively kill at least 10%, preferably at
least 75%, of the subpopulation of unstimulated blood cells in the
mammal. In one embodiment the method includes determining whether the
blood of the mammal contains a subpopulation of
unstimulated blood cells with higher than normal sensitivity to the
composition to be administered. In some embodiments, the mammal has an
injured or diseased organ that has increased normal functional activity
after administration of the composition.
In still another aspect, the invention features a method for
treating, stabilizing, or preventing a disease, disorder, or condition
in a mammal. This method (a) administering to the mammal a first
composition that selectively kills a
predetermined subpopulation of blood cells, in an amount sufficient to
selectively kill at least 10%, preferably 75%, of a first subpopulation
of blood cells in the mammal, and (b) prior to, after, or concurrently
with step (a), administering to the
mammal a second composition that selectively kills a predetermined
subpopulation of blood cells, in an amount sufficient to selectively
kill at least 10%, preferably 75%, of a second subpopulation of blood
cells in the mammal. The first subpopulation
and the second subpopulation are either partially overlapping
subpopulations or non-overlapping subpopulations. Desirably, first
subpopulation and the second subpopulation are in different stages of
differentiation. In some embodiments, the first
subpopulation and the second subpopulation are in different stages of
the cell cycle. In various embodiments, the first subpopulation and the
second subpopulation are sensitive to different inducers of cell death.
In some embodiments, the first
subpopulation and the second subpopulation undergo cell death through
different pathways. In particular embodiments, one subpopulation
undergoes cell death through apoptosis and the other subpopulation
undergoes cell death through necrosis. In some
embodiments, the patient has arthritis (e.g., rheumatoid arthritis). In
particular embodiments, a patient with arthritis is not administered
any cells. In other embodiments, the patient is administered
chondrocytes or cells that differentiate into
chondrocytes. In some embodiments, the patient has injured or damaged
cells of a predetermined cell type. Desirably, the method also involves
administering to the patient one or more cells that have the potential
to differentiate into one or more cells
of the predetermined type or that are of the predetermined cell type.
The cells can be administered prior to, after, or concurrently with the
administration of the first and/or second compositions.
In another aspect, the invention features a method for
treating, stabilizing, or preventing a disease, disorder, or condition
in a mammal. This method involves (a) administering to the mammal a
first composition that selectively kills a
predetermined subpopulation of unstimulated blood cells, in an amount
sufficient to selectively kill at least 10%, preferably at least 75%, of
the subpopulation of unstimulated blood cells in the mammal, and (b)
prior to, after, or concurrently with step
(a), administering to the mammal a second composition that selectively
kills a predetermined subpopulation of stimulated blood cells, in an
amount sufficient to selectively kill at least 10%, preferably at least
75%, of the subpopulation of stimulated
blood cells in the mammal. In some embodiments, the mammal has injured
or damaged cells of a predetermined cell type. In certain embodiments,
the method involves administering to the mammal one or more cells that
have the potential to differentiate
into one or more cells of the predetermined type or that are of the
predetermined cell type.
In another aspect, the invention a method of increasing or
maintaining the number of functional cells of a predetermined type in a
human patient. This method involves inducing damage or uncovering
endogenous damage (e.g., damage that promotes
engraftment of transplanted cells) to the cells of a predetermined type
in the patient. Endogenous damage can be measured using, e.g., blood
tests, (e.g., liver function tests, tests for glucose levels, neurologic
tests, or blood cell tests), visual or
radiographic tests, or functional tests. A first composition that
selectively kills a predetermined subpopulation of unstimulated blood
cells is administered to a patient, in an amount sufficient to
selectively kill at least 10%, preferably at least
75%, of the subpopulation of unstimulated blood cells in the patient.
Prior to, after, or concurrently with the administration of the first
composition, a second composition that kills stimulated blood cells is
administered to the patient in an amount
sufficient to selectively kill a subpopulation of stimulated blood cells
in the patient. Prior to, after, or concurrently with the prior steps,
one or more cells that have the potential to differentiate into one or
more cells of the predetermined type
or that are of the predetermined cell type cells are administered to the
patient.
In another aspect, the invention features a method for
increasing or maintaining the number of functional cells of a
predetermined type, for example, pancreas cells that produce insulin,
brain cells, heart cells, vascular tissue cells, cells of
the bile duct, chondrocytes, or skin cells, in a human patient who has
injured or damaged cells or a deficiency of cells of the predetermined
type. This method includes (a) administering to the patient MHC class I
and peptide (e.g., soluble MHC class I
and peptide or MHC class I and peptide present on the surface of a
cell); (b) prior to, after, or concurrently with step (a), administering
to the patient cells that have the potential to differentiate into the
predetermined type or that are of the
predetermined type; and (c) prior to, after, or concurrently with step
(b), inducing transient lymphopenia in the patient or in a blood sample
from the patient that is re-administered to the patient. In some
embodiments, steps (a) and (b) are performed
concurrently by administering cells that have the capacity to present
MHC class 1 and peptide and that have the potential to differentiate
into the predetermined type or that are of the predetermined type.
In another aspect, the invention features another method of
increasing or maintaining the number of functional cells of a
predetermined type in a human patient. This method involves (a)
identifying endogenous damage of or inducing damage to the
cells of a predetermined type in the patient, (b) exposing the patient
to MHC class I and peptide, (c) prior to, after, or concurrently with
step (b), administering to the patient cells that have the potential to
differentiate into the predetermined type
or that are of the predetermined cell type, and (d) prior to, after, or
concurrently with step (c), inducing transient lymphopenia in the
patient or in a blood sample from the patient that is re-administered to
the patient. In some embodiments, steps
(b) and (c) are performed concurrently by administering cells that have
the capacity to present MHC class I and peptide and that have the
potential to differentiate into the predetermined type or that are of
the predetermined type.
In desirable embodiments of any of the aspects of the
invention, the methods include administering to the mammal a cell (e.g.,
an endothelial cell or mesenchymal cell) that promotes proliferation of
the precursor cells or cells of the
predetermined cell type at the site of desired regeneration, or a cell
that can itself differentiate into the predetermined cell type.
Desirably, the methods also include administering a cytokine, chemokine,
or growth factor to the mammal.
Alternatively, the methods also can include the pretreatment of
mesenchymal or endothelial cell precursors with a cytokine, chemokine,
or growth factor prior to their administration to the mammal. Exemplary
cells of the predetermined type are islet
cells that produce insulin, blood cells, spleen cells, chondrocytes,
brain cells, heart cells, vascular tissue cells, cells of the bile duct,
epithelial cells, endothelial cells, endoderm cells, mesoderm cells,
mesenchymal cells, cells of mesenchymal
origin, and skin cells. Desirable cells that differentiate into cells
of the predetermined type in vivo are splenocytes, bone marrow derived
cells, Hoechst 33342 positive cells, brain cells, CNS positive cells,
hepatocytes, mesenchymal cells, mesodermal
cells, endothelial cells, mural cells, and fetal cells. In some
embodiments, the cells that differentiate into cells of the
predetermined type in vivo are semi-allogeneic or isogeneic. In various
embodiments, the cells that differentiate into cells of
the predetermined type in vivo fail to express Fas or FasL. Desirable
blood cells are T-cells, B-cells, or macrophages. Mesenchymal cells
that are derived from the blood, spleen, or bone marrow and defined as
Hox 11.sup.+, CD90.sup.+, Flk.sup.low,
CD34.sup.+ or CD45.sup.+ are highly desirable. Other desirable cells
are cells that do not, at the time they are administered, express MHC
class I and peptide, but which have the capacity to do so in vivo
post-transplantation, e.g., by stimulation with
the appropriate antigens. In some embodiments, the MHC class I and
peptide are semi-allogeneic or isogeneic. In certain embodiments, the
composition is a compound that crosslinks or binds to a T-cell receptor
(TCR) or other surface protein on a T-cell. In various embodiments, the
composition is TNF-alpha, a TNF-alpha agonist, or a TNF-alpha inducing
substance. In some embodiments, the composition binds or activates a
death receptor. Exemplary TNF-alpha inducing substances include
Complete Freund's
Adjuvant (CFA), ISS-ODN, microbial adjuvants, such as cell wall
components with LPS-like activity, cholera particles, E. coli heat
labile enterotoxin, E. coli heat labile enterotoxin complexed with
lecithin vesicles, ISCOMS-immune stimulating complexes,
chemical adjuvants, such as polyethylene glycol and
poly(N-2-(hydroxypropyl)methacrylamide), synthetic oligonucleotides
containing CpG or CpA motifs, lipid A derivatives, such as
monophosphoryl lipid A, MPL, muramyl dipeptide derivatives, Bacillus
Clamette-Guerin (BCG), Tissue Plasminogen Activator (TPA),
lipopolysaccharide (LPS), Interleukin-1 (IL-1), Interleukin-2 (IL-2), UV
light, lymphotoxin, cachectin, a transcription factor-like nuclear
regulator-2 (TNFR-2) agonist, a neutral blocking
antibody to a B-lymphocyte stimulator (BLyS) receptor or soluble
protein, an intracellular mediator of the TNF-alpha signaling pathway, a
NF.kappa.B inducing substance, lymphotoxin, cachectin, IRF-1, STAT1, an
agonist of an ICS-2lgAS promoter element, a
lymphokine, LPS, an agonist, such as an antibody, to a TNF-superfamily
receptor or soluble form of a TNF-superfamily member, a combination of
TNF-alpha and an anti-TNFR-1 antibody, or a combination of TNF-alpha and
a TNFR.
In desirable embodiments, the method includes administering to
the mammal a proteasome activity promoting substance, such as gamma
interferon. In some embodiments, the method includes administering to
the mammal an agent that increases Flk or
Flt expression or function. Examples include TNF-, IL-1, HAT, or NF-B
induction, or cAMP inhibition. In certain embodiments, the method
includes administering to the mammal an agent that increases VEGF,
VEGF1, VEGF2, VEGF1R, or VEGF2R expression or
function, such as a VEGF polypeptide, a nucleic acid molecule encoding a
VEGF polypeptide, or a substance that activates a promoter of the VEGF
receptor. In some embodiments, the method includes administering to the
mammal an inhibitor of Fas or FasL
expression or signaling. Desirably, the method includes maintaining the
blood glucose level in the mammal within a normal range. In a
particular embodiment, bone marrow cells or precursor (or pluripotent)
cells (e.g., cord blood cells) are administered
to hasten the healing process. These cells recapitulate the embryonic
process in adult animals by hastening the critical mesoderm to endoderm
interactions, endoderm and ectoderm interactions and mesoderm and
ectoderm interactions, all of which are
crucial for organ regeneration.
In desirable embodiments of any of the various aspects of the
invention, the composition that kills naive T-cells is MHC class I and
peptide, and the MHC class I has at least one allele that matches an MHC
class I allele expressed by the
patient. In some embodiments, the MHC class I and peptide is soluble
MHC class I and peptide or MHC class I and peptide present on the
surface of a cell. In some embodiments, the administration of cells and
the administration of MHC class I and peptide
to kills naive T-cells are performed concurrently by administering cells
that have the capacity to present MHC class I and peptide and that have
the potential to differentiate into the predetermined type or that are
of the predetermined type. In some
embodiments, the composition that kills pathologic T-cells (e.g., naive
T-cells) is a compound (e.g., an antibody or antibody fragment,
cytokine, lymphokine, small molecule antagonist, T-cell mitogen, or
co-receptor) that crosslinks a T-cell receptor
(TCR) of naive T-cells (e.g., naive T-cells that might otherwise develop
into autoimmune T-cells or pathologic cells that die if bound by an
antibody or agonist). In some embodiments, the compound that kills a
subpopulation of naive T-cells is not BCG
or is not FAS. In other embodiments, the compound that kills a
subpopulation of T-cells (e.g., naive T-cells) is BCG, FAS, or a
compound that modulates a protein kinase. An exemplary compound that
selectively kills an undesired subpopulation of naive
T-cells is -CD3 antibody, a selective TCR stimulant. Examples of
methods for the re-selection of naive/unstimulated T cells include the
direct killing of the disease-causing T cells, direct killing of the
monocyte/macrophage antigen presenting cell with
deficient MHC class I and self peptide (for example, BCG), and the
re-introduction of cells correctly presenting MHC class I and self
peptide fragments.
In desirable embodiments, the mammal has an autoimmune disease
or an increased risk for an autoimmune disease. Exemplary autoimmune
diseases include Alopecia Areata, Ankylosing Spondylitis,
Antiphospholipid Syndrome, Autoimmune Addison's
Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Behcet's
Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis,
Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic
Inflammatory Demyelinating Polyneuropathy,
Churg-Strauss Syndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold
Agglutinin Disease, Crohn's Disease, Discoid Lupus, Essential Mixed
Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease,
Guillain-Barre, Hashimoto's Thyroiditis,
Hypothyroidism, Idiopathic Pulmonary Fibrosis, Idiopathic
Thrombocytopenia Purpura (ITP), IgA Nephropathy, Insulin dependent
Diabetes, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's Disease,
Mixed Connective Tissue Disease, Multiple Sclerosis,
Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis
Nodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,
Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary
Biliary Cirrhosis, Psoriasis, Raynaud's
Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis,
Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome,
Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Ulcerative
Colitis, Uveitis, Vasculitis, Vitiligo,
Wegener's Granulomatosis, and myasthenia gravis. In some embodiments,
any metabolic disorder that is due to the injury or damage of the cells
of a predetermined type or due to the autoimmune disease is controlled.
In some embodiments, the mammal has an
established autoimmune disease (e.g., the mammal has symptoms of the
autoimmune disease). In some embodiments, the mammal does not have an
established autoimmune disease. In particular embodiments, the mammal
does not have cancer or AIDS. Desirably,
the mammal is a human.
MHC class I and peptide can be administered either
simultaneously (together or separately) or within 24 hours of each
other. In some embodiments, two or more distinct MHC class I molecules
that each contain a different allele that matches an
MHC class I allele expressed by the patient are administered to the
patient. In some embodiments, MHC class I and peptide are administered
in an amount sufficient to induce tolerance to the donor cells or to the
cells of the predetermined type.
Desirably, the number of the autoimmune cells in the patient (e.g.,
B-cells that produce a self-reacting antibody or T-cells that are
activated by presented self epitopes) decreases by at least 5, 10, 20,
30, 40, 50, 60, 80, 90, 95, or 100%.
In various embodiments of the above aspects, MHC class I and
peptide are administered to the patient by administering cells that
express MHC class I and peptide and that either have the potential to
differentiate into the predetermined type or
are of the predetermined cell type. In other embodiments, a population
of living or dead (e.g., irradiated) cells that express MHC class I
which has at least one allele that matches an allele expressed by the
patient and which presents a peptide are
administered to the patient, and another population of cells that differ
from the first population of cells and have the potential to
differentiate into the predetermined type or are of the predetermined
cell type are administered to the patient. This
latter population of cells may or may not present MHC class I and
peptide, and, if expressed, the MHC class I may or may not contain one
or more alleles that match that of the patient. In some embodiments,
two populations of cells each with a different
MHC class I that matches an allele expressed by the patient are
administered.
In certain embodiments, a complex of MHC class I and peptide is
formed by incubating an extracellular region of MHC class I (e.g., a
soluble Fab fragment) with one or more peptides (e.g., peptides from a
cell lysate or a library of random
peptides, synthetic peptides, or naturally-occurring peptides). Because
MHC class I binds peptides with high affinity, the soluble MHC class I
fragment binds peptides in solution. In other embodiments, complexes of
MHC class I and peptide are cleaved
from MHC class I-expressing cells (e.g., healthy lymphocytes with at
least one MHC class I allele that matches that of the patient) using,
e.g., a protease. In yet other embodiments, cells that express MHC
class I, which may not be complexed with
peptide due to potential problems with assembly, are isolated from the
patient. A fragment of the MHC class I is cleaved from the cells and
incubated with one or more peptides, and the resulting complex of MHC
class I and peptide is administered to the
same patient from which the cells were obtained or to a different
patient.
In some embodiments of any of the above aspects, lymphopenia is
induced by administering to the patient an agent that is nonspecific,
i.e., an agent that is toxic to lymphocytes generally, rather than
targeting a particular subset of lymphocytes
or lymphopenia due to new cellular distributions. Examples of such
inducers of lymphopenia are TNF-alpha and substances that induce
TNF-alpha, e.g., Complete Freund's Adjuvant ("CFA"), ISS-ODN, microbial
adjuvants, such as cell wall components with
LPS-like activity, cholera particles, E. coli heat labile enterotoxin,
E. coli heat labile enterotoxin complexed with lecithin vesicles,
ISCOMS-immune stimulating complexes, chemical adjuvants, such as
polyethylene glycol and
poly(N-2-(hydroxypropyl)methacrylamide), synthetic oligonucleotides
containing CpG or CpA motifs, lipid A derivatives, such as
monophosphoryl lipid A, MPL, muramyl dipeptide derivatives, Bacillus
Clamette-Guerin ("BCG"), other vaccinations, Tissue
Plasminogen Activator ("TPA"), lipopolysaccharide ("LPS"),
Interleukin-1, Interleukin-2, UV light, lymphotoxin, cachectin, a TNFR-2
agonist, a NF.kappa.B inducing substance, lymphotoxin, cachectin,
IRF-1, STAT1, an agonist of an ICS-2lgAS promoter
element, or the combination of TNF-alpha and a TNFR-1 antibody. These
inducers of lymphopenia can specifically kill a subpopulation of blood
cells (e.g., a subpopulation of T-cells) if administering in a dose that
is sufficient to specifically kill the
subpopulation but not sufficient to nonspecifically kill all blood
cells. For example, autoimmune patients have subpopulations of blood
cells with increased sensitivity to cell death: thus, low dose of these
compounds are required to kill these cells.
In cases in which non-specific lymphopenia is desired (e.g., such as the
treatment of mammals without an autoimmune disease) larger doses can be
administered. A nonspecific agent can also be an intracellular
mediator of the TNF-alpha signaling pathway,
e.g., NF.kappa.B, Jun N-terminal kinase ("JNK"), TRAILR2, FasL, TRADD,
FADD, TRAF2, RIP, MAPK, kinase activators, a caspase, or pro-caspase.
Stimulation of a signaling pathway may involve, e.g., receptors of the
TNF superfamily or intracellular
mediators of these pathways. Other examples of compounds that induce
lymphopenia include compounds that bind or activate one or more members
of the TNF receptor superfamily (e.g., TNF receptor 1 or 2, Trail-RE
Trail-R2, Trail-R3, Trail-R4, OPG, Rank,
Fn14, DR6, Hvem, LtbetaR, DcR3, Tramp, Fas, CD40, CD30, CD27, 4-1BB,
OX40, Gitr, Ngfr, BCMA, Taxi, Baff-r, EDAR, Xedar, Troy, Relt, or
CD95L). Therapeutic agents can include TNF receptor superfamily
cytokine agonists or cytokine agonist antibodies.
Additional compounds that directly or indirectly increase TNF-alpha can
be readily identified using routine screening assays for TNF-alpha
expression levels or activity. Desirably, an inducer of lymphopenia
also promotes organ formation, promotes
differentiation of donor cells (e.g., blood cells) into a desired cell
type, and/or induces damage to host cells of a predetermined cell type
to facilitate incorporation of donor cells into the desired organ. In
some embodiments, transient lymphopenia
is induced for a period of time sufficient to destroy at least 10, 20,
30, 40, 50, 60, 80, 90, 95, or 100% of the autoimmune cells in the
patient (e.g., B-cells that produce a self-reacting antibody, T-cells
that are activated by presented self epitopes,
or a subset of antigen presenting cells with defective antigen
presentation). In some embodiments, that agent that kills naive T-cells
is not BCG or FAS.
In some embodiments, one or more of the following TNF super
family ligands are administered to the mammal or are upregulated by
administration of another compound to the mammal: Trail(Apo2L), RANKL,
TWEAK, TNF, LT, LIGHT, LT, TL1A, FASL, CD40L,
CD30L, CD27L, CD27L, 4-1BBL(CD137L), OX40L(CD134L), GITRL, APRIL, BAFF,
EDA1, or EDA2. In certain embodiments, one or more of the following
TNF.alpha. superfamily receptors are activated by administration of a
compound to the mammal: TRAIL-R1(DR4),
TRAIL-R2(DR5), TRAIL-R3(DCR1), TRAIL-R4(DCR2), OPG, RANK, RN14, DR6,
THF-R2(CD120B), TNF-R1(CD120A), FVEM, LIBETAR, DCR3, TRAMP(DR3),
FAS(CD95), CD40, CD30, CD27, 4-1BB(CD137), CD134(OX40), GITR, NGFR,
BCMA, TACI, BAFFR, EDAR, XEDAR, TROY, or RELT.
Desirably, one or more of the following members of death cascades are
activated: FASL(CD95L:CD178) with FAS (CD95), TRAIL (APO-2L) with
TRAIL-R1(DR4), TRAIL(APO2L) with TRAIL R2(DR5), or TNF with TNFR1.
Desirably, one or more of the following are administered to the
mammal: IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13,
IL-18, INF-alpha, IFN-beta, IFN-gamma, TFG-beta, PDGF, and/or VEGF. A
small molecule or antibody agonist of
TLR1, TLR2, TLR6, TLR3, TLR4, TLR5, TLR7, and/or TLR9 is desirably
administered. Exemplary TNF superfamily members and their receptor
agonists include TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, OPG, RANK,
Fn14, DR6, TNF-R2, TNF-R1, HVEM, LtbetaR, DcR3,
TRAMP, Fas, CD30, CD27, 4-1BB, OX40, GITR, NGFR, BCMA, TACI, EDAR,
XEDAR, Troy, and RELT. Biologics of diverse compositions such as BCG,
BLP, fibronectin Domain A, lipoarabinomannan, LPS binding protein, LPS,
lipoteichoic acid, macrophage stimulatory
lipopeptide 2, manosylated phosphatidylinositol peptidoglycan,
respiratory syncytial virus protein F, and soluble tuberculosis factor
may also be administered, if desired.
Any of a wide variety of cells can be administered to the
patient according to the invention. The cells can be cells that,
compared to the desired functional cells, are relatively
undifferentiated; i.e., they can be stem cells derived, e.g.,
from embryonic or fetal tissue, from adult stem cell sources, from adult
tissues harboring a subset of pluripotent cells, or from cord blood.
Alternatively, the administered cells can be relatively more
differentiated cells, e.g., cells that stain
positively for the stain Hoechst 33342 which stains the nucleus of
immature hematopoietic cells; brain-derived cells; cells derived from
non-brain CNS tissue (e.g., spinal cord); hepatocytes; chondrocytes;
splenocytes; bone marrow-derived cells; cells of
blood or lymphoid origin; or parenchymal cells. In certain embodiments,
the administered cells are not islets, not beta cells, or not
insulin-producing beta cells. In various embodiments, cells other than
the predetermined cell type are administered to
the patient and form the predetermined cell type in vivo. For example,
in some embodiments, cells other than islets are administered to the
patient and form insulin-producing islets in vivo. Desirably, the cells
are administered to the same human from
which they were obtained or to another human. It is also contemplated
that donor cells from other mammals can be used. Exemplary donor
mammalian cells are from pigs or primates such as monkeys.
In some embodiments, patients (e.g., patients without an
autoimmune disease) are administered cells that have been genetically
engineered (e.g. by elimination of genes encoding cell death proteins
such as Fas, FasL, or caspases), chemically
pre-treated, or biologically pre-treated (e.g., treated with an antibody
or antibody fragment reactive with Fas or FasL) to exhibit reduced
resistance to spontaneous cell death. In certain embodiments, a
compound that decreases the expression level or
activity of Fas or FasL is administered to the donor cells or to the
host. In some embodiments, the cells are allogeneic, semi-allogeneic,
or isogeneic. Exemplary MHC class I-expressing cells that can be used
in the invention include monocytes,
macrophages, dendritic cells, B-cells, Langerhans cells, epithelial
cells, mesenchymal cells, and parenchymal cells. Other cells express
MHC class I at lower levels and can also be used in the present methods.
Desirably, at least 20, 40, 60, 80, 90,
95, or 100% of the administered cells express MHC class I complexed with
peptide. In some embodiments, cells present MHC class 1 and peptide
before and after they are administered to the patient. In other
embodiments, cells present MHC class 1 and
peptide only after they are administered to the patient. In some cases,
the cells differentiate in vivo into cells that present MHC class 1 and
peptide.
In some embodiments, one or more death receptors (e.g., the
death receptors listed in FIG. 5) are inactivated on the donor cells or
one or more intracellular signaling proteins that mediate cell death are
inactivated in the donor cell to prevent
death of the transplanted cells. For example, FLIP can be used to down
regulate Fas/FasL expression. In other embodiments, extracellular
inhibition or reduction in IL2 (e.g., inhibition due to chemicals or
antibodies) is used to upregulate FLIP which
then down regulates FAS. In other embodiments, the donor cells have a
blockage of IL2R, such as the binding of a chemical (e.g., a non-lytic
antibody fragment) to IL2R to inhibit binding of IL2 to IL2R and thus
IL2-mediated upregulation of FAS. In
other embodiments, one or more members of the intracellular pathway for
FAS activation are inhibited in the donor cells prior to transfer.
Examples include the inhibition of the translation of transcription
factors such as cFOS, cJAN, PKC, Lck, Zap70,
MAPK, Itk (IL-2 inducible T cell kinase) and JNK. In particular
embodiments, the transcription or translation of transcription factors
is transiently inhibited with antisense oligonucleotides or by RNA
interference (RNAi).
The number of functional cells maintained according to the
invention can be increased by further treating the patient with a
substance that increases proteasome activity; this treatment can
specifically increase the immuno-proteasome subunits
LMP2, LMP7, and LMP10, rendering the cells or host more responsive to
the growth promoting activities of VEGF pathways or NF B pathways, which
are important for cellular regeneration. Administration of the
proteasome-enhancing substance can be carried
out at any time during the method of the invention; e.g., prior to or
following the administration of the cells and/or MHC class I and
peptide; or prior to or following induction of transient lymphopenia.
The proteasome activity-increasing substance can
be, e.g., gamma interferon; VEGF or a substance that increases VEGF
level or activity, such as a nucleic acid molecule encoding VEGF or an
active fragment thereof; fetal liver kinase-1/kinase domain region
(Flk-1/KDR) or a substance that increases Flk
level or activity; or fms-like tyrosine kinase-1 (Flt-1) or a substance
that increases Flt level or activity. Additional proteasome
activity-increasing substances include compounds that inhibit the
expression or signaling of Fas or FasL (e.g., an
anti-Fas or anti-FasL antibody) and/or promote the viability of
endogenous or exogenously administered pluripotent cells.
Desirably, the number of cells of the desired cell type that
are present at least one day, one week, one month, or one year after
treatment using the methods of the invention increases by at least 20,
30, 50, 75, 100, 200, 500, or even 1000%
relative to the number of cells of that cell type that are present in
the patient before treatment or the number of cells present in a control
subject (e.g., a subject who received a vehicle control or a placebo).
In some embodiments, the number of
cells of the desired cell type in a treated patient is at least 50, 60,
70, 80, 90, or 100% of the amount of the corresponding cells in a
healthy patient without a deficiency in those cells. In certain
embodiments, cells of donor origin are present in
the patient at least one day, one week, one month, or one year after
treatment. For example, diabetic patients that are treated using the
present methods desirably contain cells of donor origin in their
pancreas after treatment.
In some embodiments, the methods of the invention are used to
treat damage or deficiency of cells in an organ, muscle, or other body
structure or to form an organ, muscle, or other body structure.
Desirable organs include the bladder, brain,
nervous system tissue, blood vessels, skin, eye structures, gut, bone,
muscle, ligament, esophagus, fallopian tube, heart, pancreas,
intestines, gallbladder, kidney, liver, lung, ovaries, prostate, spinal
cord, spleen, stomach, testes, thymus, thyroid,
trachea, ureter, stomach, urethra, and uterus. For these applications,
donor differentiated cells, such as cells from any of these organs, or
undifferentiated cells, such as embryonic or adult stem cells, are
administered to a patient. In a desirable
embodiment, a pancreas is regenerated, or the organelle representing the
islets of Langerhans reappears. In some embodiments, the organ,
muscle, or other body structure that is repaired or replaced was
damaged, at least in part, due to the aging
process. In some embodiments, at least 20, 40, 60, 80, 90, 95, or 100%
of the cells of the regenerated organ (e.g., the bile ducts, endocrine
portions of the pancreas, or the entire pancreas), muscle, or other body
structure are of donor origin. In
certain embodiments, the precursor cells are provided in a female by
pregnancy.
The methods of the invention can be used to treat, prevent, or
stabilize autoimmune diseases and diseases or conditions other than
autoimmune diseases, such as diseases or injuries associated with damage
to a particular class of cells. For
example, these method may be used to treat, prevent, or stabilize
autoimmune diseases including, but not limited to, Insulin dependent
Diabetes, lupus, Sjogren's disease, rheumatoid arthritis, pemphigus
vulgaris, multiple sclerosis, hypothyroidism,
graves disease, psoriasis, premature ovarian failure (POF), and
myasthenia gravis. Other examples of autoimmune diseases are described
herein. In these procedures, the cells that are attacked by the
recipient's own immune system may be replaced by
transplanted cells that either are the desired cell type or that
differentiate into the desired cell type in vivo. For the treatment of
type I or type II diabetes, insulin-producing islet cells (e.g., islet
cells expressing MHC class I that has at least
one allele that matches the patient and that presents a peptide) or
cells that differentiate into insulin-producing islet cells in vivo
(e.g., cells originating from splenocytes, bone marrow origin cells,
blood origin cells, or fibroblasts) can be
transplanted into the patient. Desirably, the patient's glucose level
decreases to less than 200 mg/dl, 150 mg/dl, or 120 mg/dl (in order of
increasing preference).
Examples of other medical applications for these methods
include the administration of neuronal cells or cells that differentiate
into neuronal cells to an appropriate area in the human nervous system
to treat, prevent, or stabilize a
neurological disease such as Alzheimer's disease, Parkinson's disease,
Huntington's disease, or ALS; or a spinal cord injury. For example,
degenerating or injured neuronal cells may be replaced by transplanted
cells. Undifferentiated donor cells may be
administered, e.g., systemically or locally.
In preferred embodiments for the treatment of conditions other
than autoimmune conditions, a compound that inhibits the destruction of
stem cells by an administered inducer of lymphopenia (e.g., TNF-alpha)
is desirably administered to the
patient. Examples of such compounds include anti-Fas antibodies. In
some embodiments, stem cells or cells that form stem cells in vivo are
administered to the patient after the administration of the inducer of
lymphopenia or lymphoid redistribution
(e.g., after the destruction of endogenous autoimmune cells, such as
after 7-14 days of lymphopenia).
With respect to the therapeutic methods of the invention, it is
not intended that the administration of one or more compounds (e.g.,
purified or unpurified donor cells, MHC class I and peptide, and an
inducer of lymphopenia) to a patient be
limited to a particular mode of administration, dosage, or frequency of
dosing; the present invention contemplates all modes of administration,
including intramuscular, intravenous, intraperitoneal, intravesicular,
intraarticular, intralesional,
subcutaneous, or any other route sufficient to provide a dose adequate
to increase the number of desired cells. The compound(s) may be
administered to the patient in a single dose or multiple doses. When
multiple doses are administered, the doses may
be separated from one another by, for example, one day, one week, one
month, or one year. For example, a compound that induces lymphopenia
may be administered once a week for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15,
20, or more weeks. It is to be understood
that, for any particular subject, specific dosage regimes should be
adjusted over time according to the individual need and the professional
judgment of the person administering or supervising the administration
of the compositions. For example, the
dosage of donor cells can be increased if more cells of a particular
cell type are needed, e.g., if the glucose levels of a diabetic patient
have not returned to normal or if an ongoing process decreases the
number or activity of cells of the
predetermined cell type. The dosage of a compound that induces
lymphopenia can also be increased if autoimmune cells remain in the
patient, for example, if a blood sample from the patient contains
autoantibodies or contains cells with increased
sensitivity to TNF-alpha, indicating that autoimmune cells are still
present in the patient. Conversely, the dosage of donor cells or
lymphopenia-inducing compounds can be decreased if a desired number of
cells are present in the patient or if
autoimmune cells are no longer present. If desired, conventional
treatments may be used in combination with the therapies of the present
invention. For example, diet and exercise can be used to facilitate the
control of glucose levels in diabetic
patients.
Other embodiments of the present methods are disclosed in U.S.
patent application Ser. No. 09/521,064, filed Mar. 8, 2000, and
09/768,769, filed Jan. 23, 2001, and PCT publication WO00/53209,
published Sep. 14, 2000, which are incorporated
by reference).
DEFINITIONS
By "treating, stabilizing, or preventing a disease, disorder,
or condition" is meant preventing or delaying an initial or subsequent
occurrence of a disease, disorder, or condition; increasing the
disease-free survival time between the
disappearance of a condition and its reoccurrence; stabilizing or
reducing an adverse symptom associated with a condition; reducing the
severity of a disease symptom; slowing the rate of the progression of a
disease; or inhibiting or stabilizing the
progression of a condition. Desirably, at least 10, 20, 30, 40, 60, 80,
90, or 95% of the treated subjects have a complete remission in which
all evidence of the disease disappears. In another preferred
embodiment, the length of time a patient remains
free of disease symptoms after being diagnosed with a condition and
treated with a therapy of the invention is at least 10, 20, 40, 60, 80,
100, 200, or even 500% greater than (i) the average amount of time an
untreated patient remains free of disease
symptoms or (ii) the average amount of time a patient treated with
another therapy remains free of disease symptoms. Desirably, the number
of disease-causing white blood cells decreases by at least 10, 20, 30,
40, 60, 80, 95, or 100%.
By "autoimmune disease" is meant a disease in which an immune
system response is generated against self epitopes. Some examples of
autoimmune diseases include Alopecia Areata, Ankylosing Spondylitis,
Antiphospholipid Syndrome, Autoimmune
Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis,
Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac
Sprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS),
Chronic Inflammatory Demyelinating Polyneuropathy,
Churg-Strauss Syndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold
Agglutinin Disease, Crohn's Disease, Discoid Lupus, Essential Mixed
Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease,
Guillain-Barre, Hashimoto's Thyroiditis,
Hypothyroidism, Idiopathic Pulmonary Fibrosis, Idiopathic
Thrombocytopenia Purpura (ITP), IgA Nephropathy, Insulin dependent
Diabetes, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's Disease,
Mixed Connective Tissue Disease, Multiple Sclerosis,
Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis
Nodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,
Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary
Biliary Cirrhosis, Psoriasis, Raynaud's
Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis,
Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome,
Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Ulcerative
Colitis, Uveitis, Vasculitis, Vitiligo,
Wegener's Granulomatosis, and myasthenia gravis.
"MHC class I and peptide" is commonly understood to refer to
the MHC/peptide complex as it is naturally presented on the surface of a
cell in connection with the normal functioning of the immune system.
Cytoplasmic antigens are processed into
peptides by cytoplasmic proteases and the proteasome, a multicatalytic
proteinase complex associated with the Lmp2, Lmp7, and Lmp10 protein.
As used herein, the term "MHC class I and peptide" includes such
naturally occurring complexes, and in addition
includes peptides that differ from native antigen-derived peptides but
which are nonetheless able to form a complex with class I that is
effective to maintain functional cells according to the invention.
Exemplary peptides that differ from native
antigen-derived peptides may contain unnatural amino acids, e.g.,
D-amino acids, as well as naturally-occurring amino acids. Preferred
MHC class I and peptide complexes are those in which a chain of amino
acids between 8 and 10 residues in length is
correctly complexed with an MHC class I molecule that is either
semi-allogeneic, i.e., at least one MHC class I allele is mismatched and
at least one MHC class I allele is matched between donor and recipient,
or syngeneic, i.e., all MHC class I alleles
are matched between donor and recipient, where the MHC class I and
peptide complex contributes to the re-education or re-selection of the
immune system.
In some embodiments, the MHC class I and peptide are present on
the surface of cells that are administered to the patient. Other MHC
class I/peptide complexes are soluble complexes that are not expressed
on the surface of a cell. In particular
embodiments, the extracellular region of MHC class I (e.g., a Fab
fragment of MHC class I) or soluble, full-length MHC class I is
incubated with one or more peptides according to known methods under
conditions that allow a peptide to bind the MHC class I
fragment, and the resulting MHC class I and peptide complex is
administered to the patient. In other embodiments, a mixture of MHC
class I and peptide are administered to the patient, and the MHC class I
and peptide bind in vivo after administration to
the patient. In some embodiments, the administered MHC class I has 1,
2, 3, or 4 alleles with at least 60, 70, 80, 90, 95, or 100% sequence
identity to that MHC class I expressed by the patient. Sequence
identity is typically measured using sequence
analysis software with the default parameters specified therein (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University Avenue,
Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to various
substitutions, deletions, and other modifications.
By "functional cell," is meant cells that carry out their
normal in vivo activity. In certain desirable embodiments of the
invention, the cells are capable of expressing endogenous self-peptide
in the context of MHC class I.
By "predetermined type," when used in reference to functional
cells, is meant a defined cell type. For example, one skilled in the
art may decide to carry out the method of the present invention in order
to increase or maintain the number of
functional islet cells in the pancreas. In this example, the cells of a
predetermined type are islet cells or islet precursor cells.
Standard assays can be used to determine whether administered
cells form cells of the predetermined cell type in vivo. For example,
cells may be analyzed for expression of particular proteins (e.g.,
proteins specific for the predetermined cell
type) using standard Western or immunofluorescence analysis or for the
expression of particular mRNA molecules (e.g., mRNA molecules specific
for the predetermined cell type) using a cDNA array (Ausubel et al.,
supra). Examples of other characteristics
of the administered cells that may be analyzed to determine whether they
have been converted into the desired cell type include the size of the
cell, cell morphology, volume of cytoplasm, and cell function (e.g.,
production of insulin or other hormones).
By "semi-allogeneic," is meant a match of at least one marker,
for example, an MHC class allele, between cells of the same type from
different individuals of the same species. Desirably at least two or
three MHC class I alleles match between
the donor and the host. Standard methods may be used to determine
whether an MHC class I allele expressed by a donor cell matches an MHC
class I allele expressed by the recipient. For example, antibodies
specific for a particular MHC class I allele can
be used to determine what alleles are expressed. Alternatively, PCR
amplification of nucleic acids encoding MHC class I alleles can be used.
By "syngeneic donor cell" or "isogeneic donor cell," is meant
(i) a donor cell that is genetically identical, or matched at the HLA
region (i.e., has at least four, and preferably all 6, of the standard
markers in common with), to a cell of the
recipient or (ii) a donor cell that is re-administered to the same
patient from which it was obtained.
A "TNF-alpha inducing agent," is desirably a compound that
results in the expression of endogenous TNF-alpha, enhances secretion of
TNF-alpha, or enhances bioavailability or stability of TNF-alpha.
However, TNF-alpha agonists, agents that
stimulate TNF-alpha signaling or enhance post-receptor TNF-alpha action,
or agents that act on pathways that cause accelerated cell death of
autoimmune cells, are also included in this definition. Stimulation of
TNF-alpha induction (e.g., by
administration of CFA) is desirably carried out prior to, after, or
during administration (via implantation or injection) of cells in vivo.
By "lymphopenia" is meant a decrease in the total number of
lymphocytes in a blood sample from a mammal. In some embodiments, this
decrease is due to death of lymphocytes, such as T-cells, B-cells,
and/or macrophages. In certain embodiments,
this decrease is due to redistribution of lymphocytes.
By "nonspecific," in reference to lymphopenia, is meant a
reduction in the total number of lymphocytes in an individual, and is
not limited to a subset of lymphocytes.
By "selectively killing blood cells" is meant directly or
indirectly reducing the number or relative percentage of a subpopulation
of blood cells (e.g., autoreactive lymphoid cells such as T- or B cells
or the defective antigen presenting cells)
such as a subpopulation of unstimulated cells or stimulated cells. In
desirable embodiments, the subpopulation is a subset of T-cells,
B-cells, or macrophages. Desirably, the killed memory T-cells are
autoimmune T-cells, i.e., T-cells that are
activated by presented self epitopes. In desirable embodiments, the
killed naive T-cells are cells that would otherwise become autoimmune
T-cells. Desirably, the number of autoimmune T-cells or cells that
would otherwise become autoimmune T-cells
decreases by at least 25, 50, 100, 200, or 500% more the number of
healthy non-autoimmune T-cells decreases. In some embodiments, the
number of autoimmune T-cells or cells that would otherwise become
autoimmune T-cells decreases by at least 25, 50, 75,
80, 90, 95, or 100%, as measured using standard methods. The T-cells
can be killed due to any pathway, such as apoptosis, necrosis, and/or
activation induced cell death. Apoptosis can be assayed by detecting
caspase-dependent cell shrinkage,
condensation of nuclei, or intranuclear degradation of DNA. Necrosis
can be recognized by caspase-independent cell swelling, cellular
degradation, or release of cytoplasmic material. Necrosis results in
late mitochondrial damage but not cytochrome C
release. In some embodiments, memory T-cell are killed by apoptosis,
and naive T-cells are killed by necrosis. For the treatment of lupus,
B-cells are desirably killed or, alternatively, they are allowed to
developmentally mature.
By "stimulated blood cell" is meant a blood cell (e.g., a
memory T-cell, a B-cell, or a macrophage) that has been exposed to an
antigen.
By "unstimulated blood cell" is meant a blood cell (e.g., a
naive T-cell, a B-cell, or a macrophage) that has not been exposed to an
antigen.
By "pathologic T cell" is meant a T cell that is involved, or
has the potential to be involved, in an autoimmune response or disorder.
Stimulated cells tend to be in later stages of maturation than
unstimulated cells, in active progression through the cell-cycle, and/or
involved in infiltrating a diseased or damaged organ or tissue.
Unstimulated cells tend to progress through
the cell-cycle more slowly or not at all. Memory T-cells tend to
express a higher density of IL-2 receptor (e.g., 10-20% higher density)
than naive T-cells. Naive T-cells tend to express a higher density
(e.g., a 5-20% higher density) of
CD45RB.sup.high, CD95, and/or CD62L than memory T-cells.
By "purified" is meant separated from other components that
naturally accompany it. Typically, a factor (e.g., a protein, small
molecule, or cell) is substantially pure when it is at least 50%, by
weight, free from proteins, antibodies, and
naturally-occurring organic molecules with which it is naturally
associated. Desirably, the factor is at least 75%, more desirably, at
least 90%, and most desirably, at least 99%, by weight, pure. A
substantially pure factor may be obtained by chemical
synthesis, separation of the factor from natural sources, or production
of the factor in a recombinant host cell that does not naturally produce
the factor. Proteins, vesicles, chromosomes, nuclei, other organelles,
and cells may be purified by one
skilled in the art using standard techniques such as those described by
Ausubel et al. (Current Protocols in Molecular Biology, John Wiley &
Sons, New York, 2000). The factor is desirably at least 2, 5, or 10
times as pure as the starting material, as
measured using polyacrylamide gel electrophoresis, column
chromatography, optical density, HPLC analysis, or western analysis
(Ausubel et al., supra). Preferred methods of purification include
immunoprecipitation, column chromatography such as
immunoaffinity chromatography, magnetic bead immunoaffinity
purification, and panning with a plate-bound antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
The application file contains drawings executed in color (FIG.
1). Copies of this patent or patent application with color drawings
will be provided by the Office upon request and payment of the necessary
fee.
FIG. 1 is a table that demonstrates the presence of
Y-chromosome positive cells in adult female NOD mouse pancreatic islets
with disease reversal. Column 1 and column 2 represent two different
female NOD mice with long term correction of their
disease defined as the reversal of hyperglycemia followed by
regeneration of the insulin-secreting cells in the pancreas. All
histology sections from each animal were stained by immunohistochemistry
and were from consecutively cut specimens. The first
row shows the stain of the pancreas with insulin antibodies,
demonstrating the reappearance of beautiful islets free of disease. The
second row demonstrates that Y-chromosome staining of the same islet
produces punctuate dark pink staining of the Y
chromosome only within the islet tissue and associated duct tissue, but
not within the associated exocrine portions of the pancreas. This
result is definitive evidence that the origin of the reappearing islets
is the male donor splenocytes. Row 3 is a
close-up picture of the associated duct that also shows that the Y cells
were not only present in the islet but also populate the duct tissue.
The duct was repopulated by the donor cells and is less uniform than the
islet repopulation. In general,
almost all the islet tissue in the pancreases of these mice was of donor
origin, as demonstrated by the uniform Y chromosome presence in all
cells. The ducts in the pancreases of these animals may be of
completely female origin or of a mixed origin
composed of both female and male cells. Row 4 demonstrates that a
developmental marker of early islet regeneration is PDX-1. The staining
of the pancreatic sections reveals bright PDX-1 staining, exclusively
in the regenerating islet but not in the
associated exocrine portions of the pancreas.
FIGS. 2A-2C are graphs demonstrating the percentage of CD8
CD45RB.sup.high, CD8 CD62L, and CD8 CD95 cells in mice treated with or
without CFA and with or without donor splenocytes.
FIGS. 3A and 3B are graphs of the percent change in the mean
antigen density in mice treated with or without CFA and with or without
donor splenocytes.
FIG. 4 is a table demonstrating the ability of multiple doses
of TNF-alpha to inhibit insulitis in 20 to 35 week old, diabetic mice.
FIG. 5 is a list of exemplary death receptors.
FIG. 6 is list of exemplary inhibitors of cell death that can
be used to prevent death of transplanted donor cells or of endogenous
healthy cells (Oncogene Research Products, 2002/2003 catalogue, San
Diego, Calif.). An exemplary inhibitor of
necrosis is geldanomycin, and an exemplary inhibitor of apoptosis is
zVAD-fmk.
FIG. 7 is a list of standard kits that can be used to measure
the level of cell death (Oncogene Research Products, 2002/2003
catalogue, San Diego, Calif.).
FIG. 8 is list of exemplary compounds that induce cell death
(Oncogene Research Products, 2002/2003 catalogue, San Diego, Calif.).
FIG. 9 is a table that summarizes the analysis of splenocytes
or PBLs from mice injected biweekly for 40 days with 10.sup.7 donor
cells. Fewer cells may also be used in each injection in the present
methods, especially if the cells are
administered more often and/or for longer. Column "Whole GFP+%" lists
the percentage of splenocytes or PBL cells that are fluorescent,
indicating they are of donor origin. Column "H-2 kb+GFP+" lists the
percentage of analyzed cells that are fluorescent
and that express the same class 1 locus as the donor cells. Column
"CD3+ + GFP+" lists the percentage of analyzed cells that are
fluorescent and express CD3, which is only expressed on T-cells. Column
"B220+ GFP+" lists the percentage of analyzed cells
that are fluorescent and express B220, which is only expressed on
B-cells. Column "Mac-1+GFP+" lists the percentage of analyzed cells
that are fluorescent and express Mac-1, which is only expressed on
macrophages.
FIG. 10 is a table that summarizes characteristics of mice
treated with various methods of the present invention. "TNF-.alpha.
low dose" refers to a 2 .mu.g dose of TNF.alpha.. "Sp" denotes
splenocytes, and "Bm" denotes bone marrow donor
cells. "(-)" indicates no significant difference compared to untreated
control mice, "(+)," "(++)," and "(+++)" denote increasing differences
(e.g., increased activity or increased number of cells) compared to
untreated control mice.
FIG. 11 is a table summarizing conditions that promote regeneration.
DETAILED DESCRIPTION
We have succeeded in regenerating a functioning organ (the
islet cells of a pancreas) in an animal with a damaged pancreas, and
have shown that almost all of the visible portions of the organ, as well
as a large portion of the bile ducts, are of
donor origin.
The present methods for increasing the number of functional
cells of a predetermined cell type in a patient have a number of
advantages. For example, the present methods are robust and durable.
The methods can be used to replace most or all of
an endogenous organ using donor cells of the same cell type or of a
different cell type as the organ. The methods can also be performed
without immunosuppression for nonspecific inactivation or death of
autoimmune cells.
These following examples are provided for the purpose of illustrating the invention and should not be construed as limiting.
Materials and Methods
Diagnosis and Treatment of Mice
Female NOD mice were obtained from either Taconic Farms
(Germantown, N.Y.) or Jackson Laboratory (Bar Harbor, Me.). C57BL/6J
mice were obtained from The Jackson Laboratory. All mice were
maintained under pathogen free conditions.
NOD mice were screened for the onset of diabetes by monitoring
their body weight and blood sugar level. The criteria for diagnosis of
diabetes included two consecutive blood sugar concentrations exceeding
400 mg/dl. Mice with blood sugar
levels greater than 400 mg/dl were given daily injections of 1.0 to 1.5
units of Neutral Protamine Hagedorn (NPH) human insulin for each 100
grams of body weight to prevent immediate death due to hyperglycemia.
The use of such severely diabetic mice
relatively late after disease onset ensures that the endogenous pancreas
islets were completely obliterated prior to transplantation of the
donor islet cells.
Splenocyte donors included normal C57 mice, C57
.beta..sub.2M.sup.-/- mice whose cells have a decreased ability to
express MHC class I and peptide on their surface due to the ablation of
the chaperone protein .beta..sub.2 microglobulin but can
express MHC class I and self peptide when exposed to normal mouse serum,
C57 .beta..sub.2M.sup.-/- TAP1.sup.-/- mice which have a decreased
ability to re-express MHC class I and self peptide, and MHC class
II.sup.-/- mice in which the I-A gene is
disrupted and the E locus for MHC class II is not expressed because of
endogenous defects in the C57 strain. Splenocytes, generally at a dose
of 9.times.10.sup.6 cells, were injected into some NOD recipients
through the tail vein twice a week.
TNF-alpha, which is commercially available from a variety of vendors
including Genentech (South San Francisco, Calif.), Hoffman-LaRoche
(Basel, Switzerland), Boehringer Ingelheim (Germany), Asahi Chemical
Industry (Japan), and Sigma-Aldrich (St. Louis,
Mo.), was administered intraperitoneally (1-20 .mu.g per dose, 2 to 3
times per week) to eliminate autoreactive cells and promote regeneration
in the host by recapitulating the embryonic program of blood vessel
endothelium, i.e. induction of NF-B and/or
VEGF. CFA (Difco Laboratory, Detroit, Mich.) was mixed with an equal
volume of physiological saline, and approximately 1-50 .mu.l were
injected into each hind-footpad at the time of the islet transplantation
or after the first splenocyte injection.
Islet Transplantation
Islets were isolated from donor C57 mice or 6 to 8 week-old
pre-diabetic female NOD mice and served as a glucose clamp and the
source of MHC class I and self-peptide. Gradient centrifugation
followed by manual selection of islets was performed
to ensure that both preparations were highly enriched for islets and to
accurately determine the number of transplanted islets. Approximately
500 to 600 specially selected islets were grafted beneath the left
kidney capsule of each diabetic NOD
recipient. For islet encapsulation, 900-1100 islets were enclosed in
0.2-0.5 cm diameter alginate spheres that were surgically inserted into
the peritoneal cavity of diabetic NOD mice. Transplantation was
considered successful if the non-fasting blood
glucose concentration returned to normal within 24 hours after surgery.
The glucose concentration of the recipient's blood was monitored three
times a week after transplantation with a Glucometer Elite instrument
(Bayer Corp., Pittsburgh, Pa.).
Allografts were considered to have been rejected if the blood glucose
concentration increased to more than 250 mg/dl at two monitoring time
points. The recipients that rejected allografts were tagged for
histological examination and flow-cytometric
studies.
To determine the effect of the endogenous pancreatic islets in
the control of blood sugar concentration, the subrenal islet transplants
were surgically removed and analyzed. Similarly, islets that had been
encapsulated in alginate spheres were
removed, as necessary, from the peritoneal cavity after direct
localization using a dissecting microscope. Histological analyses of
the pancreata and allograft were performed by (i) staining with
hematoxylin and eosin for evaluation of lymphoid
infiltrates and (ii) staining with aldehyde-fuchsin for insulin islet
content. The entire pancreas from splenic to duodenal stomach
attachments was removed, fixed, and subjected to serial sectioning,
usually at 10 .mu.m per section.
Example 1
CFA Treatment and Islet Transplantation in NOD Mice
Hosts for the transplantation experiments were severely
diabetic female NOD mice, usually greater than 20 weeks of age, which
exhibited blood glucose concentrations of greater than 400 mg/dl for at
least seven days and had been treated by daily
administration of insulin for that length of time. Islet transplants
were placed unilaterally in the kidney capsule to facilitate non-lethal
removal and histological examination. Islets from 6-8 week-old
pre-diabetic donor females or from normal C57
donor mice were rapidly rejected by diabetic NOD hosts in all cases,
usually by day 9. Although C57 donor islets with transient ablation of
class I survive indefinitely in non-immunosuppressed diabetic and
non-autoimmune diabetic hosts, the survival
time for .beta..sub.2M.sup.-/- C57 islets in diabetic NOD females is
only about two to three times that of normal C57 islets. This
observation confirms that the ablation of the donor cells is related to
the current disease, and not related to allograft
rejection. CFA treatment prolonged survival of syngeneic islet grafts
in diabetic NOD hosts, but had a minimal effect on the survival of
C57BL/6 islets, which were uniformally rejected about 11 days after
transplantation. However, the combination of
.beta..sub.2M C57 islet transplants with CFA treatment resulted in
sustained normoglycemia for more than 120 days in 5 out of 14 diabetic
hosts. Although the duration of hyperglycemia before initiation of the
therapy varied between 7 and 20 days in
these cohorts, the length of this interval was not statistically related
to the duration of sustained normoglycemia after treatment. The
animals in this study with sustained normoglycemia also had progressive
weight gain similar to that in NOD female
host cohorts that never became diabetic. The normalization of blood
sugar concentration is a measure of treatment success.
After the recurrence of hyperglycemia in NOD mice that had been
treated with CFA and syngeneic NOD transplants that do not have MHC
class I-presented self-peptide, the kidney containing the allograft was
examined histologically. Macrophage and
T-cell specific infiltrates were apparent under the kidney capsule at
the site of transplantation, a characteristic of recurring autoimmune
disease. Moreover, no intact islets were detected in the pancreas.
Although an occasional host had islet
remnants in the pancreas, these were largely obscured by dense pockets
of infiltrating lymphocytes. This recurrence of hyperglycemia after
administration of NOD transplants may be due to the inability of the MHC
class I in the NOD transplants to present
peptide because of an error in this pathway in NOD mice. This lack of
peptide presented by MHC class I cells may prevent the transplanted
cells from inducing tolerance. Alternatively, even if NOD islets
present MHC class I and peptide, it is possible
that a single exposure of a parenchymal cell is insufficient. Thus, the
host NOD mice generate autoimmune T-cells that destroy the transplanted
cells.
Similar histological characteristics, including infiltrating
lymphocytes at both the transplant site and in the pancreas, were
apparent in diabetic NOD mice that received CFA in combination with
semi-allografts from C57 donors. Unexpectedly,
for all NOD mice with long-term normoglycemia, after removing the
.beta..sub.2M.sup.-/- C57 islets, and after CFA treatment, no surviving
allografts were detected under the kidney capsule when the animals were
examined more than 129 days after
transplantation using this protocol. In contrast, the pancreas in each
of these five recipients exhibited well-formed islets that appeared
completely granulated when stained with aldehyde-fuchsin. Our results
show that the islets were free of
lymphocytes, with lymphocytes only present around the circumference of
the islets. This pattern of lymphocyte accumulation, with lymphocytes
surrounding, but not invading the islets has been associated with
non-progressing or interrupted beta cell
autoimmunity. The return to normoglycemia in the absence of detectable
transplanted islet tissues, together with the presence of islets in the
pancreas largely devoid of infiltrating lymphocytes indicates not only
that autoimmunity has been interrupted,
but also that the function of the endogenous beta cells had been
restored. This disruption of autoimmunity is thought to be due, at
least in part, to the induction of TNF-alpha by the administered CFA,
and the destruction of autoimmune T-cells by the
resulting TNF-alpha. Additionally, the expression of MHC class I with
an allele that matches the host and peptide by the donor cells induces
tolerance by T-cell re-selection perhaps both by necrosis and apoptosis
that also prevents destruction of the
transplanted cells and regenerating organ.
The relative contribution of endogenous cells and donor cells
to restoring endogenous beta cell activity was measured to determine
whether the increase in beta cell activity was a result of endogenous
regeneration from host precursor cells or,
as demonstrated below, the conversion of donor lymphoid cells or donor
islets into hosts islets. As described below, although rescue may play a
role in the reversal of early diabetes and late diabetes, the
administered donor cells were cells that could
become host pancreatic beta cells. Restoration of near normal
pancreatic islet histology was observed only in diabetic NOD mice that
received both the .beta..sub.2M.sup.-/- allograft and the CFA treatment.
Pancreatic islets were not detected in any
diabetic NOD mouse treated with CFA and syngeneic NOD islets. The
persistence of normoglycemia in recipients of syngeneic NOD islets was
apparently solely due to transplanted islets which always exhibited
invasive insulinitis. Here, treatment with CFA,
together with syngeneic NOD islets may have slowed disease recurrence,
but persistent autoimmunity remained. We also assessed the relative
contribution of restored endogenous pancreatic islets and transplanted
islets to the maintenance of normoglycemia
in NOD mice treated with CFA and allografts from .beta..sub.2M.sup.-/-
C57 donors. The relative contribution of the endogenous pancreatic
islets was determined by removing the kidney containing the islet
transplants after 120 days of normoglycemia from
a group of five animals, as well as from a control group that had not
received the allograft. All five mice which had received the allograft
remained normoglycemic after nephrectomy until they were killed 3-60
days later. Histological analysis of
kidneys that had not received the graft revealed a complete loss of
identifiable islet structures. In contrast, the pancreata for all five
allograft recipients contained well-formed islets, either without
lymphoid infiltrates or with circumferentially
distributed lymphocytes only. Normoglycemia after nephrectomy was thus
maintained solely by endogenous pancreatic islet that we now know had
reappeared in the host by both rescue as well as regeneration from
endogenous or exogenous sources.
These results were affirmed by analysis of nephrectomies
performed during the post-transplant period of normoglycemia on two mice
which had received CFA plus syngeneic NOD islets. In this case, the
treatment resulted in a rapid return to
hyperglycemia, demonstrating that the control of blood sugar in this
treatment group was mediated solely by the transplanted islet tissue.
Diabetic NOD mice were also transplanted with islets from C57 mice in
which the genes from both the .beta..sub.2M
and TAP1 genes had been deleted. Together with TAP2, TAP1 mediates
transport of endogenous peptides from the cytosol into the lumen of the
endoplasmic reticulum for the assembly with MHC class I molecules. Our
data using C57 mice in which the genes for
both .beta..sub.2M and TAP1 had been deleted showed that these mice are
more defective in the presentation of MHC class I self-antigens than
those only having a mutation in the .beta..sub.2M gene. Transplantation
of these double-knockout C57 islets
lacking both .beta..sub.2M and TAP1, combined with the injection of CFA
results in return of hyperglycemia within 14 days in some of the
animals. Two out of six NOD mice treated with the double knock out
Class I cells had normoglycemia after 40 days of
treatment. Physiological examination of the pancreas revealed a pattern
typically seen in untreated diabetic NOD mice. The decrease in
efficiency of this protocol supports the important role of correctly
assembled and administered MHC class I and
peptide complexes.
Based on these results, a transient interruption of peptide
presentation of donor MHC molecules or a transient deficient MHC class I
is important for the abrogation of autoimmunity. This transient
ablation or decreased MHC class I or permanent
presentation of donor MHC class I also appears to be a feature that
allows these cells to transform into other cell types. In contrast, the
sustained interruption of this process prevents the re-establishment of
tolerance and the restoration of
endogenous pancreatic islet integrity. The repeat administration of a
normal MHC class I and self peptide or a short lived cell showed similar
efficacy.
Given that the restoration of normoglycemia in diabetic NOD
hosts treated with CFA and .beta..sub.2M.sup.-/- C57 islets cannot
depend on the continuing secretion of insulin by the islet graft, we now
investigated whether C57 donor cell types,
other than islets, might serve a therapeutic role. We initially
performed these experiments expecting the C57 donor cell to provide MHC
class I and self-peptide. As shown by the following results, donor
lymphoid cells actually transform into insulin
secreting beta cells in the host or fuse with damaged cells of the host
or adjacent cells of the host. In these experiments, nine diabetic NOD
mice were treated with a single bilateral injection of CFA, followed by a
40-day regimen of biweekly
intravenous injections of C57 splenocytes. These lymphoid cells express
both MHC class I and MHC class II proteins and survive only transiently
in NOD hosts because of presumed graft rejection. However, contrary to
the prior assumption that this would
be only a transient treatment, these donor cells survive long-term
without a need for immunosuppression in the host. Repeat injections of
splenocytes ensured that the host was continuously exposed to antigen
presentation complexes on the surface of
these cells. Recipients were monitored for hyperglycemia every 3-4 days
and insulin was administered daily unless normoglycemia returned. A
control group of four diabetic NOD mice received daily insulin
injections only. All four control group mice
died on or before day 25 of the experimental period as a result of poor
control of blood sugar and consequent ketoacidosis. In contrast, some
of the mice injected with CFA and C57 splenocytes were alive after 40
days, and three of these animals had
become normoglycemic and insulin independent.
While the pancreata of control mice exhibited pronounced
lymphocytic infiltrates that obscured any remaining islet structures,
the pancreata of the four NOD mice that were treated with CFA and C57
splenocytes and remained alive but hyperglycemic
and insulin dependent revealed a marked decrease in the number of
lymphoid infiltrates located circumferentially or adjacent to the
infrequent islet structures. In the three NOD mice treated with C57
splenocytes and CFA that remained normoglycemic after
the discontinuation of insulin injections, the pancreata exhibited
abundant islets that were free of invasive insulinitis or islets
associated only with circumferential lymphocytes. The treatment of CFA
combined with re-exposure to C57 lymphocytes
resulted in complete reversal of diabetes in approximately 30% of NOD
recipients and partial restoration of beta cell function in
approximately an additional 40% of hosts.
Subsequent experiments were conducted to determine whether the
efficiency of the system could be improved by regulating glycemic
control in the host. The reversal of diabetes in NOD mice by CFA and
repeat exposure to C57 splenocytes indicated
that the restoration of endogenous islet function is achievable without
islet transplantation and despite the poor glycemic control attained by
insulin injection. To determine whether the restoration of endogenous
beta cell function could be achieved
more consistently with better control of blood glucose, insulin
injections were replaced with the intraperitoneal implantation of
alginate encapsulated C57 mouse islets. Alginate encapsulation prevents
direct contact between the donor tissue and the
host T-cells and such grafts have been shown to provide near
normoglycemic control for 40 days in approximately 78% of autoimmune NOD
recipients. Almost all diabetic NOD mice that received alginate
encapsulated C57 islets exhibited improved glucose
regulation or normoglycemia. The alginate spheres were removed 40-50
days after implantation, and the blood glucose concentration was
monitored. The seven mice treated only with alginate encapsulated
islets, the six mice that received a single
bilateral injections of CFA, and the three mice treated with biweekly
injections of C57 splenocytes, all exhibited a rapid return to
hyperglycemia and early death after removal of the implants. Pancreata
of NOD mice that received only alginate
encapsulated islets revealed no sign of intact islets or of lymphoid
infiltrates. The pancreata of NOD hosts treated with CFA and alginate
encapsulated islets exhibited marked invasive insulinitis and obscured
islet structures. In contrast, seven of
the nine diabetic NOD hosts that received CFA and C57 splenocytes
remained normoglycemic for more than 40 days after removal of the
alginate encapsulated islets. The pancreata of these animals contained
large islets with circumferentially distributed
lymphocytes. The islet mass after at least 80 days of disease reversal
was estimated to be approximately 50% of the original value. The
pancreata from control BALB/C mice contained approximately 25-35 islets,
and the pancreata from successfully treated
NOD mice contained approximately 12-20 islets, as determined by serial
histological sectioning.
In addition, the maintenance of normoglycemia due to the
treatment increased the percentage of diabetic mice permanently cured of
hyperglycemia. We next identified features of this treatment regimen
that contributed to the production of a
positive outcome. As is noted above, CFA was used to induce the
endogenous production of TNF-alpha, as well as other cytokines believed
beneficial for removal of autoimmunity and to promote regeneration. The
role of TNF-alpha in the treatment of
diabetes was therefore investigated by the intravenous administration of
rat IgG1 monoclonal antibodies to the cytokine TNF-alpha at a dose of
approximately 1.5 mg/day for the first 10 days in diabetic NOD hosts
treated with C57 splenocytes, CFA, and
alginate encapsulated islets. All five NOD mice so treated exhibited a
rapid return to hyperglycemia upon removal of the alginate encapsulated
islets 50-70 days after transplantation consistent with the role of
TNF-alpha in the beneficial effect of CFA. In a related experiment, an
anti-TNF-alpha antibody (clone MP6-X73) was administered at a dose of
1.5 mg/day for 10 days after administration of CFA, C57 splenocytes, and
alginate encapsulated islets into diabetic NOD mice (n=5). After
removal of the
alginate-encapsulated islets at day 40, hyperglycemia returned
immediately in all five mice. In mice treated similarly except for the
administration of the anti-TNF-alpha antibody, normoglycemia was
maintained in seven out of nine NOD mice after removal
of the alginate beads. The specificity of the effect induced by the
anti-TNF-alpha monoclonal antibody was confirmed by the failure of the
control rat IgG1 monoclonal antibody reactive with human T-cell receptor
beta 1 chain to produce a therapeutic
effect.
To demonstrate an increase in TNF-alpha levels due to
administration of CFA, levels of TNF-alpha were measured in NOD mice
after a single injection of CFA with or without donor lymphocytes. As
Table 1, slightly elevated levels of TNF-alpha were
transiently detectable in NOD mice for 2-5 days after a single injection
of CFA with or without splenocytes. NOD mice at days 2-8 after a
single dose of CFA also have decreased platelet levels from
30,000-60,000/mm.sup.3. This data indirectly supports
CFA's induction of TNF-.alpha. in NOD mice.
TABLE-US-00001 TABLE 1 Treatment of NOD mice with CFA results
in elevated plasma TNF-alpha. Donor TNF (g/mL) Group Cells CFA Day 0
Day 2 Day 5 Day 21 Day 40 1 -- - .40 .35 .41 .42 .38 2 -- + .51 20.1
18.1 .49 .30 3 C57BL/ + .35 21.7 17.5 .50
.57 6 TNF-concentrations were measured by solid phase ELISA using a
sandwich technique with two different monoclonal antibodies to
mTNF-alpha, one of which was conjugated to horse radish peroxidase
(Sigma, St. Louis, MO). The limit of detection was 0.2
units/ml.
The data presented above show that the injection of CFA, the
endogenous induction of TNF-alpha, or the administration of TNF-alpha
directly results in the permanent elimination of TNF-alpha sensitive
cells, the majority of which have previously
seen islet cell antigens. In addition to the beneficial apoptotic death
or effect on the lymphoid system, we also demonstrated that TNF-alpha
binds directly to TNF Receptor 2 on the vascular endothelium, the
complementary matrix for differentiation, and
the islet precursor cells or islet beta cells themselves, thereby
possibly also promoting regeneration in the pancreas. We further showed
that the introduction of MHC class I peptide complex expression, either
on the surface of normal islet cells or
normal lymphocytes, results in a partial, but stable, reselection of
T-cell population from the NOD hosts leading to an increase in the
abundance of long-term memory cells. MHC class I and self-peptide
reintroduction is important for the reselection of
cells that probably have less stimulation with islet cell antigen, but
the equivalent potential for autoreactivity (see, for example, U.S.
patent application Ser. No. 09/521,064, filed Mar. 8, 2000, and
09/768,769, filed Jan. 23, 3001, and PCT
publication WO00/53209, published Sep. 14, 2000, which are each
incorporated by reference). Furthermore, additional studies presented
herein show that these cells actually persist long-term in the host. We
demonstrate that without any immunosuppression
to the host, the semi-allogeneic MHC class I splenocyte origin cells or
splenocyte residing cells, as well as semi-allogeneic cells from spleens
differentiated into islets, persist long-term in the host without the
need for immunosuppression.
Furthermore, the reversal of any poorly controlled metabolic condition
appears to promote the regenerative process and the reversal of
established disease.
Example 2
Optimizing a Curative Therapy in the Diabetic NOD Mouse
In order to examine the differential effects of CFA, BCG,
TNF-.alpha., and splenocyte administration, late stage NOD mice (>15
weeks of age) were randomly assigned to one of four treatment groups,
i.e., one injection of CFA, one injection of
BCG (4 mg/kg), one injection of 10 ug TNF-.alpha., or one injection of
F1 splenocytes (1.times.10.sup.6 cells, IV) obtained from normal donors.
The treated NOD mice were serially sacrificed on day 2, day 7, and day
14 (Table 2). An examination of
pancreatic histology evaluated the effects of the interventions on
invasive insulitis (Table 3).
Table 2 shows that on day 2 both a single injection of CFA and a
single injection of low dose TNF-.alpha. (10 g) had eliminated
completely all subpopulations of cells with in vitro TNF-.alpha.
sensitivity. At day 7 and day 14, this population
of TNF-.alpha. sensitive cells was again evident. Simultaneous
pancreatic histology of these cohorts confirmed a dramatic reduction in
insulitis, as well as a lingering effect lasting beyond day 14 with
respect to insulitis (Table 3).
TABLE-US-00002 TABLE 2 Percentage of remaining TNF
-apoptotic-sensitive NOD splenocytes after various in vivo treatments
Day 0 (%) Day 2 (%) Day 7 (%) Day 14 (%) NOD mice 0 ng 20 ng 0 ng 20 ng 0
ng 20 ng 0 ng 20 ng CFA 26.6 24 29.2 34.2 26.6
35.1 10 g m-TNF 27.9 28.2 28.9 36 BCG 7.4 22.3 15.3 26.7 37.8 28.9 F1
splenocytes 4.3 27.5 3.8 23.1 0.8 33.7 untreated 22.1 32.6 27.1 41.8
30.7 41.7 29.4 38.6 Day 0 Day 2 Day 7 Day 14 C57BL/6 0 ng 20 ng 0 ng 20
ng 0 ng 20 ng 0 ng 20 ng untreated 14.1
14.7 12.3 12.8 18.8 16.2 17.5 17.2 NOD mice in this study were in a late
pre-diabetic stage of disease at 18 weeks of age with at least one
blood sugar greater than 200 mg/dL. Late apoptotic cells by flow
cytometric studies of NOD mice treated with
various immunomodulatory interventions were quantified on splenocytes
after animal sacrifice in the days indicated after treatment initiation.
For these flow cytometric studies, late apoptosis represents Annexin
V+ PI+ and Annexin V+ PI- cells after 24
hours in vitro exposure to TNF- (20 ng/mL)
TABLE-US-00003 TABLE 3 Percentage of NOD islets with remaining
invasive insulitis after various in vivo treatments Day 0 (%) Day 2 (%)
Day 7 (%) Day 14 (%) CFA 4 22 10 mg m-TNF 7 33 BCG 9 67 F1 splenocytes
26 31 untreated 100 0 100 100 The mice
in Table 3correspond to the same mice shown in Table 2
The responses to F1 splenocytes and BCG were markedly different
than that seen with CFA and TNF-.alpha., and somewhat different from
each other. The therapeutic effect of F1 splenocytes was an elimination
of the NOD lymphoid cells, which we
believe represent pathogenic naive cells, perhaps through a direct or
indirect mechanism (Table 2). TNF-.alpha. sensitivity remained and the
F1 splenocyte therapeutic impact lasted beyond day 14. The histology
revealed a less dramatic impact on the
numbers of NOD islets with remaining invasive insulitis. In addition,
F1 splenocytes eliminated "cords" of invasive insulitis per islet
instead of the more homogenous central elimination of insulitis
characteristic of either TNF-.alpha. or CFA (not
shown).
Unexpectedly, BCG, a known inducer of TNF-.alpha. had a
greater impact on the elimination of tissue culture (naive) sensitive,
pathogenic cells than it had on the elimination of pathogenic memory
cells. The therapeutic effect of a single, low
dose injection of BCG waned rapidly over the time course of 14 days
(Table 2). The histologic analysis of the NOD pancreases confirmed
partial elimination of insulitis (Table 3). Similar to the end result
when MHC class I and self-peptide were
reintroduced, the tissue culture-sensitive subpopulation of cells was
eliminated.
These data are consistent with data in our earlier publication
relating to the use of two limbs of interventions to "reverse" disease
in NOD mice (i.e., TNF-.alpha. and F1 splenocytes). Our data show that
these interventions can produce a
distinct, measurable impact on specific lymphoid cell populations in the
spleen. Each of the two limbs appears to target a different
subpopulation of pathogenic cells with heightened apoptosis sensitivity.
Importantly, the changes in T cell response to
culture and TNF-.alpha. in vitro parallel the pancreatic histology of
reduced invasive insulitis.
Although initially thought to be due to its reported induction
of endogenous TNF-alpha release, the action of BCG in NOD mice appears
to be predominately due to an indirect impact on naive cell selection by
the direct killing of
monocytes/macrophages that have defective MHC class I presentation and,
to a lesser extent, by reduction of the measurable burden of memory
cells with TNF-alpha sensitivity. BCG is known to infect
macrophages/monocytes and, if the BCG is avirulent,
induces lysis of the macrophage/monocyte, thus reducing hampering
continued production of tuberculin particles. Macrophage/monocyte lysis
releases TNF locally, with infected cells usually containing abundant
intracytoplasmic concentrations of this
cytokine. In the NOD mouse, the avirulent strain of BCG causes lysis of
certain subpopulations of monocytes/machrophages in an accelerated
fashion. These cells appear to be developmentally "immature", with
lower levels of MHC class I-self peptide
expressed on the surface. This suggests that BCG sub-strains with the
lowest virulence, derived from the in vitro selection of those that most
rapidly lyse human or murine autoimmune monocytes, would be the best
BCG strains to treat autoimmune
disorders. Furthermore, these data suggest three strategies for
identifying and eliminating naive T cells: direct death receptor
stimulation of the T cells through a susceptible receptor on the cells;
introduction of corrected antigen presenting cells
or complexes; with self peptide and or self peptide/MHC class I
complexes, or the introduction of agents or adjuvants like BCG that
cause the direct death of the monocytes/macrophages with the most severe
defects in antigen presentation/processing,
thereby eliminating the defective educator cells. In all three
strategies, endogenous monocytes/macrophages with sufficient antigen
presentation to bias the T cell repertoire back towards normal
reselection predominate.
Example 3
Functional Impact of Donor Cell Radiation on Restoration of Normoglycemia in Severely Diabetic NOD Mice
Severely hyperglycemic NOD mice were originally treated with
CFA to induce TNF-alpha and simultaneously exposed to functional
complexes of MHC class I molecules and allogeneic peptides presented on
either viable splenocytes or on islets.
Normoglycemia for a 40-day treatment period, a critical parameter of
this approach, was induced either by the implantation of temporary
alginate encapsulated islets or subrenally placed syngeneic islet
transplants. Both methods of glucose control can be
surgically removed to test for restored endogenous pancreatic function.
All original experiments were designed to treat established NOD female
mice with severe hyperglycemia and utilized a 40-day time period of
tight, artificial glucose control.
Utilization of this temporary glucose control permitted sufficient
endogenous pancreatic re-growth of the endogenous pancreas to sustain
normoglycemia in up to 78% of formerly hyperglycemic NOD cohorts and to
restore endogenous pancreatic insulin
secretion.
To dissect the mechanism to NOD disease reversal, irradiated
donor cells and live cells expressing MHC class I and self-peptide were
studied. These experiments tested the role of long-term donor cell
survival and the role of the donor cell
function (e.g., antigen processing) in the permanent reversal of
tolerance for self-antigens. NOD hosts used in these experiments were
severely diabetic NOD mice typically greater than 20 weeks of age that
exhibited blood glucose concentrations of
greater than 400 mg/dl for at least seven days. All diabetic NOD hosts
at this late stage of disease were dead within two weeks because of the
severity of the disease. Alginate encapsulated islets were implanted
into the peritoneal cavity for 40 days,
and the hosts were randomized to receive either CFA alone or CFA in
combination with intravenous biweekly injections of irradiated C57BL/6
splenocytes. At this point, the C57BL/6 splenocytes are only
semi-allogeneic to the NOD host; the splenocytes are
H2K.sup.bD.sup.b, and the NOD mouse is H2K.sup.dD.sup.b.
As we previously demonstrated, new cohorts of NOD mice
immunized with live C57BL/6 splenocytes demonstrated restored
normoglycemia. Seven of the nine newly treated NOD cohorts remained
normoglycemic after the surgical removal of the alginate
encapsulated C57 mouse islets. The reversal of autoimmunity was
permanent, and the seven NOD hosts remained euglycemic for observation
periods beyond 80 days at which time they were sacrificed. Eight
additional NOD mice were treated with the same
regimen, but with irradiated donor C57BL/6 splenocytes. In all eight
cases, hyperglycemia returned within two to seven days after the removal
of the alginate encapsulated islet that served as a glucose clamp
during the 40-day treatment period.
Pancreata of NOD mice that received live C57 splenocytes revealed
abundant islets in seven of the nine mice. No signs of invasive islet
lymphoid infiltrates, which are a sign of active autoimmunity, were
present. When an islet was present, fewer than
10% of islet circumferential lymphoid cells encircled the pancreatic
islet. Surprisingly, the pancreata of NOD mice that remained
hyperglycemic and received irradiated donor splenocytes consistently
revealed islets in the pancreas, but with decreased
overall abundance as assessed by serial pancreatic sections. The islets
in these pancreata were accompanied with sizable circumferential islet
infiltrates but lacked lymphocytes within the islet structure itself, a
histological pattern referred to as
invasive insulinitis. For instance, pancreata from NOD mice treated
with live splenocytes and having restored normoglycemia contained
approximately 6-8 islets with each serial histological section, while
pancreata from NOD mice treated with irradiated
splenocytes and having persistent hyperglycemia contained 4-6 islets per
serial histological section. Both irradiated and live splenocytes
appeared to rid the diabetic host of invasive and destructive
insulinitis. However, increased islet abundance and
restored insulin secretion were only observed in NOD cohorts treated
with live splenocytes.
In evaluating the significance of apparent histological
elimination of destructive insulinitis without sufficient insulin
secretion after the introduction of irradiated donor cells expressing
MHC class I and self-peptide, additional diabetic NOD
mice were treated. For these experiments, we used a treatment period of
40 days of donor MHC class I and self-peptides cell treatment, but
extended the "glucose clamp period" of artificially restored
normoglycemia from 40 to 120 days. The rationale for
this experiment was to give the less efficient irradiated donor
splenocyte treatment an opportunity for more complete host pancreatic
rescue and a regeneration of the pancreatic islet. A cohort of 25
severely diabetic NOD mice was randomized for the
same treatment protocol of live or irradiated intravenous MHC class I
and self-peptide expressing cells for 40 days with a glucose clamp now
implanted for 120 days. All treated NOD cohorts of both groups were
followed for extended observation times for
endogenous pancreatic function after clamp removal. Because a glucose
clamp containing alginate encapsulated islets has a greater than 50%
failure rate in an autoimmune host after 40 days of implantation, the
glucose clamps used for these diabetic hosts
were syngeneic islets transplanted under the kidney capsule. We removed
this glucose clamp by nephrectomy 120 days after implantation.
Placement of the 120-day extended glucose clamp yielded a
marked beneficial effect when combined with the formerly metabolically
ineffective therapy of irradiated MHC class I and self-peptide
expressing cells followed by an early evaluation at
day 40. Functionally, 11 of the 12 NOD hosts which received live MHC
class I and self-peptide expressing splenocytes continued to remain
normoglycemic for observation times extending beyond 180 to 250 days
after removal of the 120 day glucose clamp.
Eleven out of the thirteen NOD hosts received irradiated MHC class I and
peptide expressing cells and remained normoglycemic for a similar
observation time. Both NOD treatment groups, receiving either live or
irradiated MHC class I and self-peptide
expressing cells, had physiologically equivalent pancreatic islet
density as assayed using histological sections of the pancreas. The
pancreatic islets in all cohorts of these two latter groups were
examined after long-term follow-up of reversed disease
for 180-250 days after restored normoglycemia. Formerly diabetic NOD
cohorts with normoglycemia after irradiated donor lymphoid
injections-possessed healthy and abundant pancreatic islets, but they
were consistently accompanied by impressive
circumferential lymphoid infiltrates. This histological feature was
absent in treated NOD cohorts receiving live MHC class I and
self-peptide donor cells. Therefore, although irradiated MHC class I
and self-peptide expressing donor cells were effective
at restoring long-term normoglycemia due to pancreatic insulin
secretion, apparent non-progressive circumferential insulinitis was
evident during long-term follow-up of the pancreas. Despite the
histological differences, both experimental NOD treatment
groups (all 25 cohorts) have pancreatic islets free of invasive
insulinitis confirming the absence of active disease.
We also analyzed these cohorts to identify the actual
composition of the insulin secreting beta cells in the pancreas, the
blood, and the splenocytes at the time of autopsy to determine the
contribution of the donor cells to long-term chimerism
or conversion to pancreatic beta cells. These initial experiments
comparing live to irradiated cells suggested that live cells had some
advantages over irradiated cells. First, the live cells corrected
diabetes at a much more rapid rate than irradiated
cells. This result suggested that in the case of a long-term diabetic
in which the regenerative capacity of the pancreas was possibly less
evident, the introduction of live cells that have the ability to convert
to beta cells might be an advantage. On
the other hand, irradiated cells, which do not have the ability to
regenerate into pancreatic islets, may have an advantage in a new onset
diabetic because of greater safety. Also, islets in mice treated with
live versus dead cells appeared
histologically different--in that mice treated with live cells had
significantly less circumferential insulinitis than mice treated with
irradiated cells which, in many cases, had very impressive
circumferential insulinitis. However, we have no reason
to believe that circumferential insulinitis eventually progresses to
active disease following treatment with irradiated cells.
There may be at least one more advantage to the regenerating
tissue being of donor origin. In many of the treated NOD mice,
syngeneic islet transplantation was performed under the kidney capsule.
In all cases, even after complete disease
reversal, syngeneic transplants exhibited pronounced peri-islet
insulitis, in contrast to the regenerated semi-allogeneic islets in the
pancreas, which were almost entirely free of peri-insulitis. These data
suggest that syngeneic islets themselves have
some yet unidentified defect that promotes an abnormal immune response,
even in the presence of a fully re-educated lymphoid system.
Example 4
Reintroduction of MHC Class I Complexes to the Cell for Restored T-Cell Education
The histological impact of the introduction of cells expressing
matched and mismatched MHC class I peptide complexes on treatment
outcome was also analyzed. As is described herein, treatment with a
TNF-alpha inducing agent, e.g., CFA, and with
cells that express MHC class I peptide complexes either on the
parenchymal or lymphoid cells resulted in disease reversal in several
severely diabetic NOD mice. In contrast, immunization with cells
expressing MHC class II peptide complexes was not
obligatory.
We have demonstrated the therapeutic effectiveness of MHC class
I expressing islets or splenocytes from C57BL/6 strain carrying the
H2K.sup.b and H2D.sup.b alleles (a matched and a mismatched MHC class I
allele) and a self-peptide complex. We
investigated whether the therapeutic effect was restricted to the
matched or the mismatched MHC class I and peptide molecule or whether
the effect had allelic specificity requirements for stable autoimmune
disease reversal. The NOD mouse possesses two
different MHC class I genes carrying the H2K.sup.d and H2D.sup.b allele,
and the NOD mouse lymphoid cells fail to express a normal density of
either self-peptide MHC class I structures. Severely diabetic NOD mice
were treated with CFA in combination
with a glucose clamp of intraperitoneally placed islets encapsulated in
alginate or subrenally transplanted syngeneic islets. The diabetic NOD
hosts received concurrent biweekly immunizations with parenchymal cell
lines expressing fully NOD compatible
MHC class I complexes on fibroblasts (e.g., fibroblast cells from H-2
MHC recombinant donor cell lines) or fully MHC incompatible MHC class I
complexes on fibroblasts. All cohorts were sacrificed approximately
40-45 days after treatment initiation for
the histological evaluation of the pancreas. Since the fibroblast cell
lines represent tumor cell lines, the cells used in these experiments
were irradiated prior to intravenous immunization or injection.
The histological results in the endogenous pancreatic islets
differed in the NOD cohorts receiving fully MHC class I matched or
mismatched peptide complexes. NOD cohorts randomized to receive MHC
class I and self-peptide mismatched cells
(H2K.sup.kD.sup.k), possess pancreatic islets with massive invasive
lymphoid infiltrates in five of the five NOD mice. Not only was
invasive insulitis present throughout the pancreas, but none of cohorts
demonstrated a single islet structure without
insulinitis or islets with exclusively circumferential lymphoid
accumulation. The histological result obtained with diabetic NOD
cohorts treated with intravenous injections of matched donor fibroblasts
H2K.sup.d and H2D.sup.d class I and self-peptide
structures were dramatically different. Treatment with MHC class I
matched cells eliminated the invasive insulinitis in all 14 of the 14
NOD cohorts in the pancreatic islets; 2 of the 14 treated NOD cohorts
had pancreatic islets totally devoid of any
invasive or circumferential insulinitis, and 12 of the 14 treated NOD
cohorts possessed pancreatic islets with mild-to-moderate
circumferential infiltrates.
As presented above, the use of irradiated donor cells
frequently results in the reappearance of pancreatic islets, but
consistently these islets regardless of the cellular source were
accompanied by circumferential lymphoid infiltrates adjacent
to, but not invading, the islet structure. The ability of matched
H2K.sup.d D.sup.b fibroblasts to histologically eliminate active
islet-directed autoimmunity defined by invasive insulinitis confirmed
the therapeutic effect of C57BL/6 expressing cells
with a nonspecific effect of the allogeneic H2K.sup.b locus, but likely
also benefited from the MHC class I reintroduction due to the matched
H2D.sup.b locus through specific T-cell receptor or cell surface
receptor (e.g., CD3) engagement of the host.
Therefore, the target of the therapy is likely the host T-cells.
Furthermore, non-irradiated F1 splenocytes from BALB/C and C57 crosses
(i.e., CB6F1/J cells) similarly restored long-term normoglycemia in
about 75-80% of the formerly diabetic NOD hosts
receiving a single dose of CFA. Irradiated, mismatched MHC class I and
self-peptide expressing cells were uniformly ineffective in reversing
established diabetes. Therefore, this cellular therapy administered to
established autoimmune NOD cohorts had
(a) a demonstration that a therapeutic effect can be achieved with only a
single MHC class I allelic match; (b) a demonstration that the cells
can be administered in the presence or absence of allogeneic MHC class I
and self-peptide structures; (c) a
demonstration that the cells injected into diabetic NOD hosts can be
live or irradiated donor cells; (d) a demonstration that the cells can
be parenchymal or lymphoid in origin; and (e) a demonstration that the
cellular effect can be independent of donor
MHC class I donor cells expressing MHC class II and self-peptide
complexes since fibroblasts, an MHC class II negative cell type, and
lymphocytes showed equal efficacy.
We also performed a cytotoxic T-lymphocyte assay to define the
separate therapeutic effects of MHC class I and self-peptide and the
therapeutic effects of CFA/TNF-alpha for reversal of islet-directed
auto-reactivity. As detailed above, an
effective therapy to reverse established autoimmunity included the
introduction of both MHC class I and self-peptide matched cells and
administration of CFA or another TNF-alpha-inducing agent. Here, the
introduced MHC class I and self-peptide complexes
were proposed to reselect poorly trained naive cells with the potential
for autoreactivity. Indeed, peripheral tolerance homeostasis appears to
be maintained by peripheral MHC class I and self-peptide complexes, and
this prevents naive cell abundance or
unstimulated T-cell abundance--a feature seen in the pre-diabetic NOD
mouse or in the untreated NOD mouse after the onset of hyperglycemia.
In contrast, the data presented herein indicate that autoreactive memory
T-cells or cells with exposure to
antigen stimulated cells are selectively sensitive to CFA presumably due
to the obligatory endogenous TNF-alpha induction and subsequent
apoptosis due to defects in NF.kappa.B signaling.
To show that these observations could not simply be explained
by changes in cell number, we looked at the overall abundance of CD45,
CD62L, or CD95. The specific functional role of naive CD45RB high
density cells (CD45RB.sup.high) in memory and
the role of CD45RB low density (CD45RB.sup.low) NOD T-cells in
autoreactivity was tested in vitro in cytotoxic T-assays to islet
targets. While we refer to naive cells as CD45RB.sup.high and memory
cells as CD45RB.sup.low, these cells probably do not
represent naive versus memory cells, but rather represent cells in
different stages of activation depending on exposure to antigen. For
brevity, we refer to these cells as mostly stimulated or unstimulated
cells, but sometimes we also refer to these
cells as naive or memory cells. The splenocyte donors, the source of
the CTLs, were untreated NOD hosts, NOD hosts treated solely with CFA,
and NOD hosts treated with both MHC class I and self-peptide and CFA
with long-term disease reversal. Dispersed
NOD islets from 8 week-old NOD female donors were used as responder
cells. We used an insulin enzyme ELISA to detect T-cell lysis of
syngeneic islets with insulin release from the target, as well as
colorimetric quantitation of insulin by a
spectrophotometer. The receptor T-cells were sorted into two pools
prior to the assay: unstimulated T-cells defined as CD3 positive,
CD45RBhigh, and stimulated T-cells defined as CD3 positive,
CD45RB.sup.low. Numerous effector to target cell ratios
were tested. Based on these data, an optimized T-cell effector to islet
target ratio resulted in co-incubation assays of 24-hours of culture at
37.degree. C. Using the colorimetric readout as well as the direct
insulin readout we determined the
relative amounts of insulin released from live beta cells, and using the
ELISA assay we determined the actual amount of released insulin in the
culture supernatants. These results showed that diabetic NOD derived
CD3 cells, either of the memory type or
stimulated type or of the unstimulated type, equivalently lysed
dispersed islet cells after 24 hours of co-culture.
Both the colorimetric assay and the actual measurements of
released insulin confirmed the pathogenicity of the diabetic NOD cell
populations as showing self-reactivity to syngeneic islet cells. The
pathogenicity of stimulated CD45RB.sup.low
T-cells can be selectively altered. Indeed, splenocytes from NOD
cohorts treated with CFA alone 25 days prior to the assay showed the
selective elimination of autoreactivity of only the stimulated cell
population which has the ability to lyse syngeneic
dispersed islet cells. CFA treated NOD cohorts maintained unstimulated
CD45RB.sup.high T-cell populations with islet autoreactivity equivalent
to that seen in untreated NOD splenocyte donors. Given that CFA therapy
alone, with its resultant endogenous
induction of TNF-alpha, was not successful in a diabetic NOD mouse in
eliminating existing and late stage autoimmunity disease, the CTL
results were consistent with the idea that two identifiable
subpopulations of autoreactive cells may need to be
manipulated in vivo for disease reversal. A marked contrast is seen in
separated subpopulations of unstimulated CD45RB.sup.high and stimulated
CD45RB.sup.low cells obtained from successfully treated NOD cohorts that
received both syngeneic matched MHC
Class I self-peptide expressing cells and CFA. These mice show complete
and stable long-term elimination of both stimulated and unstimulated
autoreactive T-cells with syngeneic islet directed autoreactivity.
Taken together, the results of the CTL assay indicate that in
diabetic NOD hosts, unstimulated T-cells identifiable with CD45RBhigh
and stimulated T-cells expressing CD45RB.sup.10W have islet cytotoxicity
or the potential for islet cytotoxicity. Autoreactive T-cell memory
subpopulations were selectively eliminated with CFA alone, while both
autoreactive stimulated and unstimulated subpopulations were eliminated
with syngeneic MHC class I and self-peptide and CFA. Both aspects of
the treatment
may be required for the elimination of existing autoreactivity due to
the existence of both stimulated and unstimulated cells with
autoreactive potential. Accordingly, select treatments may be designed
to target and eliminate the separate cell
populations. Indeed this hypothesis was supported by our earlier
adoptive transfer data showing that autoreactive NOD cell populations
remained after TNF-alpha treatment of diabetic donor splenocytes,
presumably because of the ability of these cells to
change their phenotype and become TNF-alpha sensitive after islet
exposure.
Example 5
T-Cell Re-Education Due to Exposure to MHC Class I and Presented Self-Peptide
To demonstrate that reversal of established NOD autoimmunity
was linked to MHC class I education of T-cells, we monitored NOD mice
before and after diverse therapies to measure a trend towards restored
CD8 T-cell selection. As illustrated in
FIGS. 2A-2C, untreated NOD mice or NOD mice only treated with CFA have
high levels of CD62L, CD45RB.sup.high, and CD95 positive CD8 cells.
Treatment with CFA and C57BL/6 splenocytes or class II.sup.-/-
splenocytes decreased the T-cell expression level
of CD62L and partially normalized levels of CD45RB.sup.high and CD95 CD8
cells (FIGS. 2A-2C). Importantly, the apparent normalization of T-cell
education/selection was not observed in NOD mice treated with CFA
therapy alone (FIGS. 2A-2C). The
establishment of normal numbers of memory T-cells was not observed when
diabetic NOD were treated with CFA and C57BL/6 .beta..sub.2 m.sup.-/-
TAP1.sup.-/- splenocytes, a cell line with reduced peptide filled
surface class I structures (FIGS. 2A-2C).
Long-term memory requires the surface expression of self major
histocompatibility complex molecules, and this positive selection by
introduced class I expressing splenocytes restores T-cell selection
towards normal.
Previously published data supports the concept that CD8 gene
expression is maintained by proper peripheral MHC class I presentation.
If class I education is interrupted, treatment of CD8 cells with 0.4%
pronase followed by 48 hours of culture
results in low surface re-expression of CD8 levels (Pestano, Science
284:1187-1191, 1999). Indeed, C57BL/6 splenocytes fully recovered CD8
levels after in vitro pronase treatment: no change in CD8 density was
observed after pronase (FIGS. 3A and 3B).
In contrast, NOD splenocytes after pronase treatment did not adequately
re-express CD8 surface levels (FIGS. 3A and 3B). This result confirms
previously published studies of interrupted MHC class I presentation in
the NOD mouse. Splenocytes from NOD
mice, whose diabetes was successfully treated in vivo with C57BL/6 or
C57BL/6 class II.sup.-/- splenocytes and CFA, had improved CD8
re-synthesis after pronase treatment in vitro. In contrast, NOD mice
treated only with CFA or class I deficient C57BL/6
splenocytes with CFA had persistent problems with CD8 re-synthesis
similar to untreated NOD mice, confirming the persistence of interrupted
T-cell selection by MHC class I structure. Simultaneously performed
control experiments confirmed splenocytes
from NOD mice of diverse treatment groups and splenocytes from C57BL/6
mice re-synthesize CD3 surface proteins at comparable rates (FIGS. 3A
and 3B). Therefore, four established parameters of interrupted CD8
education (CD45RB.sup.high CD62L, cD95, and
CD8 resynthesis) due to faulty MHC class I presentation, confirm that
NOD mice with disease reversal have partial to complete correction of
CD8 phenotypes of T-cell selection.
Example 6
Both Subrenally Transplanted Islets and Endogenous Pancreatic Islets Show Equivalent In Situ Islet Regeneration at Both Sites
In the present studies, we demonstrate that an effective
therapy can utilize TNF-alpha induction of CFA combined with irradiated
or live MHC class I-matched cells. This combination cures diabetes in
over 78% of treated NOD hosts. In addition,
we demonstrate below that functional pancreatic recovery was slower in
the pancreas of cohorts receiving irradiated cells expressing MHC class I
self-peptide, although long-term and stable recovery occurs at equal
frequency with follow-up in excess of
120 days. To assess the long-term resistance of transplanted islets
compared to re-grown endogenous pancreas islets to disease, additional
sets of diabetic NOD cohorts received either live or dead MHC class I
and peptide expressing splenocytes combined
with syngeneic islet transplants, which served as a glucose clamp. The
ectopic islet transplants under the renal capsule allowed us to evaluate
disease recurrence and ectopic islet regeneration compared to that seen
in the pancreas. Fluorescence
immunocytochemistry was used to compare the pancreatic islets to the
subrenally placed islets. In these experiments, we utilized a
combination of staining to insulin and BrdU to quantify the
proliferating islet mass at the two sites and to determine a
possible difference in resistance of transplanted islets and endogenous
pancreatic islets to recurrent disease. We can also quantify possible
proliferation of islet cells and/or their precursors at the two sites in
two successful therapy versions.
Since there is speculation that transplanted islets without their
locally adjacent pancreatic precursor cells are end stage cells, these
experiments tested the hypothesis that long-term islet survival might be
an exclusive pancreatic trait. These
long-term NOD cohorts were compared to severely diabetic NOD mice which
had received subrenal syngeneic islet transplants 8 days prior to the
experiment, but without the desirable CFA and MHC Class I and
self-peptide therapy. Hematoxylin and eosin
staining of both subrenal and pancreatic islets of a recently diabetic
NOD mouse showed impressive and large lymphoid infiltrates, almost
totally obliterating the newly transplanted NOD islets, and similarly
invasive lymphoid obliteration of the
pancreatic islets. Moreover, the corresponding inspection of both the
renal and pancreatic sites for insulin positive cells revealed an almost
totally negative result. The staining for proliferating cells assessed
by BrdU at both sites also showed the
lack of islets in the pancreas and a lack of any proliferation, which
suggested that invasive and autoaggressive insulinitis have recurred or,
alternatively, that the ongoing disease was not due to local lymphoid
proliferation, but rather a migration of
these autoaggressive cells to the islet site. In other words, the BrdU
positive cells were not more highly positive in an active rejection
response, suggesting the active cells migrated to the site.
As presented above, each pancreatic section showed that NOD
hosts receiving irradiated MHC class I and self-peptide expressing cells
have healthy pancreatic and subrenal islet cells that are surrounded
with impressive circumferential lymphoid
infiltrates. These lymphoid accumulations do not progress to an
invasive islet pattern even with long-term follow-up, nor do they appear
to enlarge in the long-term. The immunocytochemistry of the islet
shows that successfully treated NOD mice have
insulin positive cells subrenally and in the pancreas. Furthermore,
within the islet mass of both the pancreas and subrenal site of
long-term corrected NOD hosts, infrequent but proliferating insulin
positive cells were observed as demonstrated by the
yellow cells clearly indicating co-staining with insulin and BrdU.
Since we used two different dyes to co-stain insulin and BrdU (i.e., red
and green, respectively) a co-staining cell is yellow. In addition,
based on the reported belief that fully
differentiated islet beta cells do not proliferate, and instead are
generated from progenitor cells, the insulin co-staining with BrdU
(i.e., the yellow color seen by immunocytochemistry) likely represents a
precursor cell in a proliferative phase.
These results demonstrate that long-term endogenous and ectopic subrenal
islet survival is possible after the underlying autoimmunity is
reversed. Importantly, in view of our analysis of this very late stage
after the successful reversal of disease,
islet regeneration defined by BrdU and insulin co-staining can occur,
although at a low frequency, in the pancreas and in subrenally
transplanted syngeneic islets.
Using similar immunohistochemical techniques, we also examined
the pancreata from long-term corrected cohorts to determine if the
insulin secreting beta cells in the pancreas were solely due to
regeneration of the pancreas from endogenous cells
or if the regenerated pancreatic islets could also have originated from a
donor source (e.g., from the injected splenocytes). In these
experiments, we used formerly diabetic NOD mice cohorts that, after the
onset of severe hyperglycemia, (i) were
treated with CFA and fresh F1 splenocytes from male donors administered
in biweekly injections for 40 days, (ii) were implanted with a subrenal
syngeneic islet transplant for 120 days, and (iii) remained
normoglycemic in the long-term when the transplant
was removed. These mice were subsequently sacrificed at varying time
intervals of stable normoglycemia, usually greater than 60 days. The
pancreata of these animals were compared to the pancreata of animals
that received the same treatment regimen,
except that they received irradiated male donor splenocytes administered
in biweekly injections for the 40-day treatment period. These
pancreata were then stained with two-color immunofluorescence in which
insulin was tagged with a red fluorochrome and
a Y chromosomal marker was tagged with a green marker. All splenocyte
donors were of male origin; therefore this fluorescence assay was used
to determine if any of the insulin positive cells in the pancreas were
of male Y chromosome origin. We
furthermore performed insulin co-staining to prove that the Y
chromosomes that can be seen in the islet were of islet origin, and were
not of donor lymphoid origin (FIG. 1). Yellow cells indicated the
co-staining of insulin and the Y chromosome marker
in a single positive cell: yellow cells were only seen in cohorts that
received live F1 splenocytes and not seen in the pancreatic islets of
cohorts that received irradiated donor stem cells.
Furthermore, the double-positive (yellow) islet cells of donor
origin with Y chromosome staining were only seen in the endocrine
tissues of the pancreas, and not in the exocrine tissues, suggesting
that the regeneration had occurred only in the
target tissue that was injured. Moreover, in the animals that received
irradiated cells, no green positive cells (i.e., Y chromosome containing
cells) were seen either in the insulin secreting tissues of the
pancreas or in the exocrine tissues of the
pancreas. Furthermore, the cohorts that received irradiated cells also
never expressed yellow cells in the exocrine or endocrine tissues of the
pancreas, therefore confirming that Y chromosome positive cells were
not present in animals that have
received irradiated cells as part of their curative regimen.
Histological analysis of the islets that contained cells of
donor origin revealed that, at times, whole islets were of donor origin
and at other times the peripheral beta cells of the islets were mostly
of donor origin. Overall, in a typical
pancreas, up to 30% to 50% of the entire islet population of the
pancreas appeared to be of donor origin suggesting that this was not an
occasional phenomenon of differentiation of blood into islet origin, but
was actually quite a dramatic finding. All
these immunohistochemical data were derived from cohorts with long-term
normoglycemia, as determined usually around 120 days after the original
islet transplant or after the original splenocyte injection.
Experiments performed on NOD mice for the regeneration of
pancreatic islets have revealed a number of transcription factors that
are beneficial for the methods of the invention and a number of protein
expression patterns that are signatures of
organ/tissue regeneration. NOD mice have at the site of vigorous islet
regeneration increased VEGF expression, increased Flk-1 expression, and
locally high levels of proteasome function, including high levels of
LMP-2 and INF-gamma. To accelerate the
regeneration process, agents such as TNF-, TNFR agonists, or gamma
interferon can be administered to the host prior to the initiation of
regeneration. The administration of cytokines that induce
TNF-expression, IL-1 expression, HAT, NF-B, AP-2, EGF-1,
Sp1, AP-1, GATA, PECAM-1, activator protein-2, CT-rich Sp1 binding
activity, PDGF-A, PDGF-B, monocyte chemoattractant protein-1, TF, Ets1,
SCL/Tal-1, FGF, HATs P/CAF, PDGF, CBP/p300 and HIF-2-alpha (HRF, EPAS,
HLF) can also be useful for the acceleration
of islet regeneration. In certain cases, islet regeneration can be
aided by the administration of VEGF, VEGF fragments, FGF, IGF-1, or by
BV endothelium differentiation or tissue regrowth.
In other cases, one or more death receptors (e.g., the death
receptors listed in FIG. 5) are inactivated on the donor cells or one or
more intracellular signaling proteins that mediate cell death are
inactivated in the donor cell to prevent
death of the transplanted cells. For example, FLIP can be used to down
regulate Fas/FasL expression. In other embodiments, extracellular
inhibition or reduction in IL2 (e.g., inhibition due to chemicals or
antibodies) is used to upregulate FLIP which
then down regulates FAS. In other embodiments, the donor cells have a
blockage of IL2R, such as the binding of a chemical (e.g., a non-lytic
antibody fragment) to IL2R to inhibit binding of IL2 to IL2R and thus
IL2-mediated upregulation of FAS. In
other embodiments, one or more members of the intracellular pathway for
FAS activation are inhibited in the donor cells prior to transfer.
Examples include the inhibition of the translation of transcription
factors such as cFOS, cJAN, PKC, Lck, Zap70,
MAPK, Itk (IL-2 inducible T cell kinase) and JNK. In particular
embodiments, the transcription or translation of transcription factors
is transiently inhibited with antisense oligonucleotides or by RNA
interference (RNAi).
Promotion of islet regeneration can be accomplished using one
agent, or more than one agent, administered with or without pluripotent
cells. The progress of islet regeneration can be monitored using
sequential RT-PCR analysis to probe for the
induction or suppression of transcription factors after agent
administration.
Example 7
Donor Derived Cells are Also Present in the Blood
Because of our dramatic findings in the pancreas of donor
origin F1 cells turning into pancreatic islets, we also serially
examined both the blood and splenocytes from these cohorts to see if the
blood and splenocytes were also of donor origin.
Approximately eight cohorts of this long-term description were examined
for the presence of K.sup.b positive lymphoid cells in the peripheral
blood; splenocytes at the time of sacrifice were also examined. As is
noted above, K.sup.b cells must be of B6
origin because the NOD mouse is of K.sup.d origin. We analyzed
peripheral blood lymphocytes from these cohorts using flow cytometry
analysis and found that in the peripheral blood 12.6%, 8.3%, 10%, 0.9%,
4.4%, and 5.8% of the lymphocytes were of donor
origin. In contrast, a cohort that received irradiated cells, in which
staining would only represent endogenous staining (i.e., background
staining), had 2.9% of lymphocytes of donor cell origin. Thus, many of
these cohorts had a percentage of donor
origin lymphocytes in the peripheral blood that was significantly above
background and had long term co-existence of blood cells of two
different genetic origins and pancreas cells of two distinct genetic
origins.
To better define this co-existence of donor derived and
endogenous cells without immunosuppression, skin transplants were also
performed on these long-term cohorts from the B6 donor. We had presumed
that since there was blood chimerism and now
pancreatic chimerism, the skin graft would survive long-term. To our
surprise, skin graft survival from the B6 cohorts was not prolonged, or
not visibly prolonged, in cohorts that retain stable blood and
pancreatic islet chimerism, indicating that this
sort of chimerism is distinct from the chimerism that results from total
body irradiation followed by bone marrow reconstitution.
Nonetheless, the methods described herein provide a remarkable
way to transplant cells without the need for immunosuppression. In view
of the standard knowledge in the field of transplantation prior to the
present invention, donor cells that
not only are chimeric--being of donor male origin bearing disparate MHC
genes and remarkably turning into pancreatic islets--but also are
semi-allogeneic would be expected to be rejected because, while the host
received CFA or TNF-alpha, the host did not
receive immunosuppressive treatment. However, as is shown by our
results, we were able to maintain long-term chimerism. In many ways the
stable chimerism that could persist beyond 180 days after therapy
termination mimics pregnancy where fetal origin
F1 cells can survive long-term in mothers, long after the fetus has been
removed.
Example 8
Organ Regeneration in GFP C57BL/6 Mice
As noted above, the data described herein using mice with
established diabetes (e.g., NOD mice) demonstrate the ability to re-grow
islet cells in the pancreas. The experimental results are excellent
and demonstrate a robust and sustained
ability to achieve engraftment. To try to duplicate these results, and
to determine the parameters that allow this remarkable phenomenon to
occur, we set up a test system to define the parameters that allow the
NOD mouse to re-grow its islets from donor
blood cells. The test model used cells from GFP BL/6 (B6) mice
expressing green fluorescent protein (GFP) in all tissues as donor cells
for introduction into B6 cohorts. Initially, we used GFP B6
splenocytes injected into normoglycemic hosts. We then
examined these hosts at varying intervals for pancreatic, lung, and
blood chimerism. After 90 days, no chimerism of the donor origin was
visible. Based on these findings, we decided to test the possibility
that the host pancreas needs to have an insult
(e.g., the co-administration of streptozotocin to allow the GFP positive
B6 donor lymphocyte cells to target the pancreas and also regenerate
it). Therefore, GFP positive B6 cells from splenocytes and bone marrow
(Hoechst 33342/SP positive cells)
obtained by flow cytometry and hepatocyte origin cells were administered
at doses of 5.times.10.sup.5 to 5.times.10.sup.7 cells over a 40-day
period, and the cohorts were then examined after 40 to 195 days either
by eye bleeds or by sacrifice followed by
examination of splenocytes.
In these experiments, although there was injury to the
pancreas, there was little persistence of long-term chimerism in the
host animals. Occasionally, a pancreas positive cell of GFP origin was
observed, but the data were in large part
negative suggesting that we had not properly duplicated the experiments
that were so successful in the NOD mouse. One potential reason for the
lack of success of this experiment or for the success of the experiments
in NOD mice is that although we had
induced injury in the pancreatic islets, these animals were severely
hyperglycemic. Based on our previous data, severe hyperglycemia
hampered regeneration.
To determine if severe hyperglycemia was interfering with the
regeneration of the pancreas, we repeated the experiments using
streptozotocin induced damage and a glucose clamp with subrenal islets
and then used donor splenocytes or donor bone
marrow from GFP positive B6 donors. In response to this treatment, the
chimerism was still partial, not long-term, and did not represent the
striking regeneration of the islet tissue.
We further optimized the treatment by administering
streptozotocin to another set of B6 cohorts, inducing the glucose clamp
with subrenally transplanted syngeneic islets, and co-administering
TNF-alpha or CFA concurrent with the donor lymphoid
cell injection. We used this protocol because we thought that we needed
injury to islets to result in high TNF Receptor 2 expression on the
islets or growth receptors to perhaps promote regeneration of the
endogenous pancreatic islets. Furthermore, we
thought that TNF Receptor 2 and progenitor cells from the blood might
also promote endogenous GFP positive B6 islet regeneration and that CFA
and TNF-alpha might be beneficial in another manner. We had previously
obtained data indicating that CFA or
TNF-alpha induces severe transient lymphopenia in the host, which is
similar to data obtained in human clinical trials. Therefore, we
injected the six hosts, not only with streptozotocin and an islet
transplant, but also with CFA or TNF-alpha to induce
the severe transient lymphopenia that might promote the peripheral blood
chimerism. In addition, we injected splenocytes biweekly for 40 days.
The results of these experiments looked much more promising, as 2-10%
chimerism was detected in the
peripheral blood 180 days after the completion of the injection and,
furthermore, GFP positive cells of donor origin, although rare, were
vividly expressed in the islets of the pancreas.
In short, in the syngeneic situation, splenocytes
differentiated into insulin secreting beta cells, fused with beta cells,
or provided factors for regeneration. We determined that, in the
C57BL/6 host, CFA or TNF-alpha is desirably not
administered concurrently with the donor cells. Therefore, these
experiments using syngeneic transplants instead of allogeneic
transplants and using an artificial model of islet injury suggest that
target organ injury or active disease promotes the
regenerative process after the elimination of the disease. A
metabolically normal state is also important and may need to be
maintained, as severe hyperglycemia appeared to interfere with the
effectiveness of this treatment. Our results also indicate
that TNF-alpha or CFA may facilitate the effectiveness of the treatment.
These results likely represent a dual effect, not only of CFA's
elimination of autoimmunity in the NOD mouse, but also of CFA's
induction of severe lymphopenia, which, in turn may
promote the chimerism of donor cells, as well as the subsequent
chimerism and differentiation in the pancreas. Also, induction or
administration of TNF-alpha has a beneficial effect on the target tissue
or precursor cells promoting regeneration.
Furthermore, it is known that the best induction of host regeneration,
based on percentage success rate (92% vs. 72%), as well as the percent
degree of chimerism/regeneration (approx. 87% vs. 54%), is still
obtained from the administration of CFA,
which is somewhat superior to the administration of TNF-alpha alone. It
should be noted that once animals are successfully treated with either
agent, the stability of disease reversal is equivalent. Although these
results could be due to dose response
phenomena, it is also observed that the simultaneous induction of
INF-gamma with CFA is of direct benefit in conditioning the host
vascular endothelium for recapitulating a regenerative program. Indeed,
INF-gamma induces both high LMP2 and other
proteasome subunits that promote vascular leakiness, a necessary step to
presumed mesodermal cell migration and differentiation.
While the above experiments relate to the treatment of
diabetes, these techniques obviously also are applicable to other
diseases where host repair is desirable, providing new ways to
transplant cells without the need for immunosuppression.
Example 9
Organ Regeneration in GFP C57BL/6 Mice
As noted in Example 7, additional experiments were performed
using normal mice that are not of the NOD genotype to further understand
and characterize the remarkable re-growth of islet cells observed in
NOD mice.
FIG. 9 summarizes the many C57BL/6 mice that were treated with
various therapies to achieve similar donor cell engraftment and possible
re-growth of an adult organ/organelle such as the islets of Langerhans.
All of these C57BL/6 hosts were made
diabetic with streptozotocin using standard methods. In these
experiments, glucose levels were not regulated using insulin injections
or a temporary glucose clamp. If desired, insulin injections or a
temporary glucose clamp may be used in any of the
methods described below to optimized islet cell regeneration. It has
been observed by us that in the NOD host, diabetes or late stage islet
destruction is necessary for islet regrowth and thus to create a similar
model of injury of the pancreatic islet,
injury was induced with streptozotocin prior to the introduction of
donor cells.
The Group 1 mouse was a female C57BL/6 mouse that received
donor male splenocytes from a syngeneic C57BL/6 donor with GFP-actin
fluorescence (C57BL/6-GFP). Thus, the donor cells can be distinguished
from endogenous cells because the donor cells
are of male origin (i.e., have XY chromosomes) and the endogenous cells
are of female origin (i.e., have XX chromosomes). Additionally, the
cells exhibit GFP fluorescence that is easily detectable using flow
cytometric analysis. Removal of blood from
this host and analysis of PBLs revealed that the peripheral blood only
had 0.59% GFP cells. This demonstrates that the introduction of cells
of splenocyte origin was not sufficient for establishing high levels of
chimerism under the conditions tested.
Also, the 0.59% value represents the degree of chimerism approximately
four days after the final bi-weekly injection of donor splenocytes
expressing GFP suggesting low levels of C57BL/6-GFP cells remained.
Group 2 C57BL/6 mice were treated as described for the Group 1
host. The spleen and the PBL of the treated mice were analyzed 150 days
after treatment began. This treatment regimen involved bi-weekly
injections of 10.sup.7 cells for the first
40 days after treatment. The PBLs of the group 2 hosts also had low
levels of donor cells. The spleen had slightly higher but still low
levels of C57BL/6-GFP cells. A subset analysis seemed to suggest the
blood cells expressing GFP were not confined
to any select lineage. Group 2 910, 911, and 903 NOD hosts were
reanalyzed by regating the flow pictures of fluorescence and had similar
trends of low levels of chimerism.
Group 2 mice 931, 939, 932, and 933 were also studied 87-98
days after transplantation of C57BL/6 splenocytes. The spleen of these
animals had slightly higher degrees of chimerism with ranges of
4.7-13.3%. Although this result suggests a
detectable level of chimerism, this chimerism was not long lasting
because by an additional 100 days, the degree of chimerism was again
low.
Group 3 and 4 C57BL/6 mice only differed from Group 2 and 3
mice in that Group 3 and 4 mice received donor bone marrow cells instead
of splenocytes. Splenocytes were better able to engraft into the host
than bone marrow donor cells. Group 5
C57BL/6 hosts received Hoechst333242 positive splenocytes; a cell type
that is alleged by the scientific literature to poses stem cell traits.
The transfer of these cells into the C57BL/6 hosts was only minimally
successful and less successful than
donor bone marrow or bone splenocytes.
Lastly, Group 7 and Group 9 C57BL/6 hosts received 10.sup.7 CNS
precursor cells or hepatocytes (HC), and the hosts were killed
approximately 100 days after cell transplantation. The spleen of hosts
demonstrated more engraftment than the PBL,
and donor CNS cells or donor hepatocytes may be better able to engraft
into the host than bone marrow or Hoecchst33342 cells.
It should be noted for all these experiments in all groups we
never saw with donor cell treatment reversal of the diabetes and we did
not observe above background levels a clear regeneration of the islets
in the pancreas. The pancreases
possible regrowth, even temporary, would probably not have been detected
with this experimental design because the killing of the mice was late
in most cases. Future experiments were thus conducted to see if like
the NOD a simultaneous tight control of
blood sugars was necessary to promote islet regrowth during the
experimental observation period and to perpetuate the chimerism in a
target organ.
In the NOD model of disease reversal and islet re-growth, the
data show that diabetic NOD mice that receive both CFA and donor F1
splenocytes exhibit islet regeneration (FIG. 10). A glucose clamp was
used to regulate glucose levels and enhance
islet regeneration. The data show that even very low dose TNF-alpha
(e.g., doses of 2 ug/bi-weekly) can also promote the reversal of disease
process. Further experiments revealed that the substitution of CFA
with TNF-alpha required TNF dosing of 10-20
ug/bi-weekly. The NOD data also clearly shows that CFA alone or donor
splenocytes alone were without long lasting effect at either disease
elimination or islet regrowth at the time periods examined.
The second part of FIG. 10 now attempts to translate the NOD
success story of organ regeneration into a C57BL/6 model of
regeneration. This has helped to define the critical elements that
promote regeneration. In all of the C57BL/6 mice in
these groups, streptozotocin was used to induce tissue injury and to
make the mice diabetic. As noted above, tissue injury promotes
re-growth. Again, the groups that appear to have target organ
regeneration are the groups that receive donor splencoytes
plus TNF-alpha. In these experiments we try to map the pathway or
receptor for regeneration as involving receptor I or II. At least for
receptor II stimulation with the use of a C57BL/6 mouse with a mutation
that inactivates TNF-.alpha. receptor I, we
can see the persistence of the islet regeneration to a certain degree
suggesting the islet regeneration may be promoted by this later pathway.
The complete experiment could not be done in the reciprocal fashion
because even very low dose TNF-alpha
administered to a C57BL/6 RII-/- mouse resulted in immediate death;
TNF-alpha toxicity may be through this receptor I, at least in control
mice.
The last portion of FIG. 10 examines the effect of TNF in NOD
mice in transiently promoting islet regrowth and the rapid elimination
of invasive insulitis. With escalating doses of TNF, one can see not
only the elimination of invasive insulitis
but also islet regeneration. These NOD cohorts were typically examined
about 40-50 days at the end of the TNF treatment course. Based on
examinations of histological sections for TNFR11 expression,
regenerating islets demonstrated up-regulation of this
receptor while there was still some tissue injury. This up-regulation
of TNFR11 may promote the beneficial effect of TNF in regeneration. For
example, 20 ug dosing of TNF eliminated all the insulitis and resulted
in regeneration of the islets to the
most significant degree. Examination of NOD mice being treated with 20
ug TNF at earlier times prior to the end of the 40-day period would like
reveal high TNFR11 expression that is eliminated by the end of the
40-day period because islet regeneration
is complete. Also, treatment of NOD mice with human-TNF-, an agonist of
only TNFR1 in the mouse, resulted in no islet regeneration, suggesting
the beneficial effect of TNF- on organ regeneration was a function of
the effect of TNF- as an agonist of
TNFRII.
FIG. 11 summarizes the diverse experiments and outcomes
depending upon the host representing an NOD mouse or a normal C57BL/6
host. The use of cells of splenocyte origin, blood origin or HC may
offer an advantage because these organs contain
diverse cell types and the re-introduction of mobilized, but not yet
fully differentiated, endothelium, mesoderm, or ectoderm may promote,
facilitate, or speed the necessary recapitulation of fetal tissue
interactions that promote organ regeneration in
an adult. The following data support the above hypothesis. During
normal embryonic pancreatic islet development, the mesoderm interacts
with the BV endothelium (endoderm). This interaction may promote VEGF
expression, as well as the upregulation of
Flk-1 receptors. To promote this process of organ specific regeneration
in an adult, a number of steps are desirably followed. First, cells of
the original developmental contact are desirably administered by IV
injections or applied directly to the
site of regeneration. For regenerating islet cells, blood vessel (BV)
endothelium is desirably primed at the regeneration site by promoting
the embryonic expression of VEFG, NF-B or associated events, such as
increased proteasome activity or TNFR2
expression, and then contacted with administered mesodermal cells, even
if of adult origin. For example, injected mesodermal cells may contact
endogenous endoderm (e.g., endodermal cells within the pancreas or
within other areas of the body), which
promotes the recapitulation of the fetal patterning, i.e. the BV
endothelium plus endoderm budding produces islets of liver cells.
Indeed, in this particular clinical setting, the power of donor
splenocyte origin cells in promoting regeneration may be
more attributable to the mesodermal cells of the spleen than the more
abundant blood cells. For re-growth of other tissues, administration of
ectoderm, mesoderm, and/or endoderm may be desirable. Furthermore, for
target organ re-growth, transient
up-regulation of VEGF may be desirable. This up-regulation may be
induced, e.g., by administering TNF-alpha, INF-gamma, or inhibiting
cAMP. Also, administration of IL-2 may promote TNF-alpha that
subsequently binds to BV endothelium, triggering VEGF
up-regulation and NF B up-regulation, and thus target organ
regeneration. Since TNFR2 is preferentially expressed on endothelial
cells, this receptor is desirably manipulated for target organ
regeneration. The ability of the NOD mouse to regenerate
islets as demonstrated herein may be attributable, at least in part, to
the islet specific up-regulation of the LMP2 subunits of the proteasome.
Up-regulation of LMP2 is very influential in promoting
VEGF/Flk-1/TNF-effects, with NF-B upregulation, as we
now demonstrate by its diminished effect in LMP2-/- mice. We have
demonstrated this regenerative process to be promoted in the NOD mouse
and eliminated in the LMP2-/- mouse, thus verifying this pathway.
If desired any of the above regeneration methods may be
enhanced by administering the donor cells more frequently and/or for a
longer length of time.
Example 10
Assay Development of Human Diabetic Peripheral Blood Lymphocytes
As the relative efficiency of donor NOD splenocytes in
transferring autoimmune disease is well known and NOD blood is very
inefficient as a source of lymphoid cells in transferring disease to
naive cohorts, the magnitude of apoptosis induced by
TNF-.alpha. in pathogenic NOD T cells from peripheral blood compared
with the effect in T-cells from NOD splenocytes was quantified.
As Table 4 shows, accelerated cell death in NOD splenocytes,
measured as both early and late apoptosis, resulted in 46% cell death.
The effect on peripheral blood lymphocytes (PBLs) in the same NOD mouse
was only 12% induced apoptosis. The
distribution of pathogenic apoptotic sensitive cells appears to be lower
in peripheral blood and higher in the spleen.
TABLE-US-00004 TABLE 4 TNF- sensitivity of PBLs vs.
splenocytes in NOD mice* Apoptosis of T Cells (%) TNF- Spleen PBLs 0
ng/mL 12.1 12.1 12.2 15.6 20 ng/mL 11.8 22.6 11.5 17.7 *Apoptosis of T
cells represents early and late apoptosis defined as
Annexin V+ PI+ and Annexin V+ PI- cells on CD3+ T cells using flow
cytometric studies
The data in Table 5 show the degree of accelerated T cell death
of human diabetic PBLs with culture and with TNF-.alpha., as measured
by flow cytometry. Apoptosis was quantified by flow cytometric
monitoring of Annexin V, with or without
propidium iodine staining, after a 12 hour in vitro culture or exposure
to TNF-(20 ng/mL), TNF-with Act D (1 ng/mL), or other protein synthesis
inhibitors known to amplify pro-apoptotic pathways of TNF-signaling by
inhibiting the rapid synthesis of
proteins that are anti-apoptotic. All assays were performed on freshly
isolated PBLs and simultaneously prepared control samples. Both early
and late apoptosis results were pooled for these data, but early and
late apoptosis each was sufficient by
itself in each category in Type I diabetics to yield highly
statistically significant values of accelerated death through culture
with TNF-. With flow cytometric data, profound changes in the relative
mean death can be observed on any given day, so
patient samples were always simultaneously studied and compared to
paired t tests to control samples. The magnitude of the TNF-.alpha.
induced apoptotic defect in humans is detectable with current flow
cytometry techniques (8-10%) and is consistent
with the results in PBLs in the NOD mouse. The 55 type 1 diabetic
patients had higher death of naive T cells (with culture) compared to 55
paired random (no history or family history of autoimmune disease)
controls (p=0.0008). Actinomycin D is an
accelerator of apoptosis when used with TNF-.alpha.. As shown in FIG.
5, TNF-.alpha. and TNF-.alpha. plus actinomycin D (p=0.0007) induced
apoptosis were also significantly greater in the human diabetic T cells
than in the control T cells (p=0.0154
and p=0.0007, respectively). The data suggest that the defect is
widespread in Type 1 diabetes, with the majority of patients showing a
detectable abnormality in T-cells (with a relatively larger fraction of
T-cells with heightened TNF-.alpha.
sensitivity).
It should also be mentioned that, similar to the NOD mouse,
there appears to be two death-mediated events, a spontaneous death of
cells with tissue culture preparation and a direct TNF-induced death of
select T cell subpopulations. The
spontaneous cell death maps to the monocyte/macrophage lineage of cells
and the direct TNF-death maps in both species of T cells. The
spontaneous death could be due to receptor activation of a death
receptor due to shear stress or, alternatively, the
elimination in the autoimmune patient of an abnormal serum factor that
is abnormally pro-life or anti-apoptotic.
TABLE-US-00005 TABLE 5 TNF-induced apoptosis of peripheral
blood lymphocytes of Type I and Type II diabetics compared to controls
Com- Paired Mean Mean % Paired t parison Samples Conditions (patient)
(control) Change test Type I 55 Culture -
28.8 26 10% 0.0008 diabetic 12 hrs vs. Control Type I 55 TNF 29.6 27.2
8% 0.0154 diabetic vs. Control Type I 55 TNF + 42.8 39.2 10% 0.0007
diabetic Actinomycin vs. D Control Type II 18 TNF 26.5 26.9 1% 0.9422
diabetic vs. Control Type II TNF + 38.8
38.1 -2% 0.5702 diabetic Actinomycin vs. D Control
Example 11
Treatment, Stabilization, or Prevention of Disease Other than Diabetes
NOD mice also suffer from other autoimmune diseases in addition
to diabetes, such as rheumatoid arthritis, lupus, multiple sclerosis,
Sjogren's syndrome, multiple sclerosis, and autoimmune hemolytic anemia.
In particular, the methods of the
invention also improved symptoms associated with these other autoimmune
disease and/or stopped progression of these diseases in NOD mice. The
following treatments have been tested and shown to enhance regrowth of
salvary glands, decrease hemopoetic
abnormalities, stop the progression of multiple sclerosis and rheumatoid
arthritis, and reduce levels of lupus autoantibodies: (i) biweekly
injections (i.v.) of 10.sup.7 splenocytes expressing MHC class I and
peptide for 40 days, (ii) biweekly injections
(i.p.) of 2, 4, 10, or 20 .mu.g TNF.alpha. or IL-1 for 40 days, (iii) a
single injection of 5 .mu.L in one footpad of 1 mg/mL solution of BCG,
(iv) a single injection of CFA, (v) combined treatment with splenocytes
and TNF.alpha. at the above doses,
and (vi) combined treatment of splenocytes and CFA at the above doses.
Mice such as C57BL6 mice can also be used as animal models for
the regeneration of other cells, tissues, or organs such as skin, liver,
or brain cells.
Example 12
Factors Affecting the Efficiency of Organ Regeneration
Our data using GFP mice also demonstrated that, as we repeated
these experiments with many different types of injected donor cells,
differences exist not only in the degree of peripheral blood chimerism,
but also in the persistence of peripheral
blood chimerism induced by these different donor cells. As is noted
above, the GFP positive donor cells that we obtained and injected
included splenocytes, bone marrow derived cells, Hoechst 33342 positive
cells obtained by cell-sorting, brain cells,
CNS derived cells, and hepatocytes. Based on analysis of peripheral
blood lymphocytes after sacrificing the animals for analysis of
splenocytes, the duration of the chimerism in the absence of CFA
treatment was dramatically different for different cell
types. It turns out that, of the different cell types tested,
splenocytes maintained the highest degree of chimerism for time periods
greater than 100 days. In comparison, donor bone marrow cells were less
effective, and the other cell types were least
effective, suggesting that the donor cell origin even from the adult
donors may also have an impact in the persistence of the chimerism.
In autoimmune hosts, the administration of any of a multitude
of cytokines induce death or apoptosis of a subpopulation of pathologic
lymphoid cells due to these cells having intrinsic errors in resistance
to apoptosis or cell death.
Accordingly, this treatment eliminates the pathologic cells from the
host without harming the endogenous cells. In addition, introduced and
endogenous cytokines promote the regeneration process of a damaged
target organ. If a target organ is inflamed,
is exposed to exogenous cytokines, or has increased proteasome activity,
such as increased activity due to the overexpression of the LMP2, LMP7,
or LMP10 subunits, a gamma responsive gene, or a TNF-alpha responsive
gene, the target organ regenerates at
an exponential rate. An increase in proteasome activity is likely to
play a role in the action of VEGF, which together with the VEGF
receptors Flk and Flt, functions in organ regeneration. Studies have
shown that VEGF binds to developing organs and
that this promotes end organ regeneration, possibly by binding to Flk or
Flt receptors. We have shown that augmented proteasome activity
results in augmented VEGF activity. In view of these results, in an
autoimmune host, it is likely that, once
disease is removed and in situ proliferation is desired, stem cells home
to the target organ that had been under autoimmune attack and
preferentially proliferate in this organ. The upregulation of
proteasome activity and/or the upregulation of
proteasome subunits with gamma interferon may promote this. In
addition, gamma interferon may be used in a non-autoimmune host with
tissue damage to promote targeting of this damaged tissue by stem cells.
Furthermore, other chemicals and cytokines that
also promote proteasome activity may be used in methods of organ
regeneration. For example, in such methods, a promoter of proteasome
activity may be administered concurrently with, prior to, or after
administering stem cells or lymphocytes obtained
from adult blood. After the addition of stem cells, local regeneration
may be promoted by increasing Flk-1 receptors via CREB inhibition or by
TNF-, HAT or NF B activation, or by administration of VEGF inhibitors.
VEGF secretion may be promoted by
proteasome augmentation, TNF-administration, cAMP inhibition, by the
administration of IL-1 or IL-2, or by the application of sheer stress.
Example 13
Exemplary Agents for Use in the Methods of the Present Invention
Select autoimmune cell death can be achieved by administering
agents that disrupt the pathways that normally protect autoimmune cells
from cell death, including soluble forms of antigen receptors such as
CD28 on autoreactive T-cells, CD40 on
B-cells that are involved in protection of autoimmune cells, and CD95 or
CD95L (i.e., FasL) on T-lymphocytes. Other such agents include p75N
TNF and lymphotoxin Beta receptor (LtbetaR). Also, antibodies or
fragments of antibodies reactive with these
receptors are useful therapeutics. Such agents are described in the
literature.
The present invention is not limited to a combined TNF-inducing
therapy or direct compound administration that includes the combination
of TNF-alpha and IL-1, but includes, e.g., any combination of
TNF-alpha-including therapies, e.g.,
vaccination with BCG, viral infection, LPS, activation of cells that
normally produce TNF-alpha (i.e., macrophages, B-cells, and T-cells),
administration of the chemotactic peptide fMet-Leu-Phe, administration
of bacterial and viral proteins that
activate NF.sub..kappa.B, administration of agents that induce signaling
pathways involved in adaptive immune responses (i.e., antigen receptors
on B- and T-cells, CD28 on T-cells, CD40 on B-cells), agents that
stimulate specific autoreactive cell death
receptors (i.e., TNF, Fas (CD95), CD40, p75NF, and lymphotoxin
Beta-receptor (LtbetaR), and administration of substances that stimulate
TNF-alpha converting enzyme (TACE) which cleaves the TNF-alpha
precursor (i.e., to provide biological activity capable
of stimulating enhanced production or enhanced cytokine life after
secretion). Such agents are described in the literature.
In a preferred embodiment, monoclonal antibodies that serve as
TNF-agonists can be administered. Such antibodies can be made using
tumor necrosis factor-alpha receptor 1 (TNFR1) or tumor necrosis
factor-alpha receptor 2 (TNFR2) as immunogens in
mice using the hybridoma method first described by Kohler &
Milstein, Nature 256:495 (1975). Such antibodies can also be made by
recombinant DNA methods [Cabilly, et al., U.S. Pat. No. 4,816,567].
Such antibodies have been prepared and described by
Brockhaus, et al., in Proc. Nat. Acad. Sci. USA 87:3127-31 (1990).
Among the antibodies produced, those with agonist activity are
identified by screening for TNF-like activity in assays measuring
cytotoxicity, fibroblast growth, interleukin-6
secretion, or activation of the transcription factor NF-B.
Alternatively, such antibodies can be screened in vitro using assays in
which agonists are identified by their ability to kill activated T-cells
obtained, for example, from a patient with
lymphoma or newly diagnosed type-2 diabetes.
Methods for humanizing non-human antibodies are well known in
the art. Generally, a humanized antibody has one or more amino acid
residues introduced into it from a non-human source. These non-human
amino acid residues are often referred to as
"import" residues, which are typically taken from an "import" variable
domain. In antibodies used in the methods of the invention, the import
variable domain is from the TNFR1 and TNFR2 antibodies produced above.
Humanization can be performed, for
example, following the method of Winter and co-workers [Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
and Verhoeyen et al., Science 239:1534-1536 (1988)], by substituting
rodent CDRs or CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such
"humanized" antibodies are chimeric antibodies (Cabilly, supra), wherein
substantially less than an intact human variable domain has been
substituted by the corresponding sequence from a
non-human species. In practice, humanized antibodies are typically
human antibodies in which some CDR residues and, in some cases, some FR
residues are substituted by residues from analogous sites in rodent
antibodies.
Humanized antibodies desirably retain high affinity for the
immunizing antigen, and thus are desirably prepared by known processes
involving analysis of the parental and humanized sequences by
three-dimensional modeling. Three dimensional
immunoglobulin models are commonly available and are familiar to those
skilled in the art. Computer programs are available which illustrate
and display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of the
residues in the functioning of the candidate immunoglobulin sequence,
i.e., the analysis of residues that influence the ability of the
candidate immunoglobulin to bind its antigen.
In this way, FR residues can be selected and combined from the consensus
and import sequence so that desired antibody characteristics, such as
increased affinity for the target antigen(s), are achieved. In general,
the CDR residues are directly and most
substantially involved in influencing antigen binding. For further
details see U.S. Pat. No. 5,821,337.
Alternatively, it is possible to produce transgenic animals
(e.g., mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, the homozygous
deletion of the antibody heavy chain joining region (J.sub.H) gene in
chimeric and germ-line mutant mice, resulting in complete inhibition of
endogenous antibody production has been described. Transfer of the
human germ-line immunoglobulin gene array in
such germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.
Natl. Acad. Sci. USA 90:2551-255 (1993); Jakobovits et al., Nature
362:255-258 (1993).
In the use of a TNF-receptor agonist antibody, patients are
dosed such that enough is administered to elicit a TNF-like effect. The
effective dose of such an antibody, or mixture of antibodies, is
determined by starting at a low dose to
ascertain tolerance, followed by dose escalation to produce the desired
changes in circulating lymphocytes. For example, therapeutic dosing can
be weekly or bi-weekly at levels of 0.025 mg/kg, 0.075 mg/kg, or 0.150
mg/kg (antibody/patient). Subsequent
to antibody administration, disease activity is monitored in each
patient category. For diabetes, monitoring may involve the tabulation
of the amounts of insulin necessary to maintain normoglycemia or a
positive trend in the reappearance of C-peptide
levels. All patients during and after monoclonal antibody therapy are
monitored for the presence of a human anti-murine antibody response to
the anti-TNFR antibodies, as well as a human anti-human response.
In another preferred embodiment, the invention allows for the
identification of drugs that induce cell death or selectively hamper the
autoimmune cells by binding to cell surface receptors or interacting
with intracellular proteins. For
example, drugs that stimulate the IL-1 pathway or drugs that interact
with converging pathways such as Fas, FasL, TACI, ATAR, RANK, DR5, DR4,
DCR2, DCR1, DR3, TALL-4, or THANK. Also accelerated cell death of
autoimmune cells maybe potentiated by adding
protein synthesis or kinase inhibitors. For instance, accelerated TNF
or FAS death is potentiated by brief exposure to a protein synthesis
inhibitor (e.g., ActD) that blocks a rapidly made TNF-alpha mediated
intercellular inhibitor(s). Similarly,
kinase inhibitors also potentate TNF-alpha mediated events. The drugs
of the present invention can be characterized in that they only kill
autoimmune cells having a selective defect in a cell death pathway which
can be characterized by two distinct
phenotypes, (1) defects in lymphoid education and (2) susceptibility to
apoptosis.
Other host treatment methods can be used as well to ablate
autoimmune cells, for example, administration of CFA, interleukin-1
(IL-1), proteasome inhibitors, TNF superfamily agonists, NF.kappa.B
inhibitors, anti-inflammatory drugs, tissue
plasminogen activator (TPA), lipopolysaccharide, UV light, or an
intracellular mediator of the TNF-alpha signaling pathway.
Example 14
Treatment
While the therapies described herein are likely to be effective
in treating pre-diabetics, i.e., patients diagnosed as progressing to
type I diabetes, but who are not yet hyperglycemic, we note that the
methods of the inventions also may be used
to treat a mammal, for example, a human with type I diabetes or any
other autoimmune disease. The ability to treat patients who already
have hyperglycemia and therefore have significant or total islet
destruction is a significant advantage of the
current therapy.
In general, before treating a patient, one may optionally
obtain blood from the patient to determine that the patient has two
disease phenotypes. The first disease phenotype is an increase in the
number of circulating CD45RA positive cells in
the blood (also defined as alterations in the number of cells positive
for CD95, CD62L, or other markers of naive or unstimulated cells).
CD45, CD95, and CD62L are all cell surface antigens that can be
monitored by flow cytometry and compared to age
matched controls. We expect to see an abundance of these naive or
unstimulated cells in the peripheral blood of subjects with diabetes or
any other autoimmune disease. The second phenotype is the presence of a
subpopulation of lymphocytes with
augmented sensitivity to cell death through apoptosis or necrosis. For
example, subpopulations of cells may have augmented sensitivity to cell
death caused by TNF-alpha, TCR receptor cross-linking agents, T-cell
specific antibodies (e.g., .alpha.TCR or
.alpha.CD3), or nonspecific stimulation with BCG. We may assay for the
presence of such cells by isolating lymphocytes from these patients,
treating them in vitro with TNF-alpha, and showing that the lymphocytes
contain a subpopulation that undergoes
apoptosis or necrosis when exposed to TNF-alpha, other cytokines,
chemical reagents, or antibodies to select surface proteins. Desirably,
control donor lymphocytes do not exhibit sensitivity to these agents.
This phenotype is a result of lymphoid cells
predominantly of pathogenic origin that have altered intercellular
signaling pathways, alterations which result in a heightened death
sensitivity. Elimination or conversion of all cells with this phenotype
is desirable for the permanent reversal of
autoimmunity. The penetrance of these defects is likely to be
relatively high in diabetic or other autoimmune patients, with the first
phenotype likely having a penetrance of over 95%, and the second
phenotype likely having a penetrance of over 50% in
type I diabetics.
Accordingly, before beginning to treat a subject with type I
diabetes or any other autoimmune condition, we may determine from blood
analysis alone whether the subject has either or both of these two
phenotypes and, therefore, is amenable to
therapy. To treat the first phenotype (i.e., an increase in the number
of circulating CD45RA positive cells) tolerance to MHC class I and
self-peptide may have to be re-established. We conclude from our
results that the lack of functional MHC class I
and self-peptide complexes causes the overabundance of naive T-cells in
the periphery or at least results in one of the phenotypes that causes
this. So for treating this phenotype, we can administer blood or bone
marrow that is a semi-allogeneic or
fully-allogeneic match to the MHC class I and self-peptide complex.
Furthermore, the blood or bone marrow derived cells, or even fibroblasts
that have been immortalized, desirably may have normal MHC class I and
self-peptide complex presentation; in
other words, they should not come from diseased patients. Those
phenotypes are easily monitored prior to treatment to determine the
suitability of the donor cells in this therapy. For example,
conformationally specific MHC class I and self-peptide
antibodies may be used to show that the complexes are properly filled.
In addition, we know that, in this aspect of the treatment, an increased
number of matches to the HLA class I alleles of the host results in an
increase in the duration of the
reversal of the disease. Desirably, at least two, and desirably all
four HLA class I alleles (e.g., the HLA A and HLA B alleles) from the
donor cells are matched. Accordingly, these donor cells may be
perfectly matched or they may be semi-allogeneic
(i.e., with only partial matches on individual cells).
Treatment may involve intravenous biweekly infusions of
1.times.10.sup.7 cells of any given donor of any given class I
haplotype. It is desirable for the administered cells to be freshly
isolated and not processed with preservatives or frozen.
Cells that may be used in the methods of the invention may be obtained,
for example, from a bloodbank. In addition, semi-allogeneic cells may
be obtained from a close relative of the patient, such as a parent or a
sibling. Furthermore, it would be
advantageous to have the red blood cells eliminated from the
preparations to decrease the volume of blood and lymphocytes
administered. We also determined that semi-allogeneic or
fully-allogeneic irradiated cells may be used in this therapy, but the
use
of irradiated cells results in a longer time course for correction.
As an alternative to administering MHC class I and peptide,
another agent that inactivates or kills naive T-cells can be
administered. Exemplary agents include antibodies that bind and
inactivate the T-cell receptor on naive T-cells or by
binding and triggering the selective death of only pathologic cells. In
some embodiments, the antibodies inhibit the activity of or naive
T-cells by at least 2, 5, 10, or 15-fold more than they inhibit the
activity of memory T-cells.
Simultaneously with the administration of donor cells, it is
also desirable to induce endogenous TNF-alpha production either through
stimulation with Bacillus Clamette-Guerin (BCG) or other immune
adjuvants such as CFA, or by the direct
administration of TNF-alpha. For example, one may administer BCG at
least biweekly or, desirably, three times a week. Again, one skilled in
the art can determine individually the dosing of the cells and
TNF-alpha or BCG by analyzing a blood sample
twice a week for evidence of the elimination of the phenotype of the
pathogenic cell. For instance, to determine the correct dose of donor
MHC class I expressing cells, we may look for the elimination of the
abundant naive cells in the peripheral blood
and to determine the correct dose of TNF-alpha or BCG, we may look for
the elimination of TNF-alpha in vitro sensitivity.
With regard to the second aspect of the therapy, TNF-alpha,
BCG, or another nonspecific form of immune stimulation may promote the
induction of endogenous TNF-alpha. For example, TNF-alpha may be
administered intramuscularly, intravesicularly,
or intravenously. Moreover, recombinant human TNF-alpha or new drugs
such as a TNF receptor 2 agonist may be used. Such compounds have two
effects, one is the elimination of apoptosis or death sensitive cells in
the periphery which can be monitored,
and the other is the promotion of endogenous beta cell regeneration, as
well as possibly differentiation from the new donor blood. Exemplary
doses of TNF-alpha that may be administered to a patient are
approximately 40 .mu.g/m.sup.2 or 200
.mu.g/m.sup.2. Other exemplary doses include doses between
2.times.10.sup.6 and 5.times.10.sup.6 mg daily for two doses in one
week. Patients with an autoimmune disease may tolerate higher doses of
TNF-alpha and/or may require lower doses for
treatment. As an alternative to TNF-alpha, tolerance can be gained by
cross-linking the TCR or by nonspecific vaccination through the same
pathway (e.g., BCG vaccination). As an alternative to administering an
inducer of lymphopenia (e.g., TNF-alpha)
directly to a patient, the inducer of lymphopenia can be administered to
blood obtained from the patient (e.g., blood obtained during
electrophoresis), and the treated blood can be re-administered to the
patient. For induces of lymphopenia with a short
half-life (e.g., TNF-alpha) little, if any, functional compound remains
in the blood that is re-introduced into the patient. Thus, this method
should decrease the incidence or severity of any potential adverse,
side-effects of the compound.
Any combination therapy described herein, e.g. a therapy which
uses MHC class I expressing cells and TNF-alpha induction, may be
administered until the disease is successfully treated. For example,
this therapy may be continued for
approximately 40 days; however, this time-period may readily be adjusted
based on the observed phenotypes. Additionally, the dose of TNF-alpha
can be adjusted based on the percentage of cells in blood samples from
the patient that have increased
sensitivity to TNF-alpha, indicating the amount of remaining autoimmune
cells. In addition, in treating type I diabetes, it may be desirable
that the patient maintains as close to normoglycemia as possible. The
murine data have demonstrated that marked
fluctuation in blood sugars hamper the normal regenerative potential of
the pancreas. Therefore, these patients may be placed on an insulin
pump for not only the exemplary 40 days of disease reversing therapy,
but also for a 120 day period to optimize
the regenerative process. The pancreas of long-term diabetics (i.e.,
ones having diabetes for more than 15 years) may have the regenerative
potential of the pancreas diminished to such a degree that the precursor
cells are no longer present. In these
patients, the therapy may be identical except for the length of the
treatment. For instance, the donor blood or bone marrow cells have to
be alive for these cells to convert to the correct tissue type, such as
into beta cells of the pancreas.
As is mentioned above, some embodiments of the invention employ
mesodermal cells, which can be isolated from a normal donor (e.g., from
the bone marrow, the spleen, or the peripheral blood). Typically, this
cell expresses, to a detectable
degree, CD90.sup.+, CD44.sup.+, or CD29.sup.+, but does not express
appreciable amounts of CD45 or CD34. This normal donor cell is
administered to a person, preferably intravenously or intraperitoneally,
to allow for rapid transport to the site of
inflammation, injury, or disease. Desirably, this cell is administered
to a person with active autoimmunity. Alternatively, the cell may be
administered to a person without autoimmunity or to a person with
quiescent autoimmunity. The absence of active
autoimmunity in a person (host) may require pretreatment of the host to
initiate an inflammatory response or injury at the regenerative site.
In addition, pretreatment of the donor cell may also be required. The
host may be treated with TNF-, IFN-,
IL-2, VEGF, FGF, or IGF-1 to prepare the blood vessel endothelium for
optimal interactions with the mobilized mesodermal cell. Additionally,
the pathway of VEGF-stimulated expression on endothelial cells can be
enhanced with a selective inhibitor of
PI-3'-kinase. Alternatively, the host can be pretreated with
platelet-derived growth factor derived from mural cells (e.g., from the
neural crest or epicardium) for optimal interactions with the mobilized
mesodermal cell. Additionally, the mesodermal
cell can be pretreated to optimize adherence to the endothelium. This
type of therapy is envisioned to be beneficial for the regeneration of
diverse organs or organelles, including brain, skin, islets of
Langerhans, heart, lung, liver, muscle,
intestine, pancreas, bone, cartilage, and fat.
It may also be possible to optimize the fresh mesenchymal cell
prior to injection into the host. This can be accomplished with
TNF-exposure, IL-1 exposure, or other chemical/drug treatments to
increase neogenesis.
For patients that have organ or tissue damage, but no
underlying autoimmunity, it may be beneficial to avoid prolonged
administration of an immune adjuvant, e.g., TNF-alpha, as such agents
may result in the depletion of stem cells. Instead,
desirably, one may induce transient lymphopenia with TNF-alpha or any
other nonspecific reagent, remove this reagent, and add cells (e.g.,
stem cells) to regenerate the organ or tissue. In addition, the added
stem or precursor cells may be altered to
have reduced TNF-alpha sensitivity or may have increased proteasome
activity or decreased death sensitivity through TNF or Fas.
Furthermore, the host may be preconditioned with an agent that increases
LMP2, LMP7, or proteasome activity (e.g., gamma
interferon) prior to, concurrent with, or after the administration of
stem cells. Compounds that increase Flt, Flk, VEGF expression or
activity, hypoxia, GATA-2, hypoglycemia, IL-1, or inhibition of cAMP can
also be used. Moreover, since administration
of TNF-alpha results in cell death due to the upregulation of Fas or
FasL, it may be beneficial to precondition a host with an inhibitor of
Fas/FasL expression or function during TNF-alpha or other immune
adjuvant therapy in both patients with and
without underlying autoimmunity.
In contrast, administration of TNF-alpha during treatment of
autoimmune conditions typically increases the number of stem cells and
thus does not require steps to inhibit destruction of stem cells or to
replace stem cells. TNF-alpha does not
deplete stem cells is in NOD mice because many of the stem cells in
these mice have intrinsic defects in Fas and FasL expression. In
contrast to normal cells, which may die due to Fas/FasL upregulation
that is induced by TNF-alpha, NOD stem cells
survive. In a variety of human autoimmune diseases, the Fas/FasL
downregulation enables these human cells to survive, or even expand, in
the presence of TNF-alpha.
In a host with autoimmune disease, the signaling pathways are
deranged and the administration of cytokines may have multiple effects.
First, administered cytokines induce apoptosis of a subpopulation of
pathologic lymphoid cells due to
intrinsic errors in apoptosis resistance, thus identifying these cells
as pathogenic. Furthermore, the introduced and endogenous cytokines
also promote the regeneration process presumably on the target organ.
Furthermore, if the target organ has
inflammation and is exposed to the administered cytokines or processes
endogenous errors in the overexpression of proteasome function (e.g.,
LMP2/7 subunit expression, a gamma responsive gene, or a TNF responsive
gene), the organ regeneration will be
promoted. While not meant to limit the invention to a particular
theory, a possible mechanism of in situ regeneration is that activation
of the proteasome is critical for the action of VEGF, and VEGF action is
critical for Flk activity. Endothelial
cells may promote this process and, with activation of the proteasome,
VEGF action is accelerated thus allowing augmented Flk action.
Exogenously added stem cells may exponentially promote this process,
e.g., by independent proliferation or fusion with
the cells or by differentiation to lineage-specific cell types.
Therefore, to promote in situ organ regeneration, proteasome inhibitors
are desirably avoided. A spontaneous autoimmune host in which target
organ hyperexperession of LMP2 and/or LMP7 is
frequent may also have accelerated organ regeneration. Organ
regeneration can also be accelerated by promoting LMP2/7 hyperexpression
with, e.g., gamma interferon, TNF, or a compound that activates the
promoters of these genes, e.g., Stat1, agonists of
the ICS-2/GAS elements in the LMP2 promoter, interferon regulatory
factor 1(IRF1), TNF-alpha, or NFkB promoters.
Conversely, the administration of proteasome inhibitors may
serve as a treatment for proliferative diseases. That is, a proteasome
inhibitor can be administered that affects an autoimmune response for
proliferative cells, such as, for example,
cancer cells, while generating a relatively diminished autoimmune
response for normal cells. Most desirably, the anti-proliferative
proteasome inhibitor generates no autoimmunity to normal cells upon
administration. In an additional example,
anti-autoimmune therapy can be administered concurrent or subsequent to
the administration of proteasome inhibitors. Other diseases that can be
treated by proteasome inhibitors include acute inflammatory processes,
such as, for example, sepsis or
atherosclerosis.
Vascular endothelial growth factor (VEGF) is a potent
angiogenic protein that enhances vascular permeability and promotes
endothelial cell proliferation. VEGF stimulate two types of tyrosine
kinase receptors, namely, the fms-like tyrosine
kinase-1 (Flt-1) and the fetal liver kinase-1/kinase domain region
(Flk-1/KDR). FGF (fibroblast growth factor), TNF, and highly confluent
cell culture induce Flk-1/FDR expression in cells, whereas transforming
growth factor 1 (TGF-1) reduces it. Thus,
to promote regeneration, FGF and TNF are used, and TGF is desirably
avoided. For regeneration in a normal host, the donor cells are
desirably not exposed to TNF-like substances too early because these
substances may accelerated death. In contrast, the
host tissue may be exposed to TNF like substances or inducers of NF B or
VEGF to increase Flk-1-like expression or signaling to promote the
regeneration process and/or interactions that promote in situ
regeneration. Therefore, normal donor cells may be
pretreated prior to transfer to prevent death when exposed to endogenous
TNF like substances. Alternatively, the host may be reconditioned with
TNF-like substances (e.g., TNF, VEGF, FGF, or and NF B stimulator)
prior to cell transfer to create an
environment for optimal proliferation. As noted above, VEGF action is
dependent upon a proteasome expressing LMP2, and thus agents that induce
proteasome function are beneficial for regeneration. One such agent is
INF (interferon), which upregulates
the obligatory inducible proteasome subunits (e.g., LMP2) for optimal
VEGF action.
Other Embodiments
From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adapt it to various usages and conditions. Such embodiments
are also within the scope of the following
claims.
All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication, patent, or patent application was specifically and
individually indicated to be incorporated by
reference.
* * * * *
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3. read and discuss the results of pubmed.org ristori + bcg with me and others
4. read and discuss the results of pubmed.org faustman dl and faustmanlab.org with me and others
blondes are useless?
Amy Schumer on father’s multiple sclerosis: ‘It’s so painful’
December 16, 2015
By Frank Lovece Special to Newsday
Amy Schumer attends a VH1 and Entertainment Weekly event
in West Hollywood, California, on Nov. 15, 2015. (Credit: Getty
Images / Frederick M. Brown)
Comedian
Amy Schumer,
who has spoken about her father’s multiple sclerosis and modeled her
stricken on-screen dad in “Trainwreck” after her father, spoke with
Barbara Walters about his current state.
“He’s not good,” the
Rockville Centre-raised Schumer, 34, says in a preview clip for the ABC
special “Barbara Walters Presents: The 10 Most Fascinating People of
2015,” airing Thursday. “Some days he’s really good,” she said, “and
he’s with it and we’re joking around. And some days I go to visit my dad
and it’s so painful. I can’t believe it.”
Gordon Schumer,
who sold upscale baby furniture in Manhattan before going bankrupt and
moving the family to Rockville Centre, learned of his MS shortly after
he and
Sandy Schumer divorced when Amy was 12 and sister
Kim was 8.
“For years, we didn’t really understand what it was,”
Amy Schumer told the Los Angeles Times in July. “We knew it was kind of
sad. But then when we got older, it was devastating.”
She tells Walters,
“In terms of my dad being sick, it was just confusing to me, especially
the way MS works. He was in physical pain. That’s when I kind of took
the lead and took care of everybody in my family. I would keep them — I
would keep everybody laughing,” she says. “I’m the one who ties it all
together.”
MS is an autoimmune
disease of the central nervous system that is treatable but incurable.
Its many symptoms can include loss of balance, muscle spasms and
tremors, vision loss, numbness and excessive fatigue. Approximately
400,000 Americans suffer from the disease, the precise cause of which is
unknown.
Schumer told
Alec Baldwin on his podcast, “Here’s The Thing,” last month that her father is in a hospital on Long Island.
In March, when
“Trainwreck” premiered at the 2015 SXSW Film Festival in Austin, Schumer
told Entertainment Weekly, “There is no denying that there is a lot of
me in this movie. I, as they say, went there. It’s really personal. It’s
about stuff I was struggling with and am constantly battling.”
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