Study the patents and publications ofdenise l faustman
Bcg is inexpensive and works, the bottom line
Mass general, Boston and the us inflicts slow, painful, demoralizing, destructive choices on thodse with autoimmune diseases. Bcg should be available to all who seek same.
The Arnold foundation needs to address alternatives etc
Bcg a better alternative than firearms, pressure cookers and Isis.
See eg pubmed.org faustman DL, pubmed.org RISTORI+ bcg
PHARMA & HEALTHCARE 23,740 views
Here's How Lee Iacocca Wants To Cure Diabetes
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In the late 1990s, former Chrysler CEO Lee Iacocca handed more than $10 million to Massachusetts General Hospital (MGH) scientist Denise Faustman and instructed her to transform an ancient tuberculosis vaccine into a cure for type 1 diabetes. Today Faustman announced the latest milestone in that project—FDA clearance to launch a large trial in people based on what her lab learned from that early research. And the 90-year-old auto magnate continues to fund her studies through the Iacocca Family Foundation, which he founded in 1984 in memory of his late wife, Mary, who died of complications from diabetes.
The trial, announced at the American Diabetes Association conference in Boston, will investigate whether treating patients with the vaccine, bacillus Calmette-Guérin (BCG), will improve natural insulin production in adult patients whose pancreases still produce small but detectable levels of the hormone. If it works, BCG might one day be used to essentially reverse the disease in some patients—even adults who have suffered from diabetes from childhood—says Faustman, director of MGH’s immunobiology laboratory and the study’s principal investigator. And it wouldn’t cost much, either, since BCG has been around for nearly a century and is available in generic form.
“We’re not only going for something cheap and safe, but also trying to figure out a good treatment that might reverse the most severe form of the disease in people who are 15 or 20 years out,” Faustman says.
Here’s how BCG works: The vaccine prompts the immune system to make tumor necrosis factor (TNF), a protein that destroys the abnormal T-cells that interfere with the pancreas’s ability to make insulin. That elevation of TNF has already been well-proven to be quite therapeutic in some settings—BCG, in fact, is approved by the FDA not only to prevent tuberculosis but also to treat bladder cancer.
Faustman’s lab spent years doing basic science experiments to show TNF can temporarily eliminate the abnormal T-cells that cause type 1 diabetes. Iacocca’s foundation, which had been supporting some of that work since coming across the lab’s earliest studies, invited Faustman to present the results of her research at a board meeting in 1999, she recalls.
Iacocca asked Faustman why she wasn’t using BCG to cure diabetes in mouse models of the disease. “I said, ‘It’s too early. We need to do more basic science,’” Faustman recalls. “He looked at me and said, ‘You know, it’s my money.’ We made a deal that if I would aggressively go forward in the mouse he would support me. He gets the credit for supporting the basic science that led to the discovery that TNF is needed in type 1 diabetes.”
Lee Iacocca’s foundation has supported basic diabetes research at Massachusetts General Hospital (Credit: AP Photo/Paul Sancya)
With continued funding from the foundation and other supporters, Faustman launched a small phase 1 clinical trial in people designed to prove that BCG would kill the bad T-cells and stimulate good T-cells in a way that would restore insulin secretion. It worked, though the positive effects were transient. So Faustman started planning a larger phase 2 study to prove that regular injections of BCG, followed by periodic booster shots, would produce a sustained response, and to determine whether that response might improve over time as the pancreas regenerates.
Still, Faustman’s team had to overcome one big hurdle before the FDA would approve the phase 2 trial: a massive shortage of BCG. Two of the biggest producers of the vaccine, Merck and Sanofi , have suffered production problems, leading to huge manufacturing delays. The issue has left some bladder cancer patients in the lurch, as reported recently in the Wall Street Journal. Faustman and her colleagues, who had been using Sanofi’s vaccine, had to go looking for an alternate supplier.
Still, Faustman’s team had to overcome one big hurdle before the FDA would approve the phase 2 trial: a massive shortage of BCG. Two of the biggest producers of the vaccine, Merck and Sanofi , have suffered production problems, leading to huge manufacturing delays. The issue has left some bladder cancer patients in the lurch, as reported recently in the Wall Street Journal. Faustman and her colleagues, who had been using Sanofi’s vaccine, had to go looking for an alternate supplier.
So MGH collaborated with a division of the Bill & Melinda Gates Foundation and the World Health Organization to secure the vaccine for the trial from a drug manufacturer that’s run by the Japanese government, Faustman says. “We had to get the FDA to certify that [the manufacturer's] processes are up to U.S. standards so the BCG can be used for trials,” she says. “This is not something that academics normally do, but we were determined.”
Faustman’s team has raised $19 million of the $25 million needed to complete the phase 2 study, thanks largely to the Iococca Family Foundation, which continues to be the project’s biggest source of support. “I made a promise to my late wife to find a cure for type 1 diabetes,” Iococca said in a statement. “Now my family and I look forward to the continued progress and are proud to support this effort to get closer to that goal.”
Faustman’s plan is to enroll 150 adults with diabetes, some of whom will receive BCG, with the others getting a placebo. The patients will have two injections four weeks apart and then annual injections over four years. They will continue to take insulin, though the research team will be watching closely to see if the BCG reduces the amount of insulin needed to maintain blood-sugar control, Faustman says. “We expect the metabolic effect to occur gradually over five years,” she says.
However it turns out, Faustman says, she will always be grateful to Iacocca for having the patience to continue funding the BCG research. “Many other people support us now, but the Iacocca Foundation makes a huge contribution to these trials,” she says. “He sees the big picture and is willing to look for ways to change the paradigm.”
Dr. Denise Faustman
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The Faustman Lab at Massachusetts General Hospital
Denise Faustman, MD, PhD, is Director of the Immunobiology Laboratory at the Massachusetts General Hospital (MGH) and an Associate Professor of Medicine at Harvard Medical School. Her current research focuses on discovering and developing new treatments for type 1 diabetes and other autoimmune diseases, including Crohn's disease, lupus, scleroderma, rheumatoid arthritis, Sjögren's syndrome, and multiple sclerosis. She is currently leading a human clinical trial program testing the efficacy of the BCG vaccine for reversal of long-term type 1 diabetes. Positive results from the Phase I study were reported in 2012.Dr. Faustman's type 1 diabetes research has earned her notable awards such as the Oprah Achievement Award for “Top Health Breakthrough by a Female Scientist” (2005), the "Women in Science Award" from the American Medical Women’s Association and Wyeth Pharmaceutical Company for her contributions to autoimmune disease research (2006), and the Goldman Philanthropic Partnerships/Partnership for Cures “George and Judith Goldman Angel Award” for research to find an effective treatment for type 1 diabetes (2011). Her previous research accomplishments include the first scientific description of modifying donor tissue antigens to change their foreignness. This achievement earned her the prestigious National Institutes of Health and National Library of Medicine “Changing the Face of Medicine” Award (2003) as one of 300 American physicians (one of 35 in research) honored for seminal scientific achievements in the United States.
Dr. Faustman earned her MD and PhD from Washington University School of Medicine in St. Louis, Missouri, and completed her internship, residency, and fellowships in Internal Medicine and Endocrinology at the Massachusetts General Hospital in Boston, Massachusetts.
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.
| United States Patent | 8,697,077 |
| Faustman | April 15, 2014 |
Methods and compositions for treating autoimmune diseases
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) | ||||||||||
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| Applicant: |
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| Assignee: | The General Hospital Corporation (Boston, MA) | ||||||||||
| Family ID: | 30002891 | ||||||||||
| Appl. No.: | 13/462,160 | ||||||||||
| Filed: | May 2, 2012 |
| Document Identifier | Publication Date | |
|---|---|---|
| US 20120213731 A1 | Aug 23, 2012 | |
| 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) |
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