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Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus

Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1... Abstract Context Type 1 diabetes mellitus (DM) results from a cell-mediated autoimmune attack against pancreatic beta cells. Previous animal and clinical studies suggest that moderate immunosuppression in newly diagnosed type 1 DM can prevent further loss of insulin production and can reduce insulin needs. Objective To determine the safety and metabolic effects of high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation (AHST) in newly diagnosed type 1 DM. Design, Setting, and Participants A prospective phase 1/2 study of 15 patients with type 1 DM (aged 14-31 years) diagnosed within the previous 6 weeks by clinical findings and hyperglycemia and confirmed with positive antibodies against glutamic acid decarboxylase. Enrollment was November 2003-July 2006 with observation until February 2007 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. Patients with previous diabetic ketoacidosis were excluded after the first patient with diabetic ketoacidosis failed to benefit from AHST. Hematopoietic stem cells were mobilized with cyclophosphamide (2.0 g/m2) and granulocyte colony-stimulating factor (10 μg/kg per day) and then collected from peripheral blood by leukapheresis and cryopreserved. The cells were injected intravenously after conditioning with cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (4.5 mg/kg). Main Outcome Measures Morbidity and mortality from transplantation and temporal changes in exogenous insulin requirements (daily dose and duration of usage). Secondary end points: serum levels of hemoglobin A1c, C-peptide levels during the mixed-meal tolerance test, and anti–glutamic acid decarboxylase antibody titers measured before and at different times following AHST. Results During a 7- to 36-month follow-up (mean 18.8), 14 patients became insulin-free (1 for 35 months, 4 for at least 21 months, 7 for at least 6 months; and 2 with late response were insulin-free for 1 and 5 months, respectively). Among those, 1 patient resumed insulin use 1 year after AHST. At 6 months after AHST, mean total area under the C-peptide response curve was significantly greater than the pretreatment values, and at 12 and 24 months it did not change. Anti–glutamic acid decarboxylase antibody levels decreased after 6 months and stabilized at 12 and 24 months. Serum levels of hemoglobin A1c were maintained at less than 7% in 13 of 14 patients. The only acute severe adverse effect was culture-negative bilateral pneumonia in 1 patient and late endocrine dysfunction (hypothyroidism or hypogonadism) in 2 others. There was no mortality. Conclusions High-dose immunosuppression and AHST were performed with acceptable toxicity in a small number of patients with newly diagnosed type 1 DM. With AHST, beta cell function was increased in all but 1 patient and induced prolonged insulin independence in the majority of the patients. Trial Registration clinicaltrials.gov Identifier: NCT00315133 Type 1 diabetes mellitus (DM) results from a cell-mediated autoimmune attack against pancreatic beta cells.1 The course of autodestruction is subclinical until the amount of beta-cell mass is insufficient to maintain glucose homeostasis. Thus, at the time of clinical diagnosis, approximately 60% to 80% of the beta-cell mass has been destroyed.2 Type 1 DM comprises only 5% to 10% of all diabetic etiologies but is associated with a high frequency of vascular complications and compromises quality and expectancy of life.3,4 Patients with type 1 DM depend on exogenous insulin administration for survival and for control of long-term complications. The best-established treatment is tight control of blood glucose achieved by frequent daily injections or continuous subcutaneous infusion of insulin, ie, intensive insulin therapy. This treatment reduces the risk of retinopathy, nephropathy, and neuropathy by 35% to 90% when compared with conventional therapy with 1 to 2 injections per day.5 Subgroup analysis of the Diabetes Control and Complications Trial showed that patients with a larger beta cell reserve demonstrable by serum C-peptide levels presented a slower decline of these levels during the study and experienced fewer microvascular complications than patients with low or undetectable C-peptide concentrations. Therefore, beta cell preservation is another important target in the management of type 1 DM and in the prevention of its related complications.6 Many clinical trials have evaluated the role of immunointervention in preventing residual beta cell loss by blocking the autoimmune response with prednisone,7 azathioprine,8,9 prednisone plus azathioprine,10 cyclosporine,11 antibodies against CD3,12,13 heat shock protein,14 and rabbit antithymocyte globulin.15 These therapies were shown to induce a slower decline or some improvement in C-peptide levels when compared with placebo groups. However, almost all patients required exogenous insulin use. Since 1996, organ-threatening systemic lupus erythematosus16 and other autoimmune diseases17 have been successfully treated with high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation (AHST). Organ function was salvaged and in many cases improved following AHST. In animal models, allogeneic bone marrow transplantation prevents both insulitis and the development of type 1 DM in susceptible strains of mice.18 On the basis of these observations, we initiated a phase 1/2 study in November 2003 analyzing the safety, metabolic effects, and ability of AHST to preserve beta cell function in patients with newly diagnosed type 1 DM. Here we report the first prospective trial, to our knowledge, of stem cell therapy in human DM. We describe 15 patients with type 1 DM, submitted to AHST, and observed from 7 to 36 months (mean 18.8 months) after treatment. Methods Patients Inclusion criteria were patients of both sexes, aged 12 to 35 years, with a diagnosis of type 1 DM during the previous 6 weeks confirmed by measurement of serum levels of anti–glutamic acid decarboxylase (anti-GAD) antibodies. From September 2003 to February 2007, more than 100 patients were offered screening for enrollment (most by e-mail or telephone interviews). Of those patients, 52 fulfilled the inclusion criteria and were personally interviewed, 15 patients opted to participate, and all 15 were subsequently enrolled between November 2003 and July 2006 and observed until February 2007 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. The main reasons for not fitting the inclusion criteria were the duration of type 1 DM longer than 6 weeks or previous episodes of diabetic ketoacidosis. Concerns about the probable adverse effects related to the immunosuppression were the main cause of refusing study participation. The first patient enrolled was diagnosed with diabetic ketoacidosis and received hydrocortisone (200 mg) and methylprednisolone (125 mg) to prevent rabbit antithymocyte globulin reactions. This patient's continued insulin dependence after AHST (see Results section) resulted in modification of the protocol to exclude patients with diabetic ketoacidosis-onset diabetes and to remove glucocorticoids from the immunosuppression regimen. Other exclusion criteria were positive serology for human immunodeficiency virus, hepatitis B or C, and underlying hematologic, nephrologic, cardiac, psychiatric or hepatic disease. Serum levels of β-human chorionic gonadotropin were determined in all women to exclude pregnancy. Participants were initially treated by their own physicians until admission to the present study. Race/ethnicity was self-reported and was assessed because of the diversity of the Brazilian population along with its prevalence of black/white biracialiy. HLA class II typing was performed at low/medium resolution using reverse sequence-specific oligonucleotide probes (RSSOP-One Lambda, Canoga Park, Calif), and at high resolution using sequence–specific primers (SSP, One Lambda). The study protocol was approved by the research ethics committees of both the University Hospital of the School of Medicine of Ribeirão Preto and the Brazilian Ministry of Health. An informed consent according to the Declaration of Helsinki was signed by patients or their parents. Study Design Key end points of the study were morbidity and mortality from transplantation and temporal changes in exogenous insulin requirements (daily dose and duration of usage). Secondary end points were serum levels of hemoglobin A1c, C-peptide levels during the mixed-meal tolerance test, and anti-GAD antibody titers measured before and at different times following transplantation. Blood samples for hemoglobin A1c determination were collected after an 8-hour fast at pretreatment and every 3 months thereafter. Blood samples for the determination of C-peptide, an indirect measure of endogenous insulin secretion, were collected in the fasting state and every 30 minutes during a 2-hour mixed-meal tolerance test. The morning and evening doses of insulin were withheld the day before the test at pretreatment, 6 months, 1 year and then yearly following AHST. Serum anti-GAD antibodies were titrated at the same intervals. All patients were encouraged to self-monitor blood glucose at least twice daily (before and 2 hours after different meals and/or at 3 AM) between mobilization and the conditioning phase and then indefinitely after discharge from the hospital. During hospitalization, blood glucose monitoring was performed before meals and at bedtime. Insulin titration was based on fasting before meals and 2 hours after meals with target blood glucose levels of less than 120 mg/dL (6.7 mmol/L) and less than 140 mg/dL (7.7 mmol/L), respectively. The dose of insulin was reduced by 1-2 IU/mL if patients presented clinical findings of hypoglycemia and/or blood glucose levels less than 4.9 mmol/L (90 mg/dL). Standard recommendations for lifestyle modification (performing physical activities and a low-sugar diet) after AHST were made to all patients irrespective of exogenous insulin use. Intensive insulin therapy was the treatment of choice for all patients who needed exogenous insulin. All changes in insulin doses were ordered by one of the endocrinologists of the team (C.E.B.C.). Stem Cell Mobilization Regimen Peripheral hematopoietic stem cells were mobilized with cyclophosphamide and granulocyte colony-stimulating factor (Leucin, Laboratory Bergamo, São Paulo, SP, Brazil). Cyclophosphamide (2 g/m2) was infused in 2 doses 12 hours apart in 250 mL of saline solution over 1 hour. Uroprotection was achieved with intravenous saline infusion at 250 mL/h, initiated 4 hours before cyclophosphamide infusion and continued for 16 hours. Mesna (sodium 2-mercaptoethanesulfonate), 4 g/m2, was infused over 24 hours to bind toxic cyclophosphamide metabolites in the bladder. Granulocyte colony-stimulating factor (10 μg/kg per day) was injected subcutaneously starting 1 day after cyclophosphamide infusion and continuing until leukapheresis was completed. Leukapheresis using a continuous-flow blood cell separator was initiated when the rebounding CD34+ cells reached 10 cells/μL. Apheresis was continued daily until the number of harvested progenitor cells reached a minimum of 3.0 × 106 CD34+ cells/kg body weight. Unmanipulated peripheral blood stem cells were frozen in 10% dimethyl sulfoxide in a rate-controlled freezer and stored in the vapor phase of liquid nitrogen. Conditioning (Immune Ablative) Regimen Conditioning was achieved with cyclophosphamide and antithymocyte globulin. Cyclophosphamide was given intravenously in divided doses of 50 mg/kg per day over 1 hour on days 5, 4, 3, and 2 before stem cell infusion. Rabbit antithymocyte globulin (thymoglobulin, IMTIX Sangstat, Lyon, France) was administered at a dose of 0.5 mg/kg per day on day 5 before, and at a dose of 1 mg/kg per day on days 4, 3, 2, and 1 before stem cell infusion. Except for the first patient, prophylaxis of antithymocyte globulin reactions was done with dexchlorpheniramine (6 mg by mouth) avoiding the use of glucocorticoids. Stem cell infusion was performed on day 0 and granulocyte colony-stimulating factor (5 μg/kg per day) was administered subcutaneously from day 5 after stem cell infusion until neutrophil count was greater than 1000/μL. Supportive Care Patients were isolated in rooms equipped with high-efficiency particulate air filters. After hospital admittance for conditioning, antimicrobial prophylaxis was started with ciprofloxacin (500 mg every 12 hours intravenously), acyclovir (250 mg/m2 every 8 hours by mouth until day 35), amphotericin B (0.2 mg/kg per day intravenously and 10 mg/d aerosolized). Ciprofloxacin was replaced by cefepime (2 g every 12 hours intravenously) during febrile episodes. After engraftment, antifungal prophylaxis was changed to fluconazole (400 mg/d by mouth until day 60) and sulfamethoxazole/trimethoprim (800/160 mg every 12 hours by mouth 2 times per week) or dapsone (100 mg 3 times per week) was given through day 60 for prevention of Pneumocystis jiroveci pneumonia. Weekly monitoring of cytomegalovirus antigenemia in circulating neutrophils was performed until day 60. During pretreatment evaluation, semen samples were collected and frozen in liquid nitrogen. Leuprolide acetate depot (3.75 mg by intramuscular injection) was given to female patients to prevent menstrual bleeding and to protect ovarian function. All women opted to use oral contraceptive methods after AHST. Laboratory Assessment of Diabetic Status Serum C-peptide levels were measured by radioimmunoassay using commercial kits (Diagnostic Systems Laboratories Inc, Webster, Tex). The lower limit of detection was 0.1 ng/mL and undetected values were reported as 0.1 ng/mL. Serum levels of anti-GAD antibodies were measured by radioimmunoassay using commercial kits (RSR Limited, Cardiff, UK) and the results were considered positive if greater than 1 U/mL. Hemoglobin A1c was measured by low-pressure liquid chromatography. Statistical Analysis Multiple comparisons of total area under the curve of serum C-peptide measured during the mixed-meal tolerance test (during fasting and at 30, 60, 90, and 120 minutes) were made using a model of multiple regression of mixed effects for periods 0, 6, 12, and 24 months posttransplantation. The same model was used to test anti-GAD titers. To present the mean variation of hemoglobin A1c levels with time, a model of linear regression of random effects was constructed using the following equation: y = β0 + β1 × log (time) + β2 × [log (time)]2, in which each parameter represents a random effect in each patient. These models are characterized to present residuals that are normally distributed. Data analysis was completed using PROC MIXED, SAS statistical software, version 8 (SAS Institute Inc, Cary, NC). Results Fifteen patients aged 14 to 31 years (mean 19.2 years) were enrolled in the study between November 2003 and July 2006. Individual demographic characteristics and follow-up variables are listed in Table 1 and Table 2. Mean body mass index (calculated as weight in kilograms divided by height in meters squared) at diagnosis was 19.8 (range, 16.6-23.4) and mean plasma glucose was 391 mg/dL (21.7 mmol/L) (range, 130-612 mg/dL [7.2-33.9 mmol/L]). All patients presented symptoms of hyperglycemia (polyuria, polydipsia, and weight loss) at diagnosis. Six patients presented both HLA haplotypes characteristic of high risk for type 1 DM, 7 patients presented 1 of those haplotypes and 2 patients presented 0. Time from diagnosis to mobilization ranged from 25 to 56 days (mean, 38.4) and mean duration of hospital stay for transplantation (from conditioning to discharge) was 19.2 days (range, 15-24). Mean number of infused CD34+ cells was 11.0 × 106/kg (range, 5.8-23.1 × 106/kg). Neutrophil engraftment (>500/μL) occurred between days 8 and 10 after transplantation (mean 9.1 days) and platelet engraftment (>20 000/μL) was detected between day 0 and day 15 after transplantation (mean 11.4 days). Most patients had febrile neutropenia, nausea, vomiting, alopecia, and other common transplantation-related complications due to the drugs used in the mobilization and conditioning (Table 3). Bilateral pneumonia of unidentified etiology that required supplementary oxygen therapy and responded completely to broad-spectrum antibiotics occurred in patient 2 and was the only severe acute complication of AHST. During long-term follow-up, patient 3 developed autoimmune hypothyroidism and transient renal dysfunction associated with rhabdomyolysis, a complication that was treated successfully with levothyroxine. Measurements of gonadal function (follicle-stimulating hormone and lutenizing hormone in both sexes, testosterone in men, and estradiol in women) were in the normal range in 14 of 15 patients. Patient 2 fathered a child 2 years after transplantation (by natural means) and patient 10 presented mild hypergonadotropic hypogonadism at 12 months following transplantation. There was no mortality. The first patient enrolled in the study presented few minor complications of transplantation (Table 3). However, this patient's insulin requirements increased progressively and at 12 months following transplantation when he abandoned follow-up, he was using a dose 250% higher than his initial requirement (1.7 IU/kg per day). His hemoglobin A1c levels were 7.6%, 8.2%, 8.9%, 9.7%, and 11.1% at 0, 3, 6, 9, and 12 months following transplantation, respectively, and his C-peptide levels were low at study entry (basal level, 0.4 ng/mL; peak stimulated level, not available) and did not increase after 1 year (basal, 0.3 ng/mL; peak stimulated level, 0.4 ng/mL) (Table 1 and Table 2). Anti-GAD antibody levels were 36.0, 9.9, and 7.7 U/mL at 0, 6, and 12 months following transplantation, respectively. Since the protocol was changed after treating this patient, his data were not included in the statistical analysis. Thus, hemoglobin A1c (Figure 1) and results of C-peptide levels (Figure 2) refers to 14 patients fulfilling the same selection criteria and receiving the same conditioning regimen. Before the mobilization regimen, all patients required exogenous insulin (mean, 0.38 IU/kg per day, range, 0.13-0.58). By February 2007, 13 patients were free from exogenous insulin for 1 to 35 months (mean, 16.2) (Table 2). Patient 7 used a fraction of the initial insulin dose for 20 months and discontinued insulin use in January 2007. Patient 10 discontinued insulin transiently during transplantation (from 2 days before to 7 days after), then resumed insulin use (0.34 IU/kg per day) and after progressive reduction in its dose discontinued insulin again 1 year after transplantation. Patient 11 was free from insulin from 3 days before transplantation until 360 days after,when insulin use was resumed (0.43 IU/kg per day) after an upper respiratory tract viral infection. The time course of individual insulin doses in different phases is presented in Table 2. All 14 patients treated according to the same protocol (patients 2-15) complied with blood glucose self-monitoring and scheduled medical appointments. The time course of hemoglobin A1c concentrations of those patients is presented in Figure 1. There was a statistically significant reduction of hemoglobin A1c levels after transplantation. At entry into the study, 11 of 14 patients presented values above 7% and within 3 months after AHST, hemoglobin A1c values were below this level and were maintained during follow-up (except for the relapsing patient 11). The time course of fasting and peak stimulated C-peptide levels and of the area under the curve response curve during mixed-meal tolerance test are shown in Table 2 and Figure 2. Compared with pretreatment levels, peak stimulated C-peptide levels following transplantation increased in 11 of 13 patients studied at 6 months, in 8 of 10 patients studied at 12 months, in 4 of 4 patients studied at 24 months, and in 1 patient studied at 36 months. Mean peak stimulated C-peptide levels were 1.3 ng/mL at pretreatment and following transplantation 4.0 ng/mL at 6 months, 3.7 ng/mL at 12 months, and 4.5 ng/mL at 24 months. The increase at 24 months following transplantation was statistically significant compared with all other time points (Table 2). Mean area under the curve of C-peptide levels before transplantation (92.0 ng/mL per 2 hours) showed a statistically significant increase at 6 months following transplantation (332.7 ng/mL per 2 hours), which was not different from 12 months (289.2 ng/mL per 2 hours) and 24 months (270.3 ng/mL per 2 hours) (Figure 2). Mean values of anti-GAD antibodies at diagnosis and at 6, 12, and 24 months after treatment were 31.8 U/mL, 17.3 U/mL, 12.5 U/mL, and 18.7 U/mL, respectively (Table 2). Statistical differences were observed between pre- and and post-6-month titers but not among posttreatment times. Anti-GAD titers showed as negative in only 1 patient (patient 3) at 6 months posttreatment, and continued to show as negative at the 2-year-follow-up. Comment Many clinical trials have analyzed the effect of various immunointervention regimens in blocking autoimmune response and preserving beta-cell function. Short chronic use (≤12 months) of prednisone,7 azathioprine,8,9 azathioprine plus prednisone,10 and cyclosporine11 in randomized controlled trials produced variable degrees of improvement in C-peptide levels at the end of follow-up compared with pretreatment values. However, these effects were not maintained after immunosuppression was discontinued.7-11 Recent studies using short-term treatment with anti-CD3 monoclonal antibodies or heat-shock protein showed long-lasting improvements on C-peptide levels (up to 18 months), however with only partial improvement in insulin usage.12-14 Control groups in the recent studies of immunointervention (treated with intensive insulin therapy) experienced progressive declines of C-peptide levels after study entry or after transient increase in its levels and a parallel increase in insulin needs.12-15 In our study, the increase of C-peptide levels and reduction of hemoglobin A1c were maintained 2 years after insulin discontinuation, excluding the acute effect of insulin therapy on C-peptide concentrations and metabolic control. The natural history of type 1 DM was more altered in our study than in other immunosuppression interventions because, different from those studies, 14 of 15 or 93% of our patients experienced variable periods of insulin independence and most of them maintained this status throughout the follow-up. Beta cell function in newly diagnosed type 1 DM is a measurable outcome that predicts long-term clinical status. Thus, preservation of beta-cell mass can be expected to provide long-term benefits.6,19 The first patient failed to show a clinical benefit probably because of a very low beta-cell reserve at study entry, predicted by previous ketoacidosis that was further jeopardized by the beta-cell apoptotic effect of glucocorticoids used during conditioning.20 Most of the subsequent 14 patients treated without glucocorticoids in the conditioning regimen demonstrated increased beta-cell function measured by C-peptide levels and became insulin-independent for 1 to 35 months. Two patients (identified as 7 and 10) who initially remained on insulin use shortly after transplantation developed insulin independence 20 and 12 months after AHST, respectively, probably secondary to progressive elevations in C-peptide levels over time. The reverse was seen in patient 11, who presented a decline in C-peptide levels after 1 year and resumed insulin use after that time. With the exception of patient 1, irrespective of insulin use all others achieved and maintained peak stimulated C-peptide levels greater than 0.60 ng/mL, which is known to be associated with reduced prevalence of diabetic complications.21 Area under the curve levels of C-peptide increased significantly after transplantation and remained high up to 24 months thereafter. All patients experienced common transplantation-related complications of high-dose immunosuppression and only 1 patient presented a major infectious complication. The low frequency of severe acute complications after AHST is expected in a group of young patients with early-onset type 1 DM in contrast to other advanced autoimmune diseases.16,17 On the other hand, 2 patients presented late endocrine dysfunctions that could be caused by autoimmune dysregulation associated with the transplant procedure22 or by autoimmune polyendocrine syndrome frequently associated with type 1 DM.23 We cannot exclude the occurrence of long-term complications related to high-dose cyclophosphamide use. The exact mechanism of action of AHST in autoimmune disorders is not fully understood. Whether the mechanism is active or passive tolerance, ie, T-regulatory cell suppression or clonal deletion, is unknown. In multiple sclerosis, evidence supporting post-AHST immune resetting includes an increase in thymus-derived naive T cells, decreased central-memory T cells, increased output of recent thymic emigrants, and recovery of a diverse but distinct T-cell receptor repertoire following AHST.24 Detailed studies of immune reconstitution are underway in these patients to better understand the mechanisms of action of AHST in new-onset diabetes. Preliminary data suggest a resetting of the immune system toward a tolerant phenotype beyond 1 year after transplantation, as observed in multiple sclerosis (K.C.R.M. and J.C.V., unpublished data, 2006). In the patients of this study, persistence of anti-GAD antibodies, even at low titers, shows that the conditioning regimen was not fully ablative for autoreactive B-cell clones and confirms that the magnitude of the humoral response is not predictive of beta cell reserve or clinical response.19 Improvement of beta-cell function after intensive immunosuppression could be explained by regeneration of beta cells from surviving beta cells or from pancreatic or bone marrow stem cells.25,26 However, pancreatic stem cells have not been clearly demonstrated, and significant in vivo generation of islet cells from hematopoietic stem cells was not observed in animal models of type 1 DM18 or in patients with long-term type 1 DM treated with allogeneic hematopoietic stem cell transplantation for concomitant blood disorders.27 This is, to our knowledge, the first report of high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation for human type 1 DM. Very encouraging results were obtained in a small number of patients with early-onset disease. Ninety-three percent of patients achieved different periods of insulin independence and treatment-related toxicity was low, with no mortality. Further follow-up is necessary to confirm the duration of insulin independence and the mechanisms of action of the procedure. In addition, randomized controlled trials and further biological studies are necessary to confirm the role of this treatment in changing the natural history of type 1 DM and to evaluate the contribution of hematopoietic stem cells to this change. Back to top Article Information Corresponding Author: Julio C. Voltarelli, MD, PhD, Regional Blood Center (Hemocentro), Campus USP, 14051-140 Ribeirão Preto, Brazil (jcvoltar@fmrp.usp.br). Author Contributions: Dr Voltarelli had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Voltarelli, Malmegrim, Foss, Squiers, Burt. Acquisition of data: Voltarelli, Couri, Stracieri, Oliveira, Moraes, Pieroni, Coutinho, Malmegrim, Foss-Freitas, Simões, Foss, Squiers. Analysis and interpretation of data: Voltarelli, Couri, Stracieri, Malmegrim, Foss-Freitas, Simões, Foss, Squiers, Burt. Drafting of the manuscript: Voltarelli, Couri, Stracieri, Malmegrim, Simões, Squiers. Critical revision of the manuscript for important intellectual content: Voltarelli, Couri, Oliveira, Moraes, Pieroni, Coutinho, Malmegrim, Foss-Freitas, Simões, Foss, Squiers, Burt. Statistical analysis: Couri, Malmegrim, Squiers. Obtained funding: Voltarelli, Malmegrim, Squiers, Burt. Administrative, technical, or material support: Voltarelli, Stracieri, Malmegrim, Foss, Squiers. Study supervision: Voltarelli, Malmegrim, Foss, Squiers. Financial Disclosures: None reported. Funding/Support: Research supported by the Brazilian Ministry of Health, FAEPA-HCRP, FUNDHERP, FAPESP, CNPq, FINEP, Genzyme Corporation, and Johnson & Johnson-LifeScan–Brazil. Role of the Sponsor: The funding organizations did not participate in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript. Acknowledgment: We are grateful to Edson Martinez, PhD, and Davi Aragon, MSc, Center for Quantitative Methods of the School of Medicine of Ribeirão Preto, University of São Paulo (CEMEQ-FMRP-USP) for statistical advice; to Lewis Joel Greene, PhD, and Elettra Greene, BA, for English review; and to the multiprofessional team of the Bone Marrow Transplantation Unit and the Regional Blood Center of the Hospital das Clínicas of Ribeirão Preto, University of São Paulo, Brazil. Individuals named in this acknowledgment received no compensation from a funding sponsor for their contribution to this article. References 1. American Diabetes Association. Diagnosis and classification of diabetes. Diabetes Care. 2004;27:(suppl I) S5-S1014693921Google ScholarCrossref 2. Notkins AL, Lernmark A. Autoimmune type 1 diabetes: resolved and unresolved issues. J Clin Invest. 2001;108:1247-125211696564Google Scholar 3. Nathan DM. Long term complications of diabetes mellitus. N Engl J Med. 1993;328:1676-16858487827Google ScholarCrossref 4. Rubin RR, Peyrot M. Quality of life and diabetes. Diabetes Metab Res Rev. 1999;15:205-21810441043Google ScholarCrossref 5. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long term complications in insulin-dependent diabetes mellitus. N Engl J Med. 1993;329:977-9868366922Google ScholarCrossref 6. The Diabetes Control and Complications Trial Research Group. Effect of intensive therapy on residual beta-cell function in patients with type 1 diabetes in the Diabetes Control and Complications Trial. Ann Intern Med. 1998;128:517-5239518395Google ScholarCrossref 7. Elliott RB, Crossley JR, Berryman CC, James AG. Partial preservation of pancreatic beta-cell function in children with diabetes. Lancet. 1981;19:631-6326116102Google ScholarCrossref 8. Harrison LC, Colman PG, Dean B, Baxter R, Martin FI. Increase in remission rate in newly diagnosed type 1 diabetic subjects treated with azathioprine. Diabetes. 1985;34:1306-13083905463Google ScholarCrossref 9. Cook JJ, Hudson I, Harrison LC. et al. Double-blind controlled trial of azathioprine in children with newly diagnosed type 1 diabetes. Diabetes. 1989;38:779-7832656346Google ScholarCrossref 10. Silverstein J, Maclaren N, Riley W. et al. Immunosupression with azatioprine and prednisone in recent-onset insulin-dependent diabetes mellitus. N Engl J Med. 1988;319:599-6043045545Google ScholarCrossref 11. Canadian–European Randomized Control Trial Group. Cyclosporin-induced remission of IDDM after early intervention: association of 1 yr of cyclosporin treatment with enhanced insulin secretion. Diabetes. 1988;37:1574-15822903105Google ScholarCrossref 12. Herold KC, Hagopian W, Auger JA. et al. Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med. 2002;346:1692-169812037148Google ScholarCrossref 13. Keymeulen B, Vandemeulebroucke E, Ziegler AG. et al. Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med. 2005;352:2598-260815972866Google ScholarCrossref 14. Raz I, Elias D, Avron A, Metzger M, Cohen IR. Beta-cell function in newly-onset type 1 diabetes and immunomodulation with a heat shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial. Lancet. 2001;358:1749-175311734230Google ScholarCrossref 15. Saudek F, Havrdova T, Boucek P, Novota P, Skibova J. Polyclonal anti-T-cell therapy for type 1 diabetes mellitus of recent onset. Rev Diabet Stud. 2004;1:80-8817273515Google ScholarCrossref 16. Burt RK, Traynor A, Statkute L. et al. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematous. JAMA. 2006;295:527-53516449618Google ScholarCrossref 17. Burt RK, Slavin S, Burns WH, Marmont AM. Induction of tolerance in autoimmune diseases by hematopoietic stem cell transplantation: getting closer to a cure? Blood. 2002;99:768-78411806976Google ScholarCrossref 18. Kang EM, Zickler PP, Burns S. et al. Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established. Exp Hematol. 2005;33:699-70515911094Google ScholarCrossref 19. Palmer JP, Fleming GA, Greenbaum CA. et al. C-peptide is the appropriate outcome measure for type 1 diabetes clinical trials to preserve beta-cell function. Diabetes. 2004;53:250-26414693724Google ScholarCrossref 20. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL. Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology. 2000;141:1384-139310746642Google ScholarCrossref 21. Steffes MW, Sibley S, Jackson M, Thomas W. Beta cell function and the development of diabetes-related complications in the diabetes control and complications trial. Diabetes Care. 2003;26:832-83612610045Google ScholarCrossref 22. Au WY, Lie AK, Kung AW, Liang R, Hawkins BR, Kwong YL. Autoimmune thyroid dysfunction after hematopoietic stem cell transplantation. Bone Marrow Transplant. 2005;35:383-38815640829Google ScholarCrossref 23. Eisenbarth GS, Gottlieb PA. Autoimmune polyendocrine syndromes. N Engl J Med. 2004;350:2068-207915141045Google ScholarCrossref 24. Muraro PA, Douek DC, Packer A. et al. Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients. J Exp Med. 2005;201:805-81615738052Google ScholarCrossref 25. Hussain MA, Theise ND. Stem-cell therapy for diabetes mellitus. Lancet. 2004;364:203-20515246735Google ScholarCrossref 26. Couri CEB, Foss MC, Voltarelli JC. Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting beta-cell regeneration. Braz J Med Biol Res. 2006;39:1271-128016941054Google ScholarCrossref 27. Nelson JL, Torrez R, Louie FM, Choe OS, Storb R, Sullivan KM. Pre-existing autoimmune disease in patients with long-term survival after allogeneic bone marrow transplantation. J Rheumatol Suppl. 1997;48:23-29Google Scholar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA American Medical Association

Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus

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American Medical Association
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Copyright © 2007 American Medical Association. All Rights Reserved.
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0098-7484
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1538-3598
DOI
10.1001/jama.297.14.1568
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Abstract

Abstract Context Type 1 diabetes mellitus (DM) results from a cell-mediated autoimmune attack against pancreatic beta cells. Previous animal and clinical studies suggest that moderate immunosuppression in newly diagnosed type 1 DM can prevent further loss of insulin production and can reduce insulin needs. Objective To determine the safety and metabolic effects of high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation (AHST) in newly diagnosed type 1 DM. Design, Setting, and Participants A prospective phase 1/2 study of 15 patients with type 1 DM (aged 14-31 years) diagnosed within the previous 6 weeks by clinical findings and hyperglycemia and confirmed with positive antibodies against glutamic acid decarboxylase. Enrollment was November 2003-July 2006 with observation until February 2007 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. Patients with previous diabetic ketoacidosis were excluded after the first patient with diabetic ketoacidosis failed to benefit from AHST. Hematopoietic stem cells were mobilized with cyclophosphamide (2.0 g/m2) and granulocyte colony-stimulating factor (10 μg/kg per day) and then collected from peripheral blood by leukapheresis and cryopreserved. The cells were injected intravenously after conditioning with cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (4.5 mg/kg). Main Outcome Measures Morbidity and mortality from transplantation and temporal changes in exogenous insulin requirements (daily dose and duration of usage). Secondary end points: serum levels of hemoglobin A1c, C-peptide levels during the mixed-meal tolerance test, and anti–glutamic acid decarboxylase antibody titers measured before and at different times following AHST. Results During a 7- to 36-month follow-up (mean 18.8), 14 patients became insulin-free (1 for 35 months, 4 for at least 21 months, 7 for at least 6 months; and 2 with late response were insulin-free for 1 and 5 months, respectively). Among those, 1 patient resumed insulin use 1 year after AHST. At 6 months after AHST, mean total area under the C-peptide response curve was significantly greater than the pretreatment values, and at 12 and 24 months it did not change. Anti–glutamic acid decarboxylase antibody levels decreased after 6 months and stabilized at 12 and 24 months. Serum levels of hemoglobin A1c were maintained at less than 7% in 13 of 14 patients. The only acute severe adverse effect was culture-negative bilateral pneumonia in 1 patient and late endocrine dysfunction (hypothyroidism or hypogonadism) in 2 others. There was no mortality. Conclusions High-dose immunosuppression and AHST were performed with acceptable toxicity in a small number of patients with newly diagnosed type 1 DM. With AHST, beta cell function was increased in all but 1 patient and induced prolonged insulin independence in the majority of the patients. Trial Registration clinicaltrials.gov Identifier: NCT00315133 Type 1 diabetes mellitus (DM) results from a cell-mediated autoimmune attack against pancreatic beta cells.1 The course of autodestruction is subclinical until the amount of beta-cell mass is insufficient to maintain glucose homeostasis. Thus, at the time of clinical diagnosis, approximately 60% to 80% of the beta-cell mass has been destroyed.2 Type 1 DM comprises only 5% to 10% of all diabetic etiologies but is associated with a high frequency of vascular complications and compromises quality and expectancy of life.3,4 Patients with type 1 DM depend on exogenous insulin administration for survival and for control of long-term complications. The best-established treatment is tight control of blood glucose achieved by frequent daily injections or continuous subcutaneous infusion of insulin, ie, intensive insulin therapy. This treatment reduces the risk of retinopathy, nephropathy, and neuropathy by 35% to 90% when compared with conventional therapy with 1 to 2 injections per day.5 Subgroup analysis of the Diabetes Control and Complications Trial showed that patients with a larger beta cell reserve demonstrable by serum C-peptide levels presented a slower decline of these levels during the study and experienced fewer microvascular complications than patients with low or undetectable C-peptide concentrations. Therefore, beta cell preservation is another important target in the management of type 1 DM and in the prevention of its related complications.6 Many clinical trials have evaluated the role of immunointervention in preventing residual beta cell loss by blocking the autoimmune response with prednisone,7 azathioprine,8,9 prednisone plus azathioprine,10 cyclosporine,11 antibodies against CD3,12,13 heat shock protein,14 and rabbit antithymocyte globulin.15 These therapies were shown to induce a slower decline or some improvement in C-peptide levels when compared with placebo groups. However, almost all patients required exogenous insulin use. Since 1996, organ-threatening systemic lupus erythematosus16 and other autoimmune diseases17 have been successfully treated with high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation (AHST). Organ function was salvaged and in many cases improved following AHST. In animal models, allogeneic bone marrow transplantation prevents both insulitis and the development of type 1 DM in susceptible strains of mice.18 On the basis of these observations, we initiated a phase 1/2 study in November 2003 analyzing the safety, metabolic effects, and ability of AHST to preserve beta cell function in patients with newly diagnosed type 1 DM. Here we report the first prospective trial, to our knowledge, of stem cell therapy in human DM. We describe 15 patients with type 1 DM, submitted to AHST, and observed from 7 to 36 months (mean 18.8 months) after treatment. Methods Patients Inclusion criteria were patients of both sexes, aged 12 to 35 years, with a diagnosis of type 1 DM during the previous 6 weeks confirmed by measurement of serum levels of anti–glutamic acid decarboxylase (anti-GAD) antibodies. From September 2003 to February 2007, more than 100 patients were offered screening for enrollment (most by e-mail or telephone interviews). Of those patients, 52 fulfilled the inclusion criteria and were personally interviewed, 15 patients opted to participate, and all 15 were subsequently enrolled between November 2003 and July 2006 and observed until February 2007 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. The main reasons for not fitting the inclusion criteria were the duration of type 1 DM longer than 6 weeks or previous episodes of diabetic ketoacidosis. Concerns about the probable adverse effects related to the immunosuppression were the main cause of refusing study participation. The first patient enrolled was diagnosed with diabetic ketoacidosis and received hydrocortisone (200 mg) and methylprednisolone (125 mg) to prevent rabbit antithymocyte globulin reactions. This patient's continued insulin dependence after AHST (see Results section) resulted in modification of the protocol to exclude patients with diabetic ketoacidosis-onset diabetes and to remove glucocorticoids from the immunosuppression regimen. Other exclusion criteria were positive serology for human immunodeficiency virus, hepatitis B or C, and underlying hematologic, nephrologic, cardiac, psychiatric or hepatic disease. Serum levels of β-human chorionic gonadotropin were determined in all women to exclude pregnancy. Participants were initially treated by their own physicians until admission to the present study. Race/ethnicity was self-reported and was assessed because of the diversity of the Brazilian population along with its prevalence of black/white biracialiy. HLA class II typing was performed at low/medium resolution using reverse sequence-specific oligonucleotide probes (RSSOP-One Lambda, Canoga Park, Calif), and at high resolution using sequence–specific primers (SSP, One Lambda). The study protocol was approved by the research ethics committees of both the University Hospital of the School of Medicine of Ribeirão Preto and the Brazilian Ministry of Health. An informed consent according to the Declaration of Helsinki was signed by patients or their parents. Study Design Key end points of the study were morbidity and mortality from transplantation and temporal changes in exogenous insulin requirements (daily dose and duration of usage). Secondary end points were serum levels of hemoglobin A1c, C-peptide levels during the mixed-meal tolerance test, and anti-GAD antibody titers measured before and at different times following transplantation. Blood samples for hemoglobin A1c determination were collected after an 8-hour fast at pretreatment and every 3 months thereafter. Blood samples for the determination of C-peptide, an indirect measure of endogenous insulin secretion, were collected in the fasting state and every 30 minutes during a 2-hour mixed-meal tolerance test. The morning and evening doses of insulin were withheld the day before the test at pretreatment, 6 months, 1 year and then yearly following AHST. Serum anti-GAD antibodies were titrated at the same intervals. All patients were encouraged to self-monitor blood glucose at least twice daily (before and 2 hours after different meals and/or at 3 AM) between mobilization and the conditioning phase and then indefinitely after discharge from the hospital. During hospitalization, blood glucose monitoring was performed before meals and at bedtime. Insulin titration was based on fasting before meals and 2 hours after meals with target blood glucose levels of less than 120 mg/dL (6.7 mmol/L) and less than 140 mg/dL (7.7 mmol/L), respectively. The dose of insulin was reduced by 1-2 IU/mL if patients presented clinical findings of hypoglycemia and/or blood glucose levels less than 4.9 mmol/L (90 mg/dL). Standard recommendations for lifestyle modification (performing physical activities and a low-sugar diet) after AHST were made to all patients irrespective of exogenous insulin use. Intensive insulin therapy was the treatment of choice for all patients who needed exogenous insulin. All changes in insulin doses were ordered by one of the endocrinologists of the team (C.E.B.C.). Stem Cell Mobilization Regimen Peripheral hematopoietic stem cells were mobilized with cyclophosphamide and granulocyte colony-stimulating factor (Leucin, Laboratory Bergamo, São Paulo, SP, Brazil). Cyclophosphamide (2 g/m2) was infused in 2 doses 12 hours apart in 250 mL of saline solution over 1 hour. Uroprotection was achieved with intravenous saline infusion at 250 mL/h, initiated 4 hours before cyclophosphamide infusion and continued for 16 hours. Mesna (sodium 2-mercaptoethanesulfonate), 4 g/m2, was infused over 24 hours to bind toxic cyclophosphamide metabolites in the bladder. Granulocyte colony-stimulating factor (10 μg/kg per day) was injected subcutaneously starting 1 day after cyclophosphamide infusion and continuing until leukapheresis was completed. Leukapheresis using a continuous-flow blood cell separator was initiated when the rebounding CD34+ cells reached 10 cells/μL. Apheresis was continued daily until the number of harvested progenitor cells reached a minimum of 3.0 × 106 CD34+ cells/kg body weight. Unmanipulated peripheral blood stem cells were frozen in 10% dimethyl sulfoxide in a rate-controlled freezer and stored in the vapor phase of liquid nitrogen. Conditioning (Immune Ablative) Regimen Conditioning was achieved with cyclophosphamide and antithymocyte globulin. Cyclophosphamide was given intravenously in divided doses of 50 mg/kg per day over 1 hour on days 5, 4, 3, and 2 before stem cell infusion. Rabbit antithymocyte globulin (thymoglobulin, IMTIX Sangstat, Lyon, France) was administered at a dose of 0.5 mg/kg per day on day 5 before, and at a dose of 1 mg/kg per day on days 4, 3, 2, and 1 before stem cell infusion. Except for the first patient, prophylaxis of antithymocyte globulin reactions was done with dexchlorpheniramine (6 mg by mouth) avoiding the use of glucocorticoids. Stem cell infusion was performed on day 0 and granulocyte colony-stimulating factor (5 μg/kg per day) was administered subcutaneously from day 5 after stem cell infusion until neutrophil count was greater than 1000/μL. Supportive Care Patients were isolated in rooms equipped with high-efficiency particulate air filters. After hospital admittance for conditioning, antimicrobial prophylaxis was started with ciprofloxacin (500 mg every 12 hours intravenously), acyclovir (250 mg/m2 every 8 hours by mouth until day 35), amphotericin B (0.2 mg/kg per day intravenously and 10 mg/d aerosolized). Ciprofloxacin was replaced by cefepime (2 g every 12 hours intravenously) during febrile episodes. After engraftment, antifungal prophylaxis was changed to fluconazole (400 mg/d by mouth until day 60) and sulfamethoxazole/trimethoprim (800/160 mg every 12 hours by mouth 2 times per week) or dapsone (100 mg 3 times per week) was given through day 60 for prevention of Pneumocystis jiroveci pneumonia. Weekly monitoring of cytomegalovirus antigenemia in circulating neutrophils was performed until day 60. During pretreatment evaluation, semen samples were collected and frozen in liquid nitrogen. Leuprolide acetate depot (3.75 mg by intramuscular injection) was given to female patients to prevent menstrual bleeding and to protect ovarian function. All women opted to use oral contraceptive methods after AHST. Laboratory Assessment of Diabetic Status Serum C-peptide levels were measured by radioimmunoassay using commercial kits (Diagnostic Systems Laboratories Inc, Webster, Tex). The lower limit of detection was 0.1 ng/mL and undetected values were reported as 0.1 ng/mL. Serum levels of anti-GAD antibodies were measured by radioimmunoassay using commercial kits (RSR Limited, Cardiff, UK) and the results were considered positive if greater than 1 U/mL. Hemoglobin A1c was measured by low-pressure liquid chromatography. Statistical Analysis Multiple comparisons of total area under the curve of serum C-peptide measured during the mixed-meal tolerance test (during fasting and at 30, 60, 90, and 120 minutes) were made using a model of multiple regression of mixed effects for periods 0, 6, 12, and 24 months posttransplantation. The same model was used to test anti-GAD titers. To present the mean variation of hemoglobin A1c levels with time, a model of linear regression of random effects was constructed using the following equation: y = β0 + β1 × log (time) + β2 × [log (time)]2, in which each parameter represents a random effect in each patient. These models are characterized to present residuals that are normally distributed. Data analysis was completed using PROC MIXED, SAS statistical software, version 8 (SAS Institute Inc, Cary, NC). Results Fifteen patients aged 14 to 31 years (mean 19.2 years) were enrolled in the study between November 2003 and July 2006. Individual demographic characteristics and follow-up variables are listed in Table 1 and Table 2. Mean body mass index (calculated as weight in kilograms divided by height in meters squared) at diagnosis was 19.8 (range, 16.6-23.4) and mean plasma glucose was 391 mg/dL (21.7 mmol/L) (range, 130-612 mg/dL [7.2-33.9 mmol/L]). All patients presented symptoms of hyperglycemia (polyuria, polydipsia, and weight loss) at diagnosis. Six patients presented both HLA haplotypes characteristic of high risk for type 1 DM, 7 patients presented 1 of those haplotypes and 2 patients presented 0. Time from diagnosis to mobilization ranged from 25 to 56 days (mean, 38.4) and mean duration of hospital stay for transplantation (from conditioning to discharge) was 19.2 days (range, 15-24). Mean number of infused CD34+ cells was 11.0 × 106/kg (range, 5.8-23.1 × 106/kg). Neutrophil engraftment (>500/μL) occurred between days 8 and 10 after transplantation (mean 9.1 days) and platelet engraftment (>20 000/μL) was detected between day 0 and day 15 after transplantation (mean 11.4 days). Most patients had febrile neutropenia, nausea, vomiting, alopecia, and other common transplantation-related complications due to the drugs used in the mobilization and conditioning (Table 3). Bilateral pneumonia of unidentified etiology that required supplementary oxygen therapy and responded completely to broad-spectrum antibiotics occurred in patient 2 and was the only severe acute complication of AHST. During long-term follow-up, patient 3 developed autoimmune hypothyroidism and transient renal dysfunction associated with rhabdomyolysis, a complication that was treated successfully with levothyroxine. Measurements of gonadal function (follicle-stimulating hormone and lutenizing hormone in both sexes, testosterone in men, and estradiol in women) were in the normal range in 14 of 15 patients. Patient 2 fathered a child 2 years after transplantation (by natural means) and patient 10 presented mild hypergonadotropic hypogonadism at 12 months following transplantation. There was no mortality. The first patient enrolled in the study presented few minor complications of transplantation (Table 3). However, this patient's insulin requirements increased progressively and at 12 months following transplantation when he abandoned follow-up, he was using a dose 250% higher than his initial requirement (1.7 IU/kg per day). His hemoglobin A1c levels were 7.6%, 8.2%, 8.9%, 9.7%, and 11.1% at 0, 3, 6, 9, and 12 months following transplantation, respectively, and his C-peptide levels were low at study entry (basal level, 0.4 ng/mL; peak stimulated level, not available) and did not increase after 1 year (basal, 0.3 ng/mL; peak stimulated level, 0.4 ng/mL) (Table 1 and Table 2). Anti-GAD antibody levels were 36.0, 9.9, and 7.7 U/mL at 0, 6, and 12 months following transplantation, respectively. Since the protocol was changed after treating this patient, his data were not included in the statistical analysis. Thus, hemoglobin A1c (Figure 1) and results of C-peptide levels (Figure 2) refers to 14 patients fulfilling the same selection criteria and receiving the same conditioning regimen. Before the mobilization regimen, all patients required exogenous insulin (mean, 0.38 IU/kg per day, range, 0.13-0.58). By February 2007, 13 patients were free from exogenous insulin for 1 to 35 months (mean, 16.2) (Table 2). Patient 7 used a fraction of the initial insulin dose for 20 months and discontinued insulin use in January 2007. Patient 10 discontinued insulin transiently during transplantation (from 2 days before to 7 days after), then resumed insulin use (0.34 IU/kg per day) and after progressive reduction in its dose discontinued insulin again 1 year after transplantation. Patient 11 was free from insulin from 3 days before transplantation until 360 days after,when insulin use was resumed (0.43 IU/kg per day) after an upper respiratory tract viral infection. The time course of individual insulin doses in different phases is presented in Table 2. All 14 patients treated according to the same protocol (patients 2-15) complied with blood glucose self-monitoring and scheduled medical appointments. The time course of hemoglobin A1c concentrations of those patients is presented in Figure 1. There was a statistically significant reduction of hemoglobin A1c levels after transplantation. At entry into the study, 11 of 14 patients presented values above 7% and within 3 months after AHST, hemoglobin A1c values were below this level and were maintained during follow-up (except for the relapsing patient 11). The time course of fasting and peak stimulated C-peptide levels and of the area under the curve response curve during mixed-meal tolerance test are shown in Table 2 and Figure 2. Compared with pretreatment levels, peak stimulated C-peptide levels following transplantation increased in 11 of 13 patients studied at 6 months, in 8 of 10 patients studied at 12 months, in 4 of 4 patients studied at 24 months, and in 1 patient studied at 36 months. Mean peak stimulated C-peptide levels were 1.3 ng/mL at pretreatment and following transplantation 4.0 ng/mL at 6 months, 3.7 ng/mL at 12 months, and 4.5 ng/mL at 24 months. The increase at 24 months following transplantation was statistically significant compared with all other time points (Table 2). Mean area under the curve of C-peptide levels before transplantation (92.0 ng/mL per 2 hours) showed a statistically significant increase at 6 months following transplantation (332.7 ng/mL per 2 hours), which was not different from 12 months (289.2 ng/mL per 2 hours) and 24 months (270.3 ng/mL per 2 hours) (Figure 2). Mean values of anti-GAD antibodies at diagnosis and at 6, 12, and 24 months after treatment were 31.8 U/mL, 17.3 U/mL, 12.5 U/mL, and 18.7 U/mL, respectively (Table 2). Statistical differences were observed between pre- and and post-6-month titers but not among posttreatment times. Anti-GAD titers showed as negative in only 1 patient (patient 3) at 6 months posttreatment, and continued to show as negative at the 2-year-follow-up. Comment Many clinical trials have analyzed the effect of various immunointervention regimens in blocking autoimmune response and preserving beta-cell function. Short chronic use (≤12 months) of prednisone,7 azathioprine,8,9 azathioprine plus prednisone,10 and cyclosporine11 in randomized controlled trials produced variable degrees of improvement in C-peptide levels at the end of follow-up compared with pretreatment values. However, these effects were not maintained after immunosuppression was discontinued.7-11 Recent studies using short-term treatment with anti-CD3 monoclonal antibodies or heat-shock protein showed long-lasting improvements on C-peptide levels (up to 18 months), however with only partial improvement in insulin usage.12-14 Control groups in the recent studies of immunointervention (treated with intensive insulin therapy) experienced progressive declines of C-peptide levels after study entry or after transient increase in its levels and a parallel increase in insulin needs.12-15 In our study, the increase of C-peptide levels and reduction of hemoglobin A1c were maintained 2 years after insulin discontinuation, excluding the acute effect of insulin therapy on C-peptide concentrations and metabolic control. The natural history of type 1 DM was more altered in our study than in other immunosuppression interventions because, different from those studies, 14 of 15 or 93% of our patients experienced variable periods of insulin independence and most of them maintained this status throughout the follow-up. Beta cell function in newly diagnosed type 1 DM is a measurable outcome that predicts long-term clinical status. Thus, preservation of beta-cell mass can be expected to provide long-term benefits.6,19 The first patient failed to show a clinical benefit probably because of a very low beta-cell reserve at study entry, predicted by previous ketoacidosis that was further jeopardized by the beta-cell apoptotic effect of glucocorticoids used during conditioning.20 Most of the subsequent 14 patients treated without glucocorticoids in the conditioning regimen demonstrated increased beta-cell function measured by C-peptide levels and became insulin-independent for 1 to 35 months. Two patients (identified as 7 and 10) who initially remained on insulin use shortly after transplantation developed insulin independence 20 and 12 months after AHST, respectively, probably secondary to progressive elevations in C-peptide levels over time. The reverse was seen in patient 11, who presented a decline in C-peptide levels after 1 year and resumed insulin use after that time. With the exception of patient 1, irrespective of insulin use all others achieved and maintained peak stimulated C-peptide levels greater than 0.60 ng/mL, which is known to be associated with reduced prevalence of diabetic complications.21 Area under the curve levels of C-peptide increased significantly after transplantation and remained high up to 24 months thereafter. All patients experienced common transplantation-related complications of high-dose immunosuppression and only 1 patient presented a major infectious complication. The low frequency of severe acute complications after AHST is expected in a group of young patients with early-onset type 1 DM in contrast to other advanced autoimmune diseases.16,17 On the other hand, 2 patients presented late endocrine dysfunctions that could be caused by autoimmune dysregulation associated with the transplant procedure22 or by autoimmune polyendocrine syndrome frequently associated with type 1 DM.23 We cannot exclude the occurrence of long-term complications related to high-dose cyclophosphamide use. The exact mechanism of action of AHST in autoimmune disorders is not fully understood. Whether the mechanism is active or passive tolerance, ie, T-regulatory cell suppression or clonal deletion, is unknown. In multiple sclerosis, evidence supporting post-AHST immune resetting includes an increase in thymus-derived naive T cells, decreased central-memory T cells, increased output of recent thymic emigrants, and recovery of a diverse but distinct T-cell receptor repertoire following AHST.24 Detailed studies of immune reconstitution are underway in these patients to better understand the mechanisms of action of AHST in new-onset diabetes. Preliminary data suggest a resetting of the immune system toward a tolerant phenotype beyond 1 year after transplantation, as observed in multiple sclerosis (K.C.R.M. and J.C.V., unpublished data, 2006). In the patients of this study, persistence of anti-GAD antibodies, even at low titers, shows that the conditioning regimen was not fully ablative for autoreactive B-cell clones and confirms that the magnitude of the humoral response is not predictive of beta cell reserve or clinical response.19 Improvement of beta-cell function after intensive immunosuppression could be explained by regeneration of beta cells from surviving beta cells or from pancreatic or bone marrow stem cells.25,26 However, pancreatic stem cells have not been clearly demonstrated, and significant in vivo generation of islet cells from hematopoietic stem cells was not observed in animal models of type 1 DM18 or in patients with long-term type 1 DM treated with allogeneic hematopoietic stem cell transplantation for concomitant blood disorders.27 This is, to our knowledge, the first report of high-dose immunosuppression followed by autologous nonmyeloablative hematopoietic stem cell transplantation for human type 1 DM. Very encouraging results were obtained in a small number of patients with early-onset disease. Ninety-three percent of patients achieved different periods of insulin independence and treatment-related toxicity was low, with no mortality. Further follow-up is necessary to confirm the duration of insulin independence and the mechanisms of action of the procedure. In addition, randomized controlled trials and further biological studies are necessary to confirm the role of this treatment in changing the natural history of type 1 DM and to evaluate the contribution of hematopoietic stem cells to this change. Back to top Article Information Corresponding Author: Julio C. Voltarelli, MD, PhD, Regional Blood Center (Hemocentro), Campus USP, 14051-140 Ribeirão Preto, Brazil (jcvoltar@fmrp.usp.br). Author Contributions: Dr Voltarelli had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Voltarelli, Malmegrim, Foss, Squiers, Burt. Acquisition of data: Voltarelli, Couri, Stracieri, Oliveira, Moraes, Pieroni, Coutinho, Malmegrim, Foss-Freitas, Simões, Foss, Squiers. Analysis and interpretation of data: Voltarelli, Couri, Stracieri, Malmegrim, Foss-Freitas, Simões, Foss, Squiers, Burt. Drafting of the manuscript: Voltarelli, Couri, Stracieri, Malmegrim, Simões, Squiers. Critical revision of the manuscript for important intellectual content: Voltarelli, Couri, Oliveira, Moraes, Pieroni, Coutinho, Malmegrim, Foss-Freitas, Simões, Foss, Squiers, Burt. Statistical analysis: Couri, Malmegrim, Squiers. Obtained funding: Voltarelli, Malmegrim, Squiers, Burt. Administrative, technical, or material support: Voltarelli, Stracieri, Malmegrim, Foss, Squiers. Study supervision: Voltarelli, Malmegrim, Foss, Squiers. Financial Disclosures: None reported. Funding/Support: Research supported by the Brazilian Ministry of Health, FAEPA-HCRP, FUNDHERP, FAPESP, CNPq, FINEP, Genzyme Corporation, and Johnson & Johnson-LifeScan–Brazil. Role of the Sponsor: The funding organizations did not participate in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript. Acknowledgment: We are grateful to Edson Martinez, PhD, and Davi Aragon, MSc, Center for Quantitative Methods of the School of Medicine of Ribeirão Preto, University of São Paulo (CEMEQ-FMRP-USP) for statistical advice; to Lewis Joel Greene, PhD, and Elettra Greene, BA, for English review; and to the multiprofessional team of the Bone Marrow Transplantation Unit and the Regional Blood Center of the Hospital das Clínicas of Ribeirão Preto, University of São Paulo, Brazil. Individuals named in this acknowledgment received no compensation from a funding sponsor for their contribution to this article. References 1. American Diabetes Association. Diagnosis and classification of diabetes. Diabetes Care. 2004;27:(suppl I) S5-S1014693921Google ScholarCrossref 2. Notkins AL, Lernmark A. Autoimmune type 1 diabetes: resolved and unresolved issues. J Clin Invest. 2001;108:1247-125211696564Google Scholar 3. Nathan DM. Long term complications of diabetes mellitus. N Engl J Med. 1993;328:1676-16858487827Google ScholarCrossref 4. Rubin RR, Peyrot M. Quality of life and diabetes. Diabetes Metab Res Rev. 1999;15:205-21810441043Google ScholarCrossref 5. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long term complications in insulin-dependent diabetes mellitus. N Engl J Med. 1993;329:977-9868366922Google ScholarCrossref 6. The Diabetes Control and Complications Trial Research Group. Effect of intensive therapy on residual beta-cell function in patients with type 1 diabetes in the Diabetes Control and Complications Trial. Ann Intern Med. 1998;128:517-5239518395Google ScholarCrossref 7. Elliott RB, Crossley JR, Berryman CC, James AG. Partial preservation of pancreatic beta-cell function in children with diabetes. Lancet. 1981;19:631-6326116102Google ScholarCrossref 8. Harrison LC, Colman PG, Dean B, Baxter R, Martin FI. Increase in remission rate in newly diagnosed type 1 diabetic subjects treated with azathioprine. Diabetes. 1985;34:1306-13083905463Google ScholarCrossref 9. Cook JJ, Hudson I, Harrison LC. et al. Double-blind controlled trial of azathioprine in children with newly diagnosed type 1 diabetes. Diabetes. 1989;38:779-7832656346Google ScholarCrossref 10. Silverstein J, Maclaren N, Riley W. et al. Immunosupression with azatioprine and prednisone in recent-onset insulin-dependent diabetes mellitus. N Engl J Med. 1988;319:599-6043045545Google ScholarCrossref 11. Canadian–European Randomized Control Trial Group. Cyclosporin-induced remission of IDDM after early intervention: association of 1 yr of cyclosporin treatment with enhanced insulin secretion. Diabetes. 1988;37:1574-15822903105Google ScholarCrossref 12. Herold KC, Hagopian W, Auger JA. et al. Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med. 2002;346:1692-169812037148Google ScholarCrossref 13. Keymeulen B, Vandemeulebroucke E, Ziegler AG. et al. Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med. 2005;352:2598-260815972866Google ScholarCrossref 14. Raz I, Elias D, Avron A, Metzger M, Cohen IR. Beta-cell function in newly-onset type 1 diabetes and immunomodulation with a heat shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial. Lancet. 2001;358:1749-175311734230Google ScholarCrossref 15. Saudek F, Havrdova T, Boucek P, Novota P, Skibova J. Polyclonal anti-T-cell therapy for type 1 diabetes mellitus of recent onset. Rev Diabet Stud. 2004;1:80-8817273515Google ScholarCrossref 16. Burt RK, Traynor A, Statkute L. et al. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematous. JAMA. 2006;295:527-53516449618Google ScholarCrossref 17. Burt RK, Slavin S, Burns WH, Marmont AM. Induction of tolerance in autoimmune diseases by hematopoietic stem cell transplantation: getting closer to a cure? Blood. 2002;99:768-78411806976Google ScholarCrossref 18. Kang EM, Zickler PP, Burns S. et al. Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established. Exp Hematol. 2005;33:699-70515911094Google ScholarCrossref 19. Palmer JP, Fleming GA, Greenbaum CA. et al. C-peptide is the appropriate outcome measure for type 1 diabetes clinical trials to preserve beta-cell function. Diabetes. 2004;53:250-26414693724Google ScholarCrossref 20. Weinhaus AJ, Bhagroo NV, Brelje TC, Sorenson RL. Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy. Endocrinology. 2000;141:1384-139310746642Google ScholarCrossref 21. Steffes MW, Sibley S, Jackson M, Thomas W. Beta cell function and the development of diabetes-related complications in the diabetes control and complications trial. Diabetes Care. 2003;26:832-83612610045Google ScholarCrossref 22. Au WY, Lie AK, Kung AW, Liang R, Hawkins BR, Kwong YL. Autoimmune thyroid dysfunction after hematopoietic stem cell transplantation. Bone Marrow Transplant. 2005;35:383-38815640829Google ScholarCrossref 23. Eisenbarth GS, Gottlieb PA. Autoimmune polyendocrine syndromes. N Engl J Med. 2004;350:2068-207915141045Google ScholarCrossref 24. Muraro PA, Douek DC, Packer A. et al. Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients. J Exp Med. 2005;201:805-81615738052Google ScholarCrossref 25. Hussain MA, Theise ND. Stem-cell therapy for diabetes mellitus. Lancet. 2004;364:203-20515246735Google ScholarCrossref 26. Couri CEB, Foss MC, Voltarelli JC. Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting beta-cell regeneration. Braz J Med Biol Res. 2006;39:1271-128016941054Google ScholarCrossref 27. Nelson JL, Torrez R, Louie FM, Choe OS, Storb R, Sullivan KM. Pre-existing autoimmune disease in patients with long-term survival after allogeneic bone marrow transplantation. J Rheumatol Suppl. 1997;48:23-29Google Scholar

Journal

JAMA – American Medical Association

Published: Apr 11, 2007

Keywords: diabetes mellitus, type 1,hematopoietic stem cell transplantation,follow-up,insulin,c-peptide,therapeutic immunosuppression,transplantation,pancreatic beta cell,cyclophosphamide,antithymoglobulin,conditioning (psychology),glutamate decarboxylase,hyperglycemia,granulocyte colony-stimulating factor,antibodies,leukapheresis,pneumonia,diabetic ketoacidosis,surrogate endpoints,hypogonadism,toxic effect,adverse effects,hypothyroidism,bone marrow transplantation,natural immunosuppression,hemoglobin a

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