Combined Islet and Hematopoietic Stem Cell Allotransplantation: A Clinical Pilot Trial to Induce Chimerism and Graft Tolerance
2008; Elsevier BV; Volume: 8; Issue: 6 Linguagem: Inglês
10.1111/j.1600-6143.2008.02230.x
ISSN1600-6143
AutoresDavide Mineo, Camillo Ricordi, X.-M. Xu, Antonello Pileggi, Rolando García-Morales, Aisha Khan, David A. Baidal, Dongmei Han, Kathy Monroy, J. Miller, Alberto Pugliese, Tatiana Froud, Luca Inverardi, Norma S. Kenyon, Rodolfo Alejandro,
Tópico(s)Diabetes Management and Research
ResumoAmerican Journal of TransplantationVolume 8, Issue 6 p. 1262-1274 Free Access Combined Islet and Hematopoietic Stem Cell Allotransplantation: A Clinical Pilot Trial to Induce Chimerism and Graft Tolerance D. Mineo, D. Mineo Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Internal Medicine, University Policlinic, 'Tor Vergata' University of Rome, ItalySearch for more papers by this authorC. Ricordi, C. Ricordi Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of Surgery Department of Microbiology-ImmunologySearch for more papers by this authorX. Xu, X. Xu Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorA. Pileggi, A. Pileggi Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of SurgerySearch for more papers by this authorR. Garcia-Morales, R. Garcia-Morales DeWitt Daughtry Family Department of Surgery Lillian Jean Kaplan Renal Transplant Center, Division of TransplantationSearch for more papers by this authorA. Khan, A. Khan Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorD. A. Baidal, D. A. Baidal Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorD. Han, D. Han Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorK. Monroy, K. Monroy Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorJ. Miller, J. Miller DeWitt Daughtry Family Department of Surgery Lillian Jean Kaplan Renal Transplant Center, Division of TransplantationSearch for more papers by this authorA. Pugliese, A. Pugliese Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Microbiology-ImmunologySearch for more papers by this authorT. Froud, T. Froud Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of Surgery Department of RadiologySearch for more papers by this authorL. Inverardi, L. Inverardi Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Microbiology-ImmunologySearch for more papers by this authorN. S. Kenyon, N. S. Kenyon Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of Surgery Department of Microbiology-Immunology Co-senior authors.Search for more papers by this authorR. Alejandro, Corresponding Author R. Alejandro Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Medicine-Endocrinology, L. Miller School of Medicine, University of Miami, FL Co-senior authors. *Corresponding author: Rodolfo Alejandro, ralejand@med.miami.eduSearch for more papers by this author D. Mineo, D. Mineo Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Internal Medicine, University Policlinic, 'Tor Vergata' University of Rome, ItalySearch for more papers by this authorC. Ricordi, C. Ricordi Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of Surgery Department of Microbiology-ImmunologySearch for more papers by this authorX. Xu, X. Xu Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorA. Pileggi, A. Pileggi Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of SurgerySearch for more papers by this authorR. Garcia-Morales, R. Garcia-Morales DeWitt Daughtry Family Department of Surgery Lillian Jean Kaplan Renal Transplant Center, Division of TransplantationSearch for more papers by this authorA. Khan, A. Khan Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorD. A. Baidal, D. A. Baidal Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorD. Han, D. Han Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorK. Monroy, K. Monroy Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FLSearch for more papers by this authorJ. Miller, J. Miller DeWitt Daughtry Family Department of Surgery Lillian Jean Kaplan Renal Transplant Center, Division of TransplantationSearch for more papers by this authorA. Pugliese, A. Pugliese Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Microbiology-ImmunologySearch for more papers by this authorT. Froud, T. Froud Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of Surgery Department of RadiologySearch for more papers by this authorL. Inverardi, L. Inverardi Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Microbiology-ImmunologySearch for more papers by this authorN. S. Kenyon, N. S. Kenyon Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL DeWitt Daughtry Family Department of Surgery Department of Microbiology-Immunology Co-senior authors.Search for more papers by this authorR. Alejandro, Corresponding Author R. Alejandro Clinical Islet Transplant Program (CITP), Diabetes Research Institute, L. Miller School of Medicine, University of Miami, FL Department of Medicine-Endocrinology, L. Miller School of Medicine, University of Miami, FL Co-senior authors. *Corresponding author: Rodolfo Alejandro, ralejand@med.miami.eduSearch for more papers by this author First published: 30 May 2008 https://doi.org/10.1111/j.1600-6143.2008.02230.xCitations: 34 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract To prevent graft rejection and avoid immunosuppression-related side-effects, we attempted to induce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem cell (HSC) infusion. Six patients with brittle type 1 Diabetes Mellitus received a single-donor allogeneic islet transplant (8611 ± 2113 IEQ/kg) followed by high doses of donor HSC (4.3 ± 1.9 × 106 HSC/kg), at days 5 and 11 posttransplant, without ablative conditioning. An 'Edmonton-like' immunosuppression was administered, with a single dose of anti-TNFα antibody (Infliximab) added to induction. Immunosuppression was weaned per protocol starting 12 months posttransplant. After transplantation, glucose control significantly improved, with 3 recipients achieving insulin-independence for a short time (24 ± 23 days). No severe hypoglycemia or protocol-related adverse events occurred. Graft function was maximal at 3 months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough levels, whereas 4 completed 1-year follow-up with functioning grafts. Graft failure occurred within 4 months from weaning (478 ± 25 days posttransplant). Peripheral chimerism, as donor leukocytes, was maximal at 1-month (5.92 ± 0.48%), highly reduced at 1-year (0.20 ± 0.08%), and was undetectable at graft failure. CD25+T-lymphocytes significantly decreased at 3 months, but partially recovered thereafter. Combined islet and HSC allotransplantation using an 'Edmonton-like' immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance. Introduction Recent progress in islet transplantation for brittle Type 1 Diabetes Mellitus (T1DM) has made this procedure a feasible, minimally invasive approach to avoid severe hypoglycemia and secondary complications, improving glucose control with variable insulin-independence and improved quality of life (1-4). Long-term results have shown immunosuppression-related side-effects and progressive decline in islet graft function, most of the patients requiring reintroduction of various levels of insulin therapy within 4–5 years posttransplant (1, 5-7). Donor bone marrow cell (BMC) transplantation following myelo- or lympho-ablative conditioning has been shown to induce recipient chimerism and graft tolerance in solid organ transplantation, with reduction or discontinuation of immunosuppression (8). Similar results were seen in experimental islet transplant models, after minimal or nonablative regimens (9). In animal and clinical studies, infusion of high doses of donor CD34+hematopoietic stem cells (HSC) using minimal or nonablative conditioning resulted in successful engraftment, reduced adverse events, immunomodulation and increased allograft survival (10-15). In this study, we attempted to induce recipient chimerism and graft tolerance in islet transplant recipients with brittle T1DM by infusing high doses of donor HSC without ablative conditioning to prevent graft rejection and eliminate life-long immunosuppression. An 'Edmonton-like' immunosuppression was given, with a single-dose of tumor necrosis factor-α (TNFα) monoclonal antibody (Infliximab) at induction to limit early posttransplant inflammation and maximize single-donor islet engraftment (1, 4, 16, 17). Immunosuppression was weaned per protocol starting 12 months posttransplant to evaluate islet survival and chimerism (18-21). Clinical status, islet function, glucose control, immune reactivity and chimerism were monitored to evaluate efficacy and safety of the protocol. Materials and Methods Study design and population This prospective, nonrandomized pilot study (NCT00315614) was approved by the University of Miami Institutional Review Board (IRB2000/0024) and written informed consent was obtained. The protocol included patients with long-term T1DM receiving a single-donor, freshly isolated allogeneic islet transplant followed by two infusions of high doses of cryopreserved donor HSC at days 5 and 11 posttransplant. No ablative conditioning for HSC engraftment was given to avoid related side-effects and risks. High doses of donor HSC (or BMC) have been shown to overcome the host HLA barrier, allowing for successful bone marrow (BM) engraftment, even without ablative regimens. Delayed timing of HSC (or BMC) infusions has resulted in greater engraftment and improved allograft survival (11-15). An 'Edmonton-like' steroid-free immunosuppression was administered, adding an antiinflammatory agent (Infliximab) at induction (1, 4, 16). Starting 12 months posttransplant, immunosuppression was weaned. This time-frame was considered adequate to allow for stable donor HSC engraftment (21) and sustained islet function (4). Detectable chimerism was not a prerequisite for weaning of immunosuppression, since occurrence of graft tolerance has been reported even in absence of measurable donor cells (18-20). Recipient selection and organ procurement Inclusion and exclusion criteria for islet transplantation were previously reported (1, 4, 16). Negative cross-match, recipient panel reactive antibodies (PRA) ≤20%, ABO/Rh and CMV serology but not HLA compatibility, were required (22). Islets and HSC were obtained from 15- to 45-year-old, heart-beating deceased donors. Both donor pancreata and vertebral bodies were preserved in specific cold-storage solutions and procurement media, and then shipped for processing and transplant (16, 23, 24). Bone marrow cell extraction and infusion Ten to twelve vertebral bodies were processed from each donor using a semi-automated system releasing around 3–5 × 1010BMC (25-27). To prevent graft-versus-host disease (GvHD), T-cell burden was indirectly reduced through CD34+ positive selection (Isolex 300i Magnetic Cell Separator; Baxter, Deerfield, IL) with a final cell preparation of >80% purity and >80% viability. Percentage of CD34+ and CD3+ cells was assessed by multicolor flow-cytometry (EPIX XL-MCL, Beckman Coulter, Fullerton, CA). Each BMC preparation was divided in two aliquots and stored in liquid nitrogen until infusion (28). Patients received a minimum of 2 × 106HSC/kg of recipient body weight (at least 10 × 108nucleated cells/kg), with a minimum of 2-logs T-cell reduction. All donors HSC obtained were infused (no maximum) (11-15). Islet cells isolation and transplantation Pancreata were processed using a modified automated method (29) and purified on density gradients (30), with a final islet preparation of purity >30% and viability >70% (16). Islets were infused by gravity into the portal vein (16, 31, 32) within 4 h from isolation. Patients received a minimum of 5000 IEQ/kg of recipient body weight (at least 300 000 IEQ total) in ≤5 mL of packed tissue. Immunosuppressive regimen Induction included Daclizumab (Zenapax®, Roche-Pharma, 1 mg/kg intravenously for five doses, starting the day of transplant, then every 14 days) plus Infliximab (Remicade®, Centocor, 5 mg/kg intravenously 2 h prior to islet infusion) (1). Maintenance consisted of sirolimus (Rapamune®, Wyeth-Ayerst, 0.2 mg/kg orally pretransplant then 0.15 mg/kg once daily, to attain trough levels of 12–15 ng/mL the first 3 months and 8–10 ng/mL thereafter) and low-dose tacrolimus (Prograf®, Fujisawa-Astellas, 1 mg orally pretransplant then 1 mg twice daily, to maintain trough levels of 3–6 ng/mL) (1, 4, 16). Immunosuppression weaning began by reducing tacrolimus (0.5 mg/week), then sirolimus (1 mg/week). Clinical monitoring Metabolic assessments included fasting plasma glucose and C-peptide, daily insulin requirement, HbA1c, C-peptide-to-glucose ratio (CPGR) and indexes from the mixed-meal tolerance test (MMTT), namely the 90 minute-glucose (90 min-glc) and the mixed-meal stimulation index (MMSI), as ratio of C-peptide and glucose areas under-the-curve (33, 34). Occurrences of hypoglycemic unawareness and coma were monitored. Renal function was evaluated by 24-h albumin urine excretion and estimated glomerular filtration rate (eGFR), using the modification of diet in renal disease equation (35), and the National Kidney Foundation stages for chronic kidney diseases (36). Immunosuppression trough levels were measured and drug-related adverse events evaluated. Definitions Insulin-independence: C-peptide positive recipients maintaining, without insulin therapy, an HbA1c <6.5% and a fasting and/or 2-h postprandial finger-stick (capillary) blood glucose (FBG) <140 mg/dL and 140 mg/dL and/or >180 mg/dL, respectively, and/or an HbA1c >6.5% in two consecutive measurements (33, 34). Islet graft failure: recipients with two or more consecutive fasting C-peptide <0.15 ng/mL, in the absence of hypoglycemia, and/or a stimulated peak of C-peptide <0.3 ng/mL during a MMTT within a month (34). Immune monitoring Chimerism was assessed monthly as percentage of donor cells circulating in the recipient's peripheral blood, including leukocytes, CD3+ and CD34+ cells. At 1-year posttransplant, a BM iliac aspirate was obtained and chimerism similarly assessed. A combined PCR and flow-cytometry analysis was used for detection of HLA class II DRβ gene and specific CD cell-surface epitopes on single fixed cells (37). White blood cell count was routinely measured and immunophenotyping was performed by multicolor flow-cytometry, using fluorochrome labeled antibodies and isotype matched control (38). Cell-surface markers included: total T-lymphocytes (CD45+/3+) and relative subpopulations (CD45+/3+/4+, CD45+/3+/8+, CD3+/4+/25+, CD3+/8+/25+), B-lymphocytes (CD20+/40+/19+) and natural killer (NK) cells (CD56+/16+/3−). Presence of PRA positivity and HLA class I and II alloantibodies was detected using a complement-dependent microlymphocytotoxic (CDC) technique (LCT assay, One Lambda). Recently, stored sera were retested with more modern and sensitive ELISA and flow-cytometry techniques (LAT assay and LABScreen assay plus LABScan flow analyzer, One Lambda and Luminex) (22). Recipient ability to respond to donor antigens was evaluated in a one-way mixed lymphocyte reaction (MLR). Recipient peripheral blood mononuclear cells (PBMC) were challenged with γ-irradiated donor splenocytes (stored frozen cells), third-party cells, and self-PBMC (negative control). Recipients PBMC were also cocultured with phytohemoagglutinin (PHA) (positive control). Data were calculated as mean counts per minute (cpm) of quadruplicate cultures. Results were expressed as ratio (Stimulation Index) of proliferation (cpm) observed for donor, third-party, or PHA reactions divided by recipient versus self-cpm (39). The cytotoxic T-lymphocyte genes (CLG) Granzyme B (GB), perforin (P) and Fas-Ligand (FasL) were evaluated by mRNA expression levels using real-time RT-PCR techniques (40). Results were expressed as the percentage ratio of copy number of the target genes to copy number of the control gene β-actin (40). Autoantibody levels for GAD65, IA2 and insulin were evaluated for recurrence of autoimmunity, using standard radioimmunoassay (41). Results were expressed as the ratio of values of graft recipients to values of age- and sex-matched healthy control subjects. Statistical analysis Demographics and descriptive statistics were expressed as mean ± standard deviation (SD). Due to the nonnormal distribution of some variables and the small sample size, nonparametric tests for paired (intra-group) comparisons were used (Wilcoxon Rank-Sum test, SPSS 14.0, Chicago, IL). A p-value <0.05 was considered statistically significant. Results From April through September 2000, 6 patients with T1DM were transplanted (2 males, 4 females, age 39 ± 6.9 years, disease duration 27 ± 11.8 years, weight 67.5 ± 16.9 kg, body mass index 23.8 ± 3.9 kg/m2), receiving a total of 574 475 ± 141 619 IEQ (8611 ± 2113 IEQ/kg) and 283.3 ± 126 × 106 HSC (4.3 ± 1.9 × 106 HSC/kg). Due to technical problems, patient 1 received a whole BMC infusion, a total of 3.5 × 1010 nucleated cells, resulting in a total of 227.0 × 106 HSC (2.3 × 106 HSC/kg) at flow-cytometry (Table 1). No procedure-related adverse events occurred. Table 1. Transplantation characteristics Pt Sex Weight (kg) HLA mismatch (A, B, DR) IEQ/kg (Purity%) HSCs (×106/kg) (Purity%) Days of insulin independence Tacrolimus weaning (Day) Sirolimus weaning (Day) Graft failure (Day) Begun End Begun End Posttransplant Postweaning 1 m 98.0 1, 1, 2 7046 (45) 2.3 (2.2)# – 377 440 478° 503° 451∞ 74∞ 2 f 54.5 2, 1, 2 12 485 (37) 3.7 (87) 50 387 415 438 480 480 93 3 f 60.6 2, 2, 2 8012 (45) 2.7 (95) 5 – – – – 178§ – 4 f 56.0 1, 2, 2 7039 (50) 6.7 (91) 17 388 430 438 465 471 83 5 f 59.5 1, 2, 2 7534 (30) 3.9 (95) – – – – – 158§ – 6 m 76.5 1, 1, 2 9547 (40) 6.4 (86) – 395 422 434 463 510 115 M - 67.5 5 8611 (41) 4.3 (91) 24 387 427 437 469 478 91 SD – 16.9 1 2113 (7) 1.9 (4) 23 7 11 2 9 25 18 IEQ = islet equivalent; HSC = hematopoietc stem cells; HLA = human leukocyte antigens; f = female; m = male; M = mean; SD = standard deviation. #Recipient of whole Bone Marrow Cells (purity value not included in the statistics). °Variable not included in the statistics because of off-protocol sirolimus weaning. ∞Islet graft failure after tacrolimus weaning only. §Variable not included in the statistics because of early graft rejection. Clinical monitoring Islet graft failure occurred in recipients 3 and 5 within 6 months posttransplant, while having low immunosuppressive drugs trough levels due to poor therapeutic adherence and severe drug-related psychosis requiring reduction of immunosuppression, respectively, and follow-up was accordingly interrupted. The remaining four recipients maintained graft function up to 1 year (Table 1). All recipients, except for those with early graft failure, had the greatest islet function and the best improvement in glycemic control in the first 3 months posttransplant but slowly deteriorated thereafter. Daily insulin requirements significantly decreased within this period (−65% of pretransplant value), with recipients 2, 3 and 4 achieving insulin-independence for a short time (50, 5 and 17 days, respectively). CPGR, 90 min-glc and MMSI significantly improved at 3 months posttransplant but worsened thereafter. By 12 months posttransplant, the four recipients with preserved grafts showed a relevant reduction in islet function and glycemic control, with increased daily insulin requirements (−40% of pretransplant value) (Tables 1 and 2, Figure 1). Table 2A. Metabolic parameters Pt Fasting C-peptide (ng/mL) HbA1c (%) Daily insulin requirements (UI/kg/day) Pretransplant 3 months 12 months Graft failure Pretransplant 3 months 12 months Graft failure Pretransplant 3 months 12 months Graft failure 1 0.13 0.95 0.24 0.12∞ 6.6 5.6 6.1 6.8∞ 0.80 0.38 0.51 0.45∞ 2 0.12 1.21 0.56 0.16 8.1 5.8 6.9 7.3 0.69 0.04 0.17 0.29 3 0.12 0.86§ – 0.15† 9.0 6.4§ – 8.1† 0.58 0.16§ – 0.41† 4 0.12 1.16 0.33 0.14 7.0 5.2 7.0 6.8 0.48 0.05 0.34 0.37 5 0.10 0.55§ – 0.13‡ 6.6 5.6§ – 6.3‡ 0.46 0.26§ – 0.32‡ 6 0.10 1. 11 0.43 0.18 7.1 5.7 6.8 7.1 0.73 0.39 0.46 0.59 M 0.12 1.11** 0.39 0.15 7.4 5.6** 6.7 7.1 0.62 0.22** 0.37 0.40 SD 0.01 0.11 0.14 0.02 0.7 0.3 0.4 0.6 0.14 0.20 0.15 0.11 ∞Islet graft failure after Tacrolimus weaning only. †Value at 6th and ‡ at 5th month after islet transplantation, respectively. §Variable not included in the statistics because of early graft rejection. *p < 0.05 and **p < 0.03 (Wilcoxon Rank-Sum test); M = means; SD = standard deviation. Table 2B. Renal function and immunosuppression trough levels Pt Albuminuria (mg/24 hr); (eGFR mL/min/1.73 m2) Tacrolimus (ng/mL) Sirolimus (ng/mL) Pretransplant 12 months Graft failure° 1 month 3 months 12 months Graft failure 1 month 3 months 12 months Graft failure 1 7 (62) 13 (78) 0 (59) 3.41 3.81 3.40 0.30∞ 10.08 11.38 8.81 9.85∞ 2 21(68) 29 (74) 0 (65) 4.10 5.40 5.25 0.00 12.24 12.29 10.80 6.63 3 58 (78) – 65 (86)† 1.62§ 3.58§ – 1.50† 6.93§ 12.01§ – 7.40† 4 26 (46) 80 (43) 19 (47) 3.40 3.53 4.45 0.00 12.59 12.00 10.70 7.55 5 0 (66) – 0 (64)‡ 3.19§ 1.55§ – 1.90‡ 11.91§ 8.19§ – 7.44‡ 6 5 (98) 9 (99) 10 (95) 4.14 3.95 6.10 0.00 14.91 13.05 9.50 7.00 M 19.7 (69.5) 32.9 (73.6) 15.6 (69.3) 3.76 4.17 4.80 0.62 12.46 12.18 9.95 7.64 SD 21.3 (17.3) 32.6 (26.9) 25.2 (17.8) 0.41 0.84 1.15 0.86 1.98 0.69 0.96 1.13 eGFR = estimated glomerular filtration rate; M = mean; SD = standard deviation. °Values after graft failure and completion of immunosuppression weaning. ∞Islet graft failure after tacrolimus weaning only. †Value at 6th and ‡ at 5th month after islet transplantation, respectively. §Variable not included in the statistics because of early graft rejection. Figure 1Open in figure viewerPowerPoint Clinical monitoring. (A) Metabolic parameters (B) Metabolic indexes (CPGR: C-peptide/Glucose Ratio; MMSI = mixed-meal stimulation index; 90 min-glc: 90 minute-glucose). (C) Immunosuppression trough levels. Data are expressed as mean ± SD. IS weaning: Immunosuppression weaning. Solid lines refer to left Y axis, while dotted lines refer to right Y axis; *p < 0.05 and **p < 0.03 (Wilcoxon Rank-Sum test). Following the 12-month follow-up visit, weaning of immunosuppression began for the four recipients with preserved graft function. Islet graft failure subsequently occurred within 4 months (478 ± 25 days posttransplant). A mild transient amelioration in fasting C-peptide and HbA1c was seen during weaning, with slight reduction of daily insulin requirements and improvement of CPGR, but not of 90 min-glc and MMSI. At islet graft failure, the metabolic parameters approximated pretransplant levels in all recipients, although lower daily insulin requirements were maintained (−35% of pretransplant value) (Tables 1 and 2, Figure 1). Severe hypoglycemic episodes or coma did not recur posttransplant, with all recipients claiming improved hypoglycemia awareness. Renal function was normal pre-transplant, except for recipient 3 with micro-albuminuria, and recipient 4 with reduced eGFR. At 1-year posttransplant, while on immunosuppression, the remaining four recipients remained stable, except for recipient 4 who developed micro-albuminuria, without changes in eGFR. Recipient 3 showed persistent micro-albuminuria even after the end of the study. After islet graft failure and immunosuppression weaning, no changes were seen in renal parameters except for recipient 4 who reversed to normo-albuminuria with unmodified eGFR (Table 2). Immunosuppression levels were consistently in the therapeutic range in the 4 recipients who completed 1 year of follow-up, except for recipient 1, whose sirolimus levels were below targeted level in the first 3 months posttransplant (Table 2). During follow-up, all recipients had multiple immunosuppression-related side-effects, with similar frequency and severity previously reported using 'Edmonton-like' protocols (5, 42, 43). The most important were: mucocutaneous (oral ulcers, n = 4 recipients), hematological (leucopenia, n = 5; anemia, n = 3), infective (respiratory tract infections, n = 5), metabolic (hyperlipidemia, n = 3) gynecological (dysfunctional menstrual bleeding, requiring endometrial ablation, n = 1), and neuropsychiatric (depression and anxiety, requiring reduction of immunosuppression, n = 1). All resolved satisfactorily after specific treatments without any sequelae. Immune monitoring Peripheral chimerism, measured as donor leukocytes, was maximal in the first 3 months posttransplant, then progressively decreased, highly reduced at 12 months and was undetectable at islet failure. At 12 months, donors HSC were slightly higher in the circulation than in the BM, while leukocytes and CD3+T-cells showed an opposite trend. Recipients 3 and 5 presented higher levels of chimerism at the time of islet graft failure when compared to the remaining four recipients at 1-year posttransplant, before starting immunosuppression weaning (Table 3, Figure 2). Table 3. Chimerism characteristics Pt Circulating donor cells%/Bone marrow donor cells%∞ Leukocytes CD34+cells CD3+cells 1 month 3 months 12 months 1 month 3 months 12 months 1 month 3 months 12 months 1 5.10 3.30 0.32 (0.50) 82.6 72.6 11.1 (1.08) 4.33 2.07 0.19 (0.56) 2 5.92 4.10 0.18 (0.51) 86.0 78.7 10.1 (1.35) 3.64 2.46 0.11 (0.60) 3 5.98 4.40 1.90†§ 85.9 81.9 – 3.79 2.71 – 4 5.80 4.56 0.18 (0.32) 80.3 79.4 11.0 (0.87) 4.90 3.52 0.11 (0.44) 5 6.59 4.70 1.68‡§ 84.1 78.9 – 4.16 3.52 – 6 6.10 4.00 0.12 (0.38) 85.3 80.1 5.3 (0.79) 4.11 2.57 0.08 (0.40) M 5.92 4.18 0.20 (0.43) 84.0 78.6 9.4 (1.02) 4.16 2.81 0.12 (0.50) SD 0.48 0.51 0.08 (0.09) 2.2 3.2 2.8 (0.25) 0.44 0.59 0.05 (0.07) †Value at 6th and ‡ at 5th month after islet transplantation, respectively. §Variable not included in the statistics because of early graft rejection. ∞Bone marrow donor cells from iliac aspirates at 12 months; M = mean; SD = standard deviation. Figure 2Open in figure viewerPowerPoint Recipient chimerism. Data are expressed as mean ± SD. Solid lines refer to left Y axis, while dotted lines refer to right Y axis. White blood cell counts presented a significant reduction of leukocytes, mainly neutrophils, within the first trimester posttransplant,
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