Pretreatment Of Recipients Research Articles

Impact of the Recipient's Pre-Treatment Blood Lymphocyte Count on Intended and Unintended Effects of Anti-T-Lymphocyte Globulin in Allogeneic Hematopoietic Stem Cell Transplantation.

Background: In allogeneic hematopoietic stem cell transplantation (HSCT), Anti-T-Lymphocyte Globulin (ATLG) may be used for the prevention of severe graft-versus-host disease (GVHD). ATLG targets both the recipient's lymphocytes and those transferred with the graft. Assuming an inverse relation between the recipient's absolute lymphocyte count (ALC) and exposure of remaining ATLG to the graft, we aim to evaluate the impact of the recipient's ALC before the first ATLG administration on the benefits (prevention of GVHD and GVHD-associated mortality) and potential risks (increased relapse incidence) associated with ATLG. Methods: In recipients of HLA-matched, ATLG-based HSCT (n = 311), we assessed the incidence of acute GVHD, GVHD-related mortality and relapse, as well as other transplant-related outcomes, in relation to the respective ALC (divided into tertiles) before ATLG. Results: The top-tertile ALC group had a significantly increased risk of aGVHD (subhazard ratio (sHR) 1.81; [CI 95%; 1.14-2.88]; p = 0.01) and aGVHD-associated mortality (sHR 1.81; [CI 95%; 1.03-3.19]; p = 0.04). At the highest ATLG dose level (≥45 mg/kg), recipients with lowest-tertile ALC had a trend towards increased relapse incidence (sHR 4.19; [CI 95%; 0.99-17.7]; p = 0.05, n = 32). Conclusions: ATLG dosing based on the recipient's ALC may be required for an optimal balance between GVHD suppression and relapse prevention.

Twenty Years of Gut Transplantation for Chronic Intestinal Pseudo-obstruction: Technical Innovation, Long-term Outcome, Quality of Life, and Disease Recurrence.

To define long-term outcome, predictors of survival, and risk of disease recurrence after gut transplantation (GT) in patients with chronic intestinal pseudo-obstruction (CIPO). GT has been increasingly used to rescue patients with CIPO with end-stage disease and home parenteral nutrition (HPN)-associated complications. However, long-term outcome including quality of life and risk of disease recurrence has yet to be fully defined. Fifty-five patients with CIPO, 23 (42%) children and 32 (58%) adults, underwent GT and were prospectively studied. All patients suffered gut failure, received HPN, and experienced life-threatening complications. The 55 patients received 62 allografts; 43 (67%) liver-free and 19 (33%) liver-contained with 7 (13%) retransplants. Hindgut reconstruction was adopted in 1993 and preservation of native spleen was introduced in 1999. Immunosuppression was tacrolimus-based with antilymphocyte recipient pretreatment in 41 (75%). Patient survival was 89% at 1 year and 69% at 5 years with respective graft survival of 87% and 56%. Retransplantation was successful in 86%. Adults experienced better patient (P = 0.23) and graft (P = 0.08) survival with lower incidence of post-transplant lymphoproliferative disorder (P = 0.09) and graft versus host disease (P = 0.002). Antilymphocyte pretreatment improved overall patient (P = 0.005) and graft (P = 0.069) survival. The initially restored nutritional autonomy was sustainable in 23 (70%) of 33 long-term survivors with improved quality of life. The remaining 10 recipients required reinstitution of HPN due to allograft enterectomy (n = 3) or gut dysfunction (n = 7). Disease recurrence was highly suspected in 4 (7%) recipients. GT is life-saving for patients with end-stage CIPO and HPN-associated complications. Long-term survival is achievable with better quality of life and low risk of disease recurrence.

Influenza Infection Induces RBC Alloimmunization By a Type 1 Interferon Dependent Mechanism

Red blood cell (RBC) transfusion exposes recipients to hundreds of non-ABO antigens that are not matched between donors and recipients. Alloantibodies formed against these antigens can cause potentially fatal hemolytic events and limit availability of compatible blood products, resulting in anemia-associated morbidity and mortality. Multiple studies have established that certain autoimmune diseases and inflammatory disorders are associated with an increased frequency of alloimmunization. However, underlying molecular mechanisms are poorly understood. A recent study reported that pre-treatment of the transfusion recipient with a pro-inflammatory stimulus (poly(I:C), a double-stranded RNA), induces type 1 interferons (IFNα/β) that promote RBC alloimmunization in mice. This finding suggests that IFNα/β-inducing infections may increase the risk of alloimmunization in transfusion recipients. Here, we hypothesized that infection with influenza, known to induce multiple cytokines including IFNα/β, may promote RBC alloimmunization. Wildtype mice were infected with 10 plaque forming units of PR8 influenza A virus 3 days prior to transfusion with transgenic RBCs expressing the KEL antigen (KEL RBCs). Anti-KEL IgM and IgG responses were measured by flow cytometry 5 and 14-28 days, respectively, following transfusion. Although mock-infected transfused wildtype mice produced low baseline levels of anti-KEL antibodies (IgG peak response mean MFI = 18.5 ± 5.7, standard error), influenza-infected mice produced significantly elevated levels of anti-KEL IgM and IgG (IgG, MFI = 675.7 ± 150.5, p<0.05) in 3 out of 3 (3/3) experiments. The influenza-induced anti-KEL antibody response was associated with increased KEL RBC clearance 1 hr post-transfusion (% of KEL RBCs remaining = 73.5% ± 0.06, p<0.05) compared to mock-infected transfused mice (98.6% ± 0.01). Influenza infection prior to transfusion also led to enhanced RBC-specific T cell proliferation and T follicular helper (Tfh) cell differentiation (% of RBC-specific T cells, Tfh = 8.9% ± 0.9, p<0.05) compared to mock-infected transfused controls (Tfh = 4.1% ± 1.1). To determine whether IFNα/β signaling contributes to influenza-induced alloimmunization, the alloimmune response to KEL RBCs was examined in influenza-infected mice lacking the receptor for IFNα/β (IFNAR1-/-) or transcription factors required for IFNα/β production (IRF3-/- IRF7-/-). As shown in Figure 1a, both IFNAR1-/- and IRF3-/- IRF7-/- mice produced significantly lower levels of anti-KEL IgG following transfusion, compared to infected wildtype mice, in 3/3 experiments. Finally, treatment of infected wildtype mice with an IFNAR1-blocking antibody 1 day prior to infection and 1 day prior to KEL-RBC transfusion profoundly inhibited anti-KEL IgG production, compared to isotype control treated mice in 3/3 experiments (Figure 1b). Collectively, these results indicate that influenza infection of mice promotes alloimmunization to an RBC antigen by an IFNα/β dependent mechanism. These data contribute to a growing appreciation of the role that IFNα/β plays in RBC alloimmunization in animal models, and suggest that an investigation into the role of type 1 interferons in human alloimmunization may be informative. DisclosuresNo relevant conflicts of interest to declare.

Recipient hypertonic saline infusion prevents cardiac allograft dysfunction

ObjectiveHypertonic saline (HTS) has potent immune and vascular effects. We assessed recipient pretreatment with HTS on allograft function in a porcine model of heart transplantation and hypothesized that HTS infusion would limit endothelial and left ventricular (LV) dysfunction following transplantation. MethodsHeart transplants were performed after 6 hours of cold ischemic storage. Recipient pigs were randomized to treatment with or without HTS (7.5% NaCl) before cardiopulmonary bypass (CPB). Using a myograft apparatus, coronary artery endothelial-dependent (Edep) and -independent (Eind) relaxation was assessed. LV performance was determined using pressure-volume loop analysis. Pulmonary interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α expression was measured. ResultsWeaning from CPB and LV performance after transplantation were improved in HTS-treated animals. Successful weaning from CPB was greater in the HTS-treated hearts (8 of 8 vs 2 of 8; P < .05). Mean LV functional recovery was improved in the HTS-treated animals, as assessed by preload recruitable stroke work (65 ± 10% vs 27 ± 10%; P < .001) and end-systolic elastance (55 ± 7% vs 37 ± 4%; P < .001). Treatment with HTS resulted in improved Edep (mean maximum elastance [Emax], 56 ± 5% vs 37 ± 7%; P < .001) and Eind (mean Emax%, 77 ± 6% vs 52 ± 4%; P < .001) vasorelaxation compared with control. Pulmonary expression of IL-2, IL-6, and TNF-α increased following transplantation, whereas HTS therapy attenuated IL production (P < .001). Transplantation increased plasma TNF-α levels and LV TNF-α expression, whereas HTS prevented this up-regulation (P < .001). ConclusionsRecipient HTS pretreatment preserves allograft vasomotor and LV function, and HTS therapy limits CPB-induced injury. HTS may be a novel recipient intervention to prevent graft dysfunction.

Photodynamic Therapy Leads to Time-Dependent Regression of Pathologic Corneal (Lymph) Angiogenesis and Promotes High-Risk Corneal Allograft Survival.

Pathologic corneal (lymph) angiogenesis is a known risk factor for immune-mediated allograft rejections after corneal transplantation. However, there is no established treatment to regress pre-existing pathological corneal blood and lymphatic vessels. This study assessed the possibility to regress both vessel types by photodynamic therapy (PDT) after intravenous (i.v.) verteporfin injection, the influence of timing of PDT after verteporfin injection, and the effect on graft survival in high-risk keratoplasty. BALB/c mice were used for suture-induced inflammatory corneal neovascularization to induce combined hem- and lymphangiogenesis. The treated group received PDT 3 minutes, 1 hour, and 24 hours after an i.v. verteporfin injection (control group: phosphate buffered saline). Corneal flatmounts were excised 3 days, 1 week, and 2 weeks after corneal PDT and stained with cluster of differentiation 31 (CD31) and lymphatic vessel endothelial hyaluronan receptor 1 antibodies (LYVE-1) to quantify hem- and lymphangiogenesis. Graft survival rates were compared between high-risk recipients with and without preoperative PDT. Corneal blood vessels were significantly reduced when PDT was performed 3 minutes after i.v. verteporfin injection, whereas lymphatic vessels showed no significant difference. Both blood and lymphatic vessels were regressed when PDT was performed 1 hour or 24 hours after i.v. verteporfin application. Long-term allograft survival increased significantly in PDT-pretreated eyes when compared with controls. PDT after i.v. verteporfin injection can selectively regress pre-existing corneal blood vessels or both blood and lymphatic vessels depending on the timing of PDT after verteporfin injection. The pretreatment of recipients with PDT and i.v. verteporfin might be a promising new method to improve graft survival in high-risk eyes.

The composition of the microbiota modulates allograft rejection.

Transplantation is the only cure for end-stage organ failure, but without immunosuppression, T cells rapidly reject allografts. While genetic disparities between donor and recipient are major determinants of the kinetics of transplant rejection, little is known about the contribution of environmental factors. Because colonized organs have worse transplant outcome than sterile organs, we tested the influence of host and donor microbiota on skin transplant rejection. Compared with untreated conventional mice, pretreatment of donors and recipients with broad-spectrum antibiotics (Abx) or use of germ-free (GF) donors and recipients resulted in prolonged survival of minor antigen-mismatched skin grafts. Increased graft survival correlated with reduced type I IFN signaling in antigen-presenting cells (APCs) and decreased priming of alloreactive T cells. Colonization of GF mice with fecal material from untreated conventional mice, but not from Abx-pretreated mice, enhanced the ability of APCs to prime alloreactive T cells and accelerated graft rejection, suggesting that alloimmunity is modulated by the composition of microbiota rather than the quantity of bacteria. Abx pretreatment of conventional mice also delayed rejection of major antigen-mismatched skin and MHC class II-mismatched cardiac allografts. This study demonstrates that Abx pretreatment prolongs graft survival, suggesting that targeting microbial constituents is a potential therapeutic strategy for enhancing graft acceptance.

Visceral Transplantation for End-Stage Crohnʼs Disease: 25 Years of Experience at a Single Center

Introduction: Intestinal and multivisceral transplantation has recently evolved and more frequently utilized for patients with irreversible intestinal and TPN failure. End-stage Crohn's disease (CD) has been the second leading indication in adults. This is the largest worldwide series of CD patients who received visceral transplantation at a single center. Long-term efficacy is addressed with special reference to disease recurrence. Methods: Over 25 years, 57 CD patients underwent visceral transplantation for a recalcitrant disease with a mean duration of 5+5 years. All patients failed TPN due to multiple line infection (94%), limited venous access (83%) and significant liver damage (80%). Male to female ratio was 1:1.7 with a mean age of 43+10 years. All patients underwent multiple abdominal operations with proctocolectomy in 37 (65%). Simultaneous hepatic replacement was required in 12 (21%) patients with a mean serum bilirubin of 9+11mg/dl. Remaining 45 (79%) received liver-free visceral allografts with intestine alone in 43 and modified multivisceral graft including stomach, duodenum, pancreas, and intestine in 2. Rejection prophylaxis was tacrolimus based with induction therapy in 8 (14%) and recipient pretreatment in 37 (65%) recipients. Results: With a mean follow-up of 54+48 months, 33 (58%) patients were alive with a retransplantation rate of 7%. Rejection was the leading cause of graft loss with an overall incidence of 56%. Actuarial patient survival was 90% at 1 year, 56% at 5 years, and 43% at 10 years with respective graft survival of 86%, 53%, and 42%. Inclusion of donor liver was associated with better outcome with a 10-year survival rate of 57%. Recipient pretreatment significantly improved patient survival with respective 1, 5, and 10-year survival rates of 92%, 61% and 61%. All survivors achieved full nutritional autonomy. Disease recurrence was histologically documented in 4 (7%) allografts at 3, 15, 18, and 19 months from date of transplant with no impact on graft function. There was no significant (P=0.6) difference in survival between CD and non-CD patients with a higher cumulative risk of rejection induced graft loss among CD patients. Conclusion: Visceral transplantation is an effective life-saving treatment for patients with end-stage CD. Disease recurrence is low with no significant impact on functional survival.

Study of biological markers for radiation pretreatment of hematopoietic stem cell transplant recipients

目的研究小鼠受60Co γ射线照射后血液和免疫系统的早期生物参数变化，为造血干细胞移植(HSCT)受者放疗预处理寻找灵敏可靠的生物剂量标志物。方法采用纯品系BALB/c雄性小鼠，随机分为1个对照组和3个照射组。应用60Coγ射线全身照射，吸收剂量分别为0、2、4和6 Gy。照射后24 h，外周血白细胞计数及淋巴细胞百分率应用血常规计数测定；外周血和骨髓嗜多染红细胞微核(mn-PCE)应用Giemsa染色镜下计数观察；骨髓细胞、脾脏和胸腺早期凋亡率应用AnnexinⅤ/PI双染法流式细胞仪检测。结果与对照组比较，各照射组小鼠外周血白细胞计数及淋巴细胞百分率随辐射剂量增加而显著降低(P<0.01)，其回归方程分别为E=0.1750D2−1.7440D+5.2020和E=84.9390−3.4255D；外周血和骨髓mn-PCE随辐射剂量增加而显著增加(P<0.01)，骨髓mn-PCE与辐射剂量呈正相关，其回归方程为E=3.9725D+2.9700；骨髓细胞、脾脏和胸腺早期凋亡率随辐射剂量增加而显著增高(P<0.01)，呈线性正相关，回归方程分别为E=3.42D+8.36，E=3.0645D+3.1840和E=2.5620D+2.5090。结论外周血淋巴细胞计数和骨髓嗜多染红细胞微核率与辐射剂量呈线性相关，其回归方程可作为放射预处理剂量评估灵敏可靠的早期生物剂量标志物；骨髓细胞、脾脏和胸腺淋巴细胞早期凋亡率与辐射剂量呈线性正相关，其回归方程可用于HCST受者放射预处理免疫系统抑制程度的判定指标。

Pre-treatment of allogeneic bone marrow recipients with the CXCR4 antagonist AMD3100 transiently enhances hematopoietic chimerism without promoting donor-specific skin allograft tolerance

Hematopoietic chimerism established by allogeneic bone marrow transplantation is known to promote donor-specific organ allograft tolerance; however, clinical application is limited by the need for toxic host conditioning and “megadoses” of donor bone marrow cells. A potential solution to this problem has been suggested by the observation that recipient bone marrow mobilization by the CXCR4 antagonist AMD3100 promotes chimerism in congenic bone marrow transplantation experiments in mice. Here we report that a single subcutaneous dose of 10mg/kg AMD3100 in recipient C57BL/6 mice was able to enhance hematopoietic chimerism when complete MHC-mismatched BALB/c donor bone marrow cells were transplanted 1h after drug dosing. However, levels of chimerism measured 30days post-transplantation were not sustained when mice were reexamined on day 90 post-transplantation. Moreover, transient chimerism induced by this protocol did not support robust donor-specific skin allograft tolerance. Using the same transient immunosuppression protocol, we confirmed that “megadoses” of donor bone marrow cells could induce durable chimerism associated with donor-specific skin allograft tolerance without AMD3100 pre-treatment. We conclude that in this protocol AMD3100 pretreatment may empty bone marrow niches that become reoccupied by allogeneic donor hematopoietic progenitor cells but not by true long-lived donor hematopoietic stem cells, resulting in short-lived chimerism and failure to support durable donor-specific allograft tolerance.

Intensive Perioperative Simvastatin Treatment Protects from Chronic Kidney Allograft Injury

Background/Aims: Ischemia-reperfusion injury (IRI) and innate immune response augment adaptive immunity and may also trigger repair processes that lead to uncontrolled fibrosis and atherosclerosis as seen in chronic allograft injury. Simvastatin has been shown to protect from renal IRI in several experimental studies. The aim of this study was to examine the effect of donor simvastatin pretreatment and early initiation of recipient simvastatin treatment on chronic kidney allograft injury. Methods: A rat renal transplantation model was used. Simvastatin was administered perorally for donor (5 mg/kg) and/or for recipient (2 mg/kg) 2 hours before transplantation and/or as daily treatment starting on the first postoperative day (2 mg/kg/day). The study included 5 groups: (1) no simvastatin, (2) donor pretreatment, (3) daily recipient treatment, (4) donor pretreatment + daily recipient treatment and (5) donor pretreatment + recipient pretreatment + daily recipient treatment. The grafts were recovered at day 90 for histopathological and immunohistochemical analysis. Kidney function was followed weekly with serum creatinine, and 24-hour urine protein was measured 60 and 90 days after transplantation. Results: We found that donor and recipient simvastatin pretreatment combined with daily recipient treatment reduced graft inflammation and chronic allograft injury. Treatment using only statins started after transplantation reduced inflammation to some extent, but did not affect chronic kidney allograft injury. Pretreatment using only donor statins impaired graft function and increased proteinuria. Conclusion: Our data suggest that perioperative recipient statin treatment reduces inflammation and may protect the graft in the long term.

T-regulatory cell treatment prevents chronic rejection of heart allografts in a murine mixed chimerism model

BackgroundThe mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection.MethodsWe recently developed a murine “Treg bone marrow (BM) transplantation (BMT) protocol” that is devoid of cytoreductive recipient pre-treatment. The protocol consists of a moderate dose of fully mismatched allogeneic donor BM under costimulation blockade, together with polyclonal recipient Tregs and rapamycin. Control groups received BMT under non-myeloablative irradiation and costimulation blockade without Treg therapy. Multilineage chimerism was followed by flow cytometry, and tolerance was assessed by donor-specific skin and heart allografts.ResultsDurable multilineage chimerism and long-term donor skin and heart allograft survival were successfully achieved with both protocols. Notably, histologic examination of heart allografts at the end of follow-up revealed that chronic rejection is prevented only in chimeras induced with the Treg protocol.ConclusionsIn a mouse model of mixed chimerism, additional Treg treatment at the time of BMT prevents chronic rejection of heart allografts. As the Treg-chimerism protocol also obviates the need for cytoreductive recipient treatment it improves both efficacy and safety over previous non-myeloablative mixed chimerism regimens. These results may significantly impact the development of protocols for tolerance induction in cardiac transplantation.

Attenuation of Transplant Arteriosclerosis by Oral Feeding of MHC Encoding Chitosan-DNA Nanoparticles Is Mediated by Humoral Immunity

Purpose One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Methods and Materials We performed fully allogeneic mouse abdominal aortic transplantats using C57BL/6 (H2 b ) mice as donors and CBA.J (H2 k ) mice as recipients Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Results Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K b , one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K b encoding nanoparticles (K b -NP) or Balb/c (H2 d ) grafts were used instead of C57BL/6 (H2 b ) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K b -NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. Conclusions These results demonstrate the potential of chitosan-DNA nanoparticles to induce K b -specific tolerance and to reduce the development of transplant arteriosclerosis.

Treg Treatment Prevents Heart Allograft Vasculopathy in a Murine Mixed Chimerism Model

Purpose The mixed chimerism approach has outstanding potency in the induction of donor-specific tolerance in both preclinical animal models and clinical pilot trials. However, some chimeras develop a state of “split tolerance”, an incompletely understood phenomenon. Cardiac allograft vasculopathy (CAV) and skin graft rejection were observed in chimeric animals that are tolerant to donor antigen in vitro . We hypothesized that Tregs therapy is able to prevent “split tolerance” and chronic rejection. Methods and Materials Groups of B6 mice received 2x10 7 unseparated Balb/c BMC (d0, iv) and costimulation blockade consisting of antiCD40LmAb (1mg, d0) and CTLA4Ig (0.5mg, d2). Control groups received 3 GyTBI to facilitate BM engraftment, whereas the “Treg protocol” received 5x10 6 Tregs (d0) and a short course of rapamycin (0.1mg/mouse, d-1/d0/d2). Multilineage chimerism was followed by flowcytometry, heterotopic heart transplantation was used to assess donor-specific tolerance. Results Treg therapy reliably induced longterm multilineage chimerism without the need for recipient TBI (8/8 chimeras Treg protocol vs 13/18 control group, p=0.28). Heart allografts survived longterm in both groups (>100 days, Treg group n=4; control group n=7), whereas third-party allografts were promptly rejected. Histological examination of donor heart allografts revealed that Treg treatment potently avoids CAV (all grafts were scored with ISHLT grade 0 or 1; 0: n=2, 1: n=2). In the control group 4/7 mice were classified with a rejection rate 2 or higher (0: n=1, 1: n=2, 2:n=2, 3:n=2). Conclusions In this study we demonstrate that Treg treatment enhances the potency of the mixed chimerism approach for the induction of donor-specific tolerance. Treg treatment obviates the need for cytoreductive recipient pretreatment and prevents CAV. Thus, both efficiency and safety of the mixed chimerism approach are improved. We think that these results will have significant impact on the development of new protocols for tolerance induction in cardiac transplantation.

Attenuation of transplant arteriosclerosis by oral feeding of MHC encoding chitosan-DNA nanoparticles is mediated by humoral immunity

Introduction: One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation.

Attenuation of transplant arteriosclerosis by oral feeding of major histocompatibility complex encoding chitosan-DNA nanoparticles

One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the alloimmune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Therefore, we performed fully allogeneic mouse abdominal aortic transplants using C57BL/6 (H2b) mice as donors and CBA.J (H2k) mice as recipients. Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for Kb, one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of Kb encoding nanoparticles (Kb-NP) or Balb/c (H2d) grafts were used instead of C57BL/6 (H2b) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the Kb-NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. These results demonstrate the potential of chitosan-DNA nanoparticles to induce Kb-specific tolerance and to reduce the development of transplant arteriosclerosis.

Intestinal Transplantation for End Stage Crohnʼs Disease: Therapeutic Efficacy and Disease Recurrence

Intestinal transplantation has recently evolved and more frequently utilized to rescue patients with irreversible intestinal failure who no longer can be maintained on TPN therapy. End stage Crohn's disease has been the second leading indication for transplantation in the adult population. This is the first study to address the procedure's therapeutic efficacy and the impact of disease recurrence on long-term outcome. Between May 2, 1990 and June 30, 2012, a total of 309 consecutive adult patients received 342 intestinal and multivisceral transplantations. Of these, 57 (18%) suffered recalcitrant Crohn's disease with irreversible intestinal failure for a mean duration of 5+5 years. All patients failed TPN therapy with multiple line infections (94%), limited venous access (83%), and significant liver damage (80%). The male to female ratio was 1:1.7 with a mean age of 43 + 10 years. All patients underwent multiple abdominal surgeries with proctocolectomy in 36 (63%). Simultaneous hepatic replacement was required in 12 (21%) patients due to end stage liver failure with a mean serum bilirubin of 9 + 11 mg/dL. The remaining 45 (79%) patients received liver-free visceral allografts with intestine alone in 43 and modified multivisceral graft including the stomach, duodenum, pancreas, and intestine in 2. Positive lymphocytotoxic crossmatch was documented in 14 (25%) recipients. All donors were cadaveric, ABO identical with random HLA crossmatch. Rejection prophylaxis was tacrolimus based in all patients with utilization of induction (cyclophosphamide/daclizumab) therapy in 8 (14%) and recipient pretreatment with rATG/alemtuzumab in 37 (65%) recipients. With a mean follow-up of 54 + 48 months, 33 (58%) of the Crohn's disease patients are currently alive with a retransplantation rate of 7%. Acute and chronic rejection was the leading cause of graft loss with an overall incidence of 56%. The actuarial patient survival rate was 90% at 1 year, 74% at 3 years, 56% at 5 years, and 43% at 10 years with respective graft survival of 86%, 65%, 53%, and 42%. Inclusion of the donor liver was associated with better long-term survival outcome with a 10-year survival rate of 57% for both patient and graft. Recipient pretreatment significantly improved patient survival with 1, 3, 5, and 10 year survival rates of 92%, 79%, 61% and 61%, respectively. All survivors achieved full nutritional autonomy enjoying unrestricted oral diet. Disease recurrence was histologically documented in 4 (7%) allografts at 3, 15, 18, and 19 months from the time of transplantation with no impact on graft function. With similar distribution of type of transplanted organs and immunosuppression, there was no significant (P = 0.6) difference in survival between the Crohn's and non-Crohn's patients. However, the cumulative risk of graft loss due to acute and chronic rejection was modestly higher in the Crohn's disease compared to the non-Crohn's disease patients. Intestinal and multivisceral transplantation is a life-saving and an effective therapy for patients with end stage Crohn's disease. Disease recurrence is very low and at best histologic with no significant impact on survival outcome and graft functions.

Development of a Murine Model of Weak Kel: Similarities to Weak Rh(D)

Abstract 842 Introduction:Aspects of RBC antigens that define their immunogenicity are not fully understood. Multiple studies in humans have demonstrated Rh(D) to be the most immunogenic RBC antigen, yet the same antigen present in lower copy number (weak Rh(D)) is, with rare exception, considered non-immunogenic. A better understanding of factors influencing RBC alloimmunogenicity would be helpful in predicting antibody responses in transfusion and pregnancy situations alike, as well as in managing donor RBC inventory. Herein, we describe the generation of transgenic mice with different levels of RBC specific expression of the clinically significant human KEL2 antigen, and test the hypothesis that antigen density impacts recipient immune response post-transfusion. Materials and Methods:Mice with RBC specific expression of KEL2 were generated utilizing constructs containing the human KEL2 sequence expressed behind a B-globin promoter, using a random integration approach. TER119+, CD45+, and CD41+ cells were evaluated by flow cytometry for KEL expression using monoclonal anti-Jsa and anti-Kpb, and RBC antigen density was estimated utilizing QIFIKIT beads. MuMT recipients were transfused with RBCs labeled with a lipophilic dye, and post-transfusion RBC recovery and antigen expression were evaluated by flow cytometry. To determine the immunogenicity of KEL2 or weak KEL2 RBCs, RBCs were transfused into C57BL/6 recipients every 2–3 weeks in the presence or absence of poly (I:C) pre-treatment. Recipient serum was analyzed by flow cytometric crossmatch with KEL2 or C57BL/6 RBC targets, using IgM or IgG secondary antibodies. To determine the effect of recipient RBC expression of weak KEL2 on the immunogenicity of KEL2 RBCs, weak KEL2 animals were transfused with KEL2 RBCs, the clearance of lipophilic labeled RBCs was tracked, and anti-KEL was evaluated on the transfused RBCs and also in the serum. Results:KEL2 RBCs have approximately 1200 antigenic sites per cell, whereas weak KEL2 RBCs have fewer than 200 sites; flow cytometric studies of TER 119+, CD45+, and CD41+ cells suggest both strains have RBC specific KEL expression. Transfusion of KEL2 or weak KEL2 RBCs into muMT animals resulted in stable post-transfusion RBC recovery and antigen expression. In 3/3 experiments (n=30 animals), all C57BL/6 recipients of KEL2 RBCs generated detectable anti-KEL IgM and IgG, which boosted with subsequent transfusions and which was enhanced in the presence of recipient inflammation with poly (I:C). However, in 2/2 experiments (n=20 animals), weak KEL RBCs led to no detectable antibody (IgM or IgG) in C57BL/6 recipients following 3 transfusions, even in the presence of recipient pre-treatment with poly (I:C). Furthermore, weak KEL2 recipients of KEL2 RBCs generated no detectable IgG and demonstrated no clearance of KEL2 RBCs, though low levels of anti-KEL IgM were detectable on the transfused RBCs and in the serum from approximately 5–12 days post-transfusion. Discussion:As hypothesized, antigen density significantly impacts the immunogenicity of KEL RBCs in this reductionist murine alloimmunization model. C57BL/6 recipients, like Rh(D) negative recipients, lack the human antigen in question (KEL2 in this case), and recipient antibody responses to weak KEL2 are, like most recipient responses to weak Rh(D), undetectable. Strengths of the KEL2 and weak KEL2 system include the fact that these animals are, to the best of our knowledge, genetically identical except for RBC KEL antigen copy number. Thus, this system allows for detailed analyses of the immune response to KEL RBCs with different antigen densities, on both the donor and recipient side of the equation, without confounding factors encountered in human studies (such as HLA presentation issues or considerations of the molecular basis of a particular type of weak Rh(D)). A better understanding of primary, secondary, and other immune responses in the KEL2 and weak KEL2 system may lay the groundwork for strategies to induce non-responsiveness to RBCs in humans, not only in the setting of transfusion medicine but also potentially in the setting of hemolytic disease of the fetus and newborn. Furthermore, translation of these and future findings in the KEL and weak KEL systems to Rh(D), weak Rh(D), and other human antigen systems may ultimately allow for creative solutions in blood inventory management. Disclosures:No relevant conflicts of interest to declare.

Preformed and De Novo Donor Specific Antibodies in Visceral Transplantation: Long-Term Outcome With Special Reference to the Liver

Despite improvement in early outcome, rejection particularly chronic allograft enteropathy continues to be a major barrier to long-term visceral engraftment. The potential role of donor specific antibodies (DSA) was examined in 194 primary adult recipients. All underwent complement-dependent lymphocytotoxic crossmatch (CDC-XM) with pre- and posttransplant solid phase HLA-DSA assay in 156 (80%). Grafts were ABO-identical with random HLA-match. Liver was included in 71 (37%) allografts. Immunosuppression was tacrolimus-based with antilymphocyte recipient pretreatment in 150 (77%). CDC-XM was positive in 55 (28%). HLA-DSA was detectable before transplant in 49 (31%) recipients with 19 continuing to have circulating antibodies. Another 19 (18%) developed de novo DSA. Ninety percent of patients with preformed DSA harbored HLA Class-I whereas 74% of recipients with de novo antibodies had Class-II. Gender, age, ABO blood-type, cold ischemia, splenectomy and allograft type were significant DSA predictors. Preformed DSA significantly (p < 0.05) increased risk of acute rejection. Persistent and de novo HLA-DSA significantly (p < 0.001) increased risk of chronic rejection and associated graft loss. Inclusion of the liver was a significant predictor of better outcome (p = 0.004, HR = 0.347) with significant clearance of preformed antibodies (p = 0.04, OR = 56) and lower induction of de novo DSA (p = 0.07, OR = 24). Innovative multifaceted anti-DSA strategies are required to further improve long-term survival particularly of liver-free allografts.

The Potency of Allospecific Tregs Cells Appears to Correlate With T Cell Receptor Functional Avidity

CD4(+) CD25(+) regulatory T cells (T(reg) cells) are an attractive adoptive cell therapy in mediating transplantation tolerance. T-cell receptor (TcR) activation is critical for T(reg) function, suggesting that the TcR avidity of T(reg) cells used in therapy may affect the therapeutic outcome. To address this, we compared the regulatory capacity of T(reg) lines expressing TcRs derived from two TcR transgenic mice shown to have the same specificity but different functional avidities. T(reg) lines generated from CD4(+)CD25(+) T cells from C57BL/6 mice were transduced with one of either of these TcRs. The antigen specificity of the transduced T(reg) lines was confirmed in vitro. T(reg) lines expressing the TcR with higher functional avidity showed stronger suppressive capacity in a linked suppression model in vitro. Furthermore, the same T(reg) lines demonstrated a stronger proliferation in vivo following antigen exposure. Pretreatment of recipient BL/6 mice with these T(reg) cells, together with anti-CD8 antibody and Rapamycin therapies, prolonged survival of BALB/c skins, as compared with mice that received T(reg) lines with lower TcR avidity. Taken together, these data suggest that the TcR functional avidity may be important for T(reg) function. It highlights the fact that strategies to select T(reg) with higher functional avidity might be beneficial for immunotherapy in transplantation.

Need for xenotransplantation in the treatment of type 1 diabetes

Type 1 diabetes (T1D) currently affects approximately 28 million patients with a rising incidence worldwide. Despite improvement of intensive insulin therapy, only a subgroup of patients reached near-normal blood glucose control. Prospective studies have shown that 30–40% of patients with T1D are at risk to develop devastating micro- or macrovascular complications. In the intensive insulin treatment arm of DCCT/EDIC study the cumulative incidences of proliferative retinopathy, nephropathy and cardiovascular diseases were 21%, 9% and 9% after 30 years of diabetes, respectively [1]. In addition, about 15% of patients with a disease history of 20–25 years have the problem of hypoglycemia unawareness due to lack of hormonal counter-regulation [2]. These serious complications lead to a significant reduction of life-expectancy as compared to non-diabetic individuals. Thus, there is an urgent need for more effective and durable management of diabetes. Transplantation of human beta cells using isolated islets or pancreas provided evidence that T1D can be cured by cell replacement therapies. Pancreas transplantation results in a significant reduction of vascular complication and reduced total mortality [3]. After islet cell transplantation 3-year insulin independency, preserved graft function (detectable C-peptide) and elimination of severe hypoglycemia have been reported in 23%, 52% and 70% by the collaborative islet transplant registry [4]. However, the total number of islet allografts (<100/years) and pancreas transplants (approximately 1800/year) worldwide is insufficient in relation to the number of potential recipients in whom islet/pancreas transplantation may be beneficial or even life-saving. In the last years there have been impressive advances in the field of beta cell generation from induced pluripotent stem cells/embryonic stem cells and xenograft transplantation. It has been shown that novel immunosuppressive regimen can induce long-lasting pig islet survival in nonhuman primates [5,6] and the use of fetal pig pancreas reduces immunogenicity [7]. Transgenic pigs expressing immunosuppressive and cytoprotective factors in beta cells have been generated. In addition, novel techniques of islet encapsulation in biochambers may allow long-term islet function and pathogen-free pig herds have been recently established to overcome the problem of zoonosis. The use of pig islets has additional advantages: (i) pig insulin is safe since it has been used for decades in the treatment of T1D; (ii) porcine pancreas is easily available on demand making it possible to reduce ischemia injury and pretreatment of recipients; (iii) pig islets do respond to glucose in a similar way as human islets. The last two points strongly favor pig islets as compared to neo-beta cells derived from stem cells, which were reported to be not fully matured and possess the risk of tumor formation (teratomas) [8,9]. The striking advances in pig islet transplantation suggest that it may be possible to overcome the major immunological barriers to pig islets in recipients so that clinical trials can be started in humans within the next years. Therefore, porcine islet xenografts may be the most realistic option to offer cell replacement therapy to a higher number of patients with T1D at risk for severe diabetes-associated complications. When this approach is successful the large-scale availability of pig islets would also open the window for other applications such as transplantation in insulin-treated patients with type 2 diabetes in whom insulin resistance currently excludes islet cells transplantation due to the demand of high insulin levels/high number of islets.