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1.  Delaying DLA-Haploidentical Hematopoietic Cell Transplantation After Total Body Irradiation 
Exposure to accidental or deliberate radiation poses a threat to public health, proving lethal at higher doses in large part due to deleterious effects on marrow. In those cases, allogeneic hematopoietic cell transplantation (HCT) might be required to restore marrow function. Most radiation accident victims will have HLA-haploidentical relatives who could serve as HCT donors. Here, we assessed in a canine HCT model the total body irradiation (TBI) doses after which transplants might be required and successful engraftment would be possible. In an attempt at mimicking the logistical problems likely to exist after radiation accidents, 4-, 8- or 10-day intervals were placed between TBI and HCT. In order to keep the experimental readout simple, no graft-vs-host disease (GVHD) prevention was administered. All dogs transplanted after a 4-day delay following 700 or 920 cGy TBI successfully engrafted while virtually all those given 450 or 600 cGy rejected their grafts. Transplant delays of 8 and 10 days following 920 cGy TBI also resulted in successful engraftment in most dogs, while a delay of 8 days after 700 cGy resulted in virtually uniform graft failure. The time courses of acute GVHD and rates of granulocyte recovery in engrafting dogs were comparable among dogs regardless of the lengths of delay. In other studies, we showed that most dogs not given HCT survived 700 cGy TBI with intensive supportive care while those given 800 cGy TBI and higher died with marrow aplasia. Thus, DLA-haploidentical HCT was successful even when carried out 4, 8 or 10 days after TBI at or above radiation exposures where dogs survived with intensive care alone.
PMCID: PMC2757126  PMID: 19747631
Hematopoietic cell transplantation; Total body irradiation; Engraftment; Dogs
2.  Adoptive Immunotherapy against Allogeneic Kidney Grafts in Dogs with Stable Hematopoietic Trichimerism 
Dogs given nonmyeloablative conditioning and marrow grafts from two dog leukocyte antigen- (DLA) identical littermate donors developed stable trichimerism and stably accepted a subsequent kidney graft from one of the marrow donors without the need for immunosuppression. Here, we used trichimeras to evaluate strategies of adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from two DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy total body irradiation and given a short course of postgrafting immunosuppression. After confirming stable hematopoietic engraftment, a kidney was transplanted from one of the two marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes (PBL) from each kidney donor were then used to sensitize the alternate marrow donor. Donor lymphocyte infusions (DLI) from the sensitized dogs were given to the trichimeric recipients, whereupon chimerism, graft-versus-host disease (GvHD), and kidney rejection were monitored. After DLI, we observed both prompt rejection of the transplanted marrow-donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably, owing to shared minor histocompatibility antigens, host chimerism also disappeared and GvHD in skin, gut, and liver developed. The native kidneys, while showing lymphocytic infiltration, remained functionally normal. The current study demonstrated that under certain experimental conditions, the kidney, an organ ordinarily not involved in graft-versus-host reactions, can be targeted by sensitized donor lymphocytes.
PMCID: PMC2603466  PMID: 18940673
marrow transplantation; multiple donors; kidney transplant; canine; nonmyeloablative; GvHD
3.  Graft-Versus-Host Disease and Graft-Versus-Tumor Effects After Allogeneic Hematopoietic Cell Transplantation 
Journal of Clinical Oncology  2012;31(12):1530-1538.
We designed a minimal-intensity conditioning regimen for allogeneic hematopoietic cell transplantation (HCT) in patients with advanced hematologic malignancies unable to tolerate high-intensity regimens because of age, serious comorbidities, or previous high-dose HCT. The regimen allows the purest assessment of graft-versus-tumor (GVT) effects apart from conditioning and graft-versus-host disease (GVHD) not augmented by regimen-related toxicities.
Patients and Methods
Patients received low-dose total-body irradiation ± fludarabine before HCT from HLA-matched related (n = 611) or unrelated (n = 481) donors, followed by mycophenolate mofetil and a calcineurin inhibitor to aid engraftment and control GVHD. Median patient age was 56 years (range, 7 to 75 years). Forty-five percent of patients had comorbidity scores of ≥ 3. Median follow-up time was 5 years (range, 0.6 to 12.7 years).
Depending on disease risk, comorbidities, and GVHD, lasting remissions were seen in 45% to 75% of patients, and 5-year survival ranged from 25% to 60%. At 5 years, the nonrelapse mortality (NRM) rate was 24%, and the relapse mortality rate was 34.5%. Most NRM was a result of GVHD. The most significant factors associated with GVHD-associated NRM were serious comorbidities and grafts from unrelated donors. Most relapses occurred early while the immune system was compromised. GVT effects were comparable after unrelated and related grafts. Chronic GVHD, but not acute GVHD, further increased GVT effects. The potential benefit associated with chronic GVHD was outweighed by increased NRM.
Allogeneic HCT relying on GVT effects is feasible and results in cures of an appreciable number of malignancies. Improved results could come from methods that control progression of malignancy early after HCT and effectively prevent GVHD.
PMCID: PMC3625710  PMID: 23478054
4.  A pre-clinical model of double versus single unit unrelated cord blood transplantation 
Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5×107/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained ≤ 1.7×107 TNC/kg. Recipients were given 9.2 Gy total body irradiation and DLA-matched unrelated cord blood with post-grafting cyclosporine and mycophenolate mofetil. After double-unit CBT, 5 dogs engrafted and 4 survived long term with one dominant engrafting unit and prompt immune reconstitution. In contrast, 0 of 5 dogs given single-unit CBT survived beyond 105 days (p=0.03, log-rank test); neutrophil and platelet recovery was delayed (both p=0.005) and recipients developed fatal infections. This new large animal model showed that outcomes were improved after double-unit compared to single-unit CBT. After double-unit CBT, the non-engrafted unit facilitates engraftment of the dominant unit.
PMCID: PMC2897972  PMID: 20304085
5.  Long-Term Tolerance to Kidney Allografts after Induced Rejection of Donor Hematopoietic Chimerism in a Preclinical Canine Model 
Transplantation  2012;94(6):562-568.
Allogeneic hematopoietic cell transplantation provides a reliable method for inducing tolerance towards solid organ grafts. However, this procedure can result in graft-versus-host disease (GVHD) thereby limiting its application. Here we test the hypothesis that mixed chimerism can be intentionally reverted to host hematopoiesis without rejection of a kidney graft.
Recipient dogs were given 2 Gy total body irradiation (TBI) before and a short course of immunosuppression after marrow infusion from dog leukocyte antigen-identical littermates. All dogs achieved stable mixed chimerism. After a mean of 20 weeks, one cohort of dogs received kidney transplants from their respective marrow donors. Subsequently, recipients were reconditioned with 2 Gy TBI and given autologous granulocyte-colony stimulating factor-mobilized leukocytes (recipient leukocyte infusion) that had been collected before marrow transplant.
Dogs receiving a second TBI and recipient leukocyte infusion without a kidney transplant rejected their donor hematopoietic graft within 3 weeks. Dogs that received kidney grafts, followed by a second TBI and recipient leukocyte infusion, rejected their marrow graft without rejecting their transplanted kidneys for periods greater than one year.
Mixed chimerism may be clinically reverted to 100% recipient without rejection of a kidney allograft. This finding may have application towards minimizing the risk of GVHD in solid organ transplant patients given hematopoietic cell transplantation from HLA-identical donors.
PMCID: PMC3448791  PMID: 22929594
dog; nonmyeloablative conditioning regimen; mixed hematopoietic chimerism; kidney allograft; recipient lymphocyte infusion; tolerance
6.  Pulmonary Administration of a Water-Soluble Curcumin Complex Reduces Severity of Acute Lung Injury 
Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin’s association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.
PMCID: PMC3488693  PMID: 22312018
cyclodextrin; LPS; turmeric; Calu-3; oxidative stress; TEER
7.  Nonmyeloablative Unrelated Donor Hematopoietic Cell Transplantation for the Treatment of Patients with Poor-Risk, Relapsed or Refractory Multiple Myeloma 
The purpose of this study was to determine long-term outcome of unrelated donor nonmyeloablative hematopoietic cell transplantation in patients with poor-risk multiple myeloma. Twenty-four patients were enrolled. Seventeen patients (71%) had chemotherapy-refractory disease and 14 patients (58%) had disease relapse or progression after prior autologous transplantation. Thirteen patients underwent planned autologous transplantation followed 43–135 days later with unrelated transplantation, while 11 proceeded directly to unrelated transplantation. All 24 patients were treated with fludarabine (90 mg/m2) and 2 Gray total body irradiation and human leukocyte antigen (HLA)-matched unrelated peripheral blood stem cell transplantation. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. The median follow-up was 3 years after allografting. One patient experienced non-fatal graft rejection. The incidences of acute grades II, III and chronic graft-versus-host disease were 54%, 13% and 75%, respectively. The 3-year non-relapse mortality was 21%. Complete responses were observed in 10 patients (42%) and partial responses in 4 (17%). At 3 years, overall and progression-free survival rates were 61% and 33%, respectively. Patients receiving tandem autologous-unrelated transplantation had superior overall and progression-free survivals, 77% and 51%, compared to patients proceeding directly to unrelated donor transplantation, 44% and 11%, respectively (progression-free survival p-value, 0.03). In summary, for patients with poor-risk, relapsed or refractory multiple myeloma, cytoreductive autologous transplantation followed with nonmyeloablative conditioning and unrelated hematopoietic cell transplantation is effective treatment with low non-relapse mortality, high complete remission rates and prolonged disease-free survival.
PMCID: PMC1950939  PMID: 17287157
Multiple myeloma; nonmyeloablative conditioning; allogeneic hematopoietic cell transplantation; unrelated donor; graft-versus-tumor effects; chronic graft-versus-host disease; peripheral blood stem cell transplantation
8.  Blood-Based Detection of Radiation Exposure in Humans Based on Novel Phospho-Smc1 ELISA 
Radiation Research  2010;175(3):266-281.
The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1Ser-957 and phospho-Smc1Ser-966. Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1Ser-957 and phospho-Smc1Ser-966 in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to 131I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident.
PMCID: PMC3123689  PMID: 21388270
9.  Long-Term Outcomes Among Older Patients Following Nonmyeloablative Conditioning and Allogeneic Hematopoietic Cell Transplantation for Advanced Hematologic Malignancies 
A minimally toxic nonmyeloablative regimen was developed for allogeneic hematopoietic cell transplantation (HCT) to treat patients with advanced hematologic malignancies who are older or have comorbidities.
To describe outcomes of patients ≥ 60 years.
Design, Setting, and Participants
From 1998 to 2008, 372 patients, 60–75 years old were enrolled in prospective clinical HCT trials at 18 collaborating institutions using conditioning with low-dose total body irradiation alone or combined with fludarabine 90 mg/m2 before related (n=184) or unrelated (n=188) donor transplants. Post-grafting immunosuppression included mycophenolate mofetil and a calcineurin inhibitor.
Main Outcome Measures
Overall and progression-free survivals were estimated by Kaplan-Meier method. Cumulative incidence estimates were calculated for acute and chronic GVHD, toxicities, achievement of full donor chimerism, complete remission, relapse, and non-relapse mortality. Hazard ratios (HR) were estimated from Cox regression models.
Overall, 5-year cumulative incidences of non-relapse mortality and relapse were 27% (95% CI, 22%–32%) and 41% (95% CI, 36%–46%), respectively, leading to overall and progression-free 5-year survivals of 35% (95% CI, 30%–40%) and 32% (95% CI, 27%–37%), respectively. These outcomes were not statistically significantly different when stratified by age groups. Furthermore, increasing age was not associated with increases in acute or chronic graft-versus-host disease (GVHD) or organ toxicities. In multivariate models, HCT-CI scores of 1–2 [HR, 1.58 (95% CI,1.08–2.31)] and ≥3 [HR, 1.97 (95% CI,1.38–2.80)] were associated with worse survival compared to HCT-CI score of 0 (overall P = 0.003). Similarly, standard relapse risk [HR, 1.67 (95% CI, 1.10–2.54)] and high relapse risk [HR, 2.22 (95% CI, 1.43–3.43)] were associated with worse survival compared to low relapse risk (overall P = 0.0008).
Among patients aged 60–75 years and treated with nonmyeloablative allogeneic HCT, 5-year overall and progression-free survivals were 35% (95% CI, 30%–40%) and 32% (95% CI, 27%–37%), respectively.
PMCID: PMC3217787  PMID: 22045765
Marrow transplantation; hematopoietic cell transplantation; age; comorbidities; toxicities; infections; performance status; graft-versus-host disease; withdrawal of immunosuppression; mortality; survival; comparative outcomes research
10.  Antagonistic and agonistic anti-canine CD28 monoclonal antibodies: tools for allogeneic transplantation1 
Transplantation  2011;91(8):833-840.
It has been widely presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to CTLA4-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, adding further to the down-regulation of T-cell function. These characteristics of CD28 are likely to be important in efforts to improve upon hematopoietic cell transplantation. Here, we developed for use in the dog model one agonistic and seven antagonistic monoclonal antibodies (mAb) to canine (ca)CD28. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T-cells. The antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, while the agonistic antibody to caCD28 enhanced MLR. 5B8 was superior to 1C6 when either was combined with anti-canine CD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T-cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig inhibited cytotoxic T lymphocyte (CTL)-mediated killing of alloreactive target cells after a secondary MLR equivalently. These studies demonstrated that mouse anti-caCD28 mAb’s with either agonistic or antagonistic function can be generated.
PMCID: PMC3180996  PMID: 21343872
Costimulatory molecule; blockade; CD28; CTLA4-Ig; CTL; regulatory T-cells
11.  Identification of Radiation-Induced Expression Changes in Nonimmortalized Human T Cells 
Radiation Research  2010;175(2):172-184.
In the event of a radiation accident or attack, it will be imperative to quickly assess the amount of radiation exposure to accurately triage victims for appropriate care. RNA-based radiation dosimetry assays offer the potential to rapidly screen thousands of individuals in an efficient and cost-effective manner. However, prior to the development of these assays, it will be critical to identify those genes that will be most useful to delineate different radiation doses. Using global expression profiling, we examined expression changes in nonimmortalized T cells across a wide range of doses (0.15–12 Gy). Because many radiation responses are highly dependent on time, expression changes were examined at three different times (3, 8, and 24 h). Analyses identified 61, 512 and 1310 genes with significant linear dose-dependent expression changes at 3, 8 and 24 h, respectively. Using a stepwise regression procedure, a model was developed to estimate in vitro radiation exposures using the expression of three genes (CDKN1A, PSRC1 and TNFSF4) and validated in an independent test set with 86% accuracy. These findings suggest that RNA-based expression assays for a small subset of genes can be employed to develop clinical biodosimetry assays to be used in assessments of radiation exposure and toxicity.
PMCID: PMC3136539  PMID: 21268710
12.  Mesenchymal Stromal Cells Fail to Prevent Acute Graft-versus-Host Disease and Graft Rejection after Dog-Leukocyte-Antigen Haploidentical Bone Marrow Transplantation 
Mesenchymal stromal cells (MSC) have been shown to have immunosuppressive effects in vitro. To test the hypothesis that these effects can be harnessed to prevent graft-versus-host disease (GVHD) and graft rejection after hematopoietic cell transplantation (HCT), we administered a combination of three different immortalized marrow-derived MSC lines (15–30×106 MSC/kg/day, 2–5 times/week) or third-party primary MSC (1.0×106 MSC/kg/day, 3 times/week) to canine recipients (n=15) of dog-leukocyte antigen-haploidentical marrow grafts prepared with 9.2 Gy total body irradiation. Additional pharmacological immunosuppression was not given after HCT. Prior to their in vivo use, the MSC products were shown to suppress alloantigen-induced T cell proliferation in a dose-dependent, major histocompatibility complex-unrestricted and cell contact-independent fashion in vitro. Among 14 dogs evaluable, 7 (50%) rejected their grafts and 7 engrafted with ensuing rapidly fatal acute GVHD (50%). These observations were not statistically different from outcomes obtained with historical controls (n=11) not given MSC infusions (p=0.69). Hence, survival curves for MSC-treated dogs and controls were virtually superimposable (median survival, 18 vs. 15 days, respectively). Finally, outcomes of dogs given primary MSC (n=3) did not appear to be different from those given clonal MSC (n=12). In conclusion, our data fail to demonstrate MSC-mediated protection against GVHD and allograft rejection in this model.
PMCID: PMC3026853  PMID: 20816818
Hematopoietic cell transplantation; mesenchymal stromal cells; MSC; graft-versus-host disease (GVHD); rejection; canine model
13.  A Novel Monoclonal Antibody Specific for Canine CD25 (P4A10): Selection and Evaluation of Canine Tregs 
A monoclonal antibody (mAb), P4A10, was made to the canine interleukin-2 receptor alpha chain (IL-2Rα; p55; Tac antigen; CD25) to facilitate studies of canine regulatory T-cells (Treg). By non-reduced western blot, P4A10 bound to a 55 kD protein, the expected size of IL-2Rα. In flow cytometry assays, it reacted with a minor population of circulating dog CD3+CD4+ T cells and the majority (>60%) of in vitro PMA-Ionomycin (PMA-IO)-activated canine CD3+ T-cells. P4A10 recognized a hematopoietic cell population enriched for FoxP3+ cells as measured by flow cytometry. The P4A10-selected fractions of T-cells had significantly increased copy numbers of CD25, FoxP3, IL-10, and TGFβ as detected by RT-PCR (reverse transcriptase PCR) compared to the negative fractions. The P4A10-selected cells inhibited 3H (tritiated) thymidine incorporation in a mixed leukocyte reaction (MLR) containing responders of the same origin. P4A10-selected T cells from fresh peripheral blood mononuclear cells had less FoxP3 (p = 0.07) by qRT-PCR (quantitative RT-PCR) and were less suppressive (p=0.01) than in vitro alloantigen-activated Treg. The mAb P4A10 is specific for canine CD25 and can be used to facilitate studies of CD25+FoxP3+ Treg in this clinically relevant large animal model.
PMCID: PMC2864801  PMID: 20060595
Monoclonal antibody; CD25; regulatory T cells; dog
14.  Animal Models for Medical Countermeasures to Radiation Exposure 
Radiation research  2010;173(4):557-578.
Since September 11, 2001, there has been the recognition of a plausible threat from acts of terrorism, including radiological or nuclear attacks. A network of Centers for Medical Countermeasures against Radiation (CMCRs) has been established across the U.S.; one of the missions of this network is to identify and develop mitigating agents that can be used to treat the civilian population after a radiological event. The development of such agents requires comparison of data from many sources and accumulation of information consistent with the “Animal Rule” from the Food and Drug Administration (FDA). Given the necessity for a consensus on appropriate animal model use across the network to allow for comparative studies to be performed across institutions, and to identify pivotal studies and facilitate FDA approval, in early 2008, investigators from each of the CMCRs organized and met for an Animal Models Workshop. Working groups deliberated and discussed the wide range of animal models available for assessing agent efficacy in a number of relevant tissues and organs, including the immune and hematopoietic systems, gastrointestinal tract, lung, kidney and skin. Discussions covered the most appropriate species and strains available as well as other factors that may affect differential findings between groups and institutions. This report provides the workshop findings.
PMCID: PMC3021126  PMID: 20334528
15.  Immunomodulatory Effects of Mixed Hematopoietic Chimerism: Immune Tolerance in Canine Model of Lung Transplantation 
Long-term survival after lung transplantation is limited by acute and chronic graft rejection. Induction of immune tolerance by first establishing mixed hematopoietic chimerism (MC) is a promising strategy to improve outcomes. In a preclinical canine model, stable MC was established in recipients after reduced-intensity conditioning and hematopoietic cell transplantation from a DLA-identical donor. Delayed lung transplantation was performed from the stem cell donor without pharmacological immunosuppression. Lung graft survival without loss of function was prolonged in chimeric (n=5) vs. nonchimeric (n=7) recipients (p≤0.05, Fisher’s test). There were histological changes consistent with low grade rejection in 3/5 of the lung grafts in chimeric recipients at ≥1 year. Chimeric recipients after lung transplantation had a normal immune response to a T-dependent antigen. Compared to normal dogs, there were significant increases of CD4+INFγ+, CD4+IL-4+ and CD8+ INFγ+ T-cell subsets in the blood (p <0.0001 for each of the 3 T-cell subsets). Markers for regulatory T-cell subsets including foxP3, IL10 and TGFβ were also increased in CD3+ T cells from the blood and peripheral tissues of chimeric recipients after lung transplantation. Establishing MC is immunomodulatory and observed changes were consistent with activation of both the effector and regulatory immune response.
PMCID: PMC3005612  PMID: 19422333
16.  High-dose immunosuppressive therapy and autologous peripheral blood stem cell transplantation for severe multiple sclerosis 
Blood  2003;102(7):2364-2372.
There were 26 patients enrolled in a pilot study of high-dose immunosuppressive therapy (HDIT) for severe multiple sclerosis (MS). Median baseline expanded disability status scale (EDSS) was 7.0 (range, 5.0–8.0). HDIT consisted of total body irradiation, cyclophosphamide, and antithymocyte globulin (ATG) and was followed by transplantation of autologous, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34-selected stem cells. Regimen-related toxicities were mild. Because of bladder dysfunction, there were 8 infectious events of the lower urinary tract. One patient died from Epstein-Barr virus (ESV)-related posttransplantation lymphoproliferative disorder (PTLD) associated with a change from horse-derived to rabbit-derived ATG in the HDIT regimen. An engraftment syndrome characterized by noninfectious fever with or without rash developed in 13 of the first 18 patients and was associated in some cases with transient worsening of neurologic symptoms. There were 2 significant adverse neurologic events that occurred, including a flare of MS during mobilization and an episode of irreversible neurologic deterioration after HDIT associated with fever. With a median follow-up of 24 (range, 3–36) months, the Kaplan-Meier estimate of progression (≥ 1.0 point EDSS) at 3 years was 27%. Of 12 patients who had oligoclonal bands in the cerebrospinal fluid at baseline, 9 had persistence after HDIT. After HDIT, 4 patients developed new enhancing lesions on magnetic resonance imaging of the brain. The estimate of survival at 3 years was 91%. Important clinical issues in the use of HDIT and stem cell transplantation for MS were identified; however, modifications of the initial approaches appear to reduce treatment risks. This was a heterogeneous high-risk group, and a phase 3 study is planned to fully assess efficacy.
PMCID: PMC2963562  PMID: 12763935
17.  Allogeneic Marrow Transplantation in Patients With Severe Systemic Sclerosis 
Arthritis and rheumatism  2006;54(6):1982-1986.
To evaluate the safety and efficacy of allogeneic hematopoietic cell transplantation (HCT) after myeloablative conditioning in patients with severe systemic sclerosis (SSc).
Eligibility criteria for the study included SSc patients with features indicative of a poor prognosis. The myeloablative conditioning regimen included busulfan, cyclophosphamide, and antithymocyte globulin. Prophylaxis for graft-versus-host disease (GVHD) consisted of cyclosporine and methotrexate. Bone marrow was transplanted from HLA-identical siblings.
Two patients with diffuse cutaneous SSc and lung involvement who were refractory to conventional immunosuppressive treatment were enrolled in the study. In patient 1, there were no complications related to the conditioning regimen, and GVHD did not develop after transplantation. At 5 years after HCT, there was nearly complete resolution of the scleroderma and marked improvement in physical functioning. Internal organ function improved (lung) or remained stable. On examination of serial skin biopsy samples, there was resolution of the dermal fibrosis. Patient 2 experienced skin toxicity from the conditioning regimen and hypertensive crisis that was likely related to high-dose corticosteroids given for treatment of GVHD. Although this patient experienced an improvement in scleroderma and overall functioning, a fatal opportunistic infection developed 17 months after HCT.
Allogeneic HCT may result in sustained remission of SSc. GVHD and opportunistic infections are the major risks associated with allogeneic HCT for SSc, as for allogeneic HCT in general.
PMCID: PMC2956579  PMID: 16732546
18.  Epstein-Barr Virus–Associated Posttransplantation Lymphoproliferative Disorder after High-Dose Immunosuppressive Therapy and Autologous CD34-Selected Hematopoietic Stem Cell Transplantation for Severe Autoimmune Diseases 
High-dose immunosuppressive therapy followed by autologous hematopoietic stem cell transplantation (HSCT) is currently being evaluated for the control of severe autoimmune diseases. The addition of antithymocyte globulin (ATG) to high-dose chemoradiotherapy in the high-dose immunosuppressive therapy regimen and CD34 selection of the autologous graft may induce a higher degree of immunosuppression compared with conventional autologous HSCT for malignant diseases. Patients may be at higher risk of transplant-related complications secondary to the immunosuppressed state, including Epstein-Barr virus (EBV)–associated posttransplantation lymphoproliferative disorder (PTLD), but this is an unusual complication after autologous HSCT. Fifty-six patients (median age, 42 years; range, 23–61 years) with either multiple sclerosis (n = 26) or systemic sclerosis (n = 30) have been treated. The median follow-up has been 24 months (range, 2–60 months). Two patients (multiple sclerosis, n = 1; systemic sclerosis, n = 1) had significant reactivations of herpesvirus infections early after HSCT and then developed aggressive EBV-PTLD and died on days +53 and +64. Multiorgan clonal B-cell infiltrates that were EBV positive by molecular studies or immunohistology were identified at both autopsies. Both patients had positive screening skin tests for equine ATG (Atgam) and had been converted to rabbit ATG (Thymoglobulin) from the first dose. Of the other 54 patients, 2 of whom had partial courses of rabbit ATG because of a reaction to the intravenous infusion of equine ATG, only 1 patient had a significant clinical reactivation of a herpesvirus infection (herpes simplex virus 2) early after HSCT, and none developed EBV-PTLD. The T-cell count in the peripheral blood on day 28 was 0/μL in all 4 patients who received rabbit ATG; this was significantly less than in patients who received equine ATG (median, 174/μL; P = .001; Mann-Whitney ranked sum test). Although the numbers are limited, the time course and similarity of the 2 cases of EBV-PTLD and the effect on day 28 T-cell counts support a relationship between the development of EBV-PTLD and the administration of rabbit ATG. The differences between equine and rabbit ATG are not yet clearly defined, and they should not be considered interchangeable in this regimen without further study.
PMCID: PMC2956744  PMID: 14506660
EBV; Lymphoma; Hematopoietic stem cell transplantation; CD34 selection; Autoimmune diseases; Systemic sclerosis; Multiple sclerosis
19.  Methylguanine methyltransferase–mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model 
Journal of Clinical Investigation  2003;112(10):1581-1588.
Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase–mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.
PMCID: PMC259127  PMID: 14617759

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