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2.
Bone Marrow Transplant ; 48(10): 1335-41, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23604009

ABSTRACT

Because of the toxicity associated with myeloablative conditioning, nonmyeloablative regimens are increasingly being used in vulnerable patient populations. For patients with sickle cell disease, stable mixed chimerism has proven sufficient to reverse the phenotype. Because the vast majority of patients do not have an HLA-matched sibling, a safe nonmyeloablative regimen that could be applied to the haploidentical setting would be ideal. We employed a mismatched mouse model using BALB/c donors and C57BL/6 recipients. Recipient mice were conditioned with 200 cGy TBI and sirolimus or CSA with or without post transplant Cy (PT-Cy). Our data show that when sirolimus or PT-Cy alone is given to C57BL/6 recipients, donor cells are not detected. However, when sirolimus is administered for 15 or 31 days starting 1 day before or up to 6 days after transplant with PT-Cy, all mice maintain stable mixed chimerism. In contrast, conventional therapy employing CSA with or without PT-Cy does not result in stable mixed chimerism. Lastly, mice with stable mixed chimerism after sirolimus display decreased reactivity to donor Ag both in vitro and in vivo. These data identify a novel strategy for inducing mixed chimerism for the treatment of nonmalignant hematologic diseases.


Subject(s)
Cyclophosphamide/pharmacology , Immunosuppressive Agents/pharmacology , Sirolimus/pharmacology , Skin Transplantation/methods , Animals , Disease Models, Animal , Drug Synergism , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Transplantation Chimera
4.
Ann Oncol ; 21(6): 1203-1210, 2010 Jun.
Article in English | MEDLINE | ID: mdl-19880437

ABSTRACT

BACKGROUND: Rituximab may improve transplant outcomes but may delay immunologic recovery. PATIENTS AND METHODS: Seventy-seven patients with low-grade or mantle cell lymphoma received autologous stem-cell transplantation (ASCT) on a phase II study. Rituximab 375 mg/m(2) was administered 3 days before mobilization-dose cyclophosphamide, then weekly for four doses after count recovery from ASCT. Immune reconstitution was assessed. RESULTS: Sixty percent of transplants occurred in first remission. Actuarial event-free survival (EFS) and overall survival (OS) were 60% and 73%, respectively, at 5 years, with 7.2-year median follow-up for OS in surviving patients. Median EFS was 8.3 years. Older age and transformed lymphomas were independently associated with inferior EFS, whereas day 60 lymphocyte counts did not predict EFS or late infections. Early and late transplant-related mortality was 1% and 8%, with secondary leukemia in two patients. B-cell counts recovered by 1-2 years; however, the median IgG level remained low at 2 years. Late-onset idiopathic neutropenia, generally inconsequential, was noted in 43%. CONCLUSION: ASCT with rituximab can produce durable remissions on follow-up out to 10 years. Major infections do not appear to be significantly increased or to be predicted by immune monitoring.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Immune System/physiology , Lymphoma, Mantle-Cell , Lymphoma , Recovery of Function/immunology , Stem Cell Transplantation/methods , Adult , Aged , Antibodies, Monoclonal, Murine-Derived , Antineoplastic Agents/administration & dosage , Combined Modality Therapy , Drug Administration Schedule , Female , Humans , Immunotherapy , Lymphoma/immunology , Lymphoma/pathology , Lymphoma/rehabilitation , Lymphoma/therapy , Lymphoma, Mantle-Cell/immunology , Lymphoma, Mantle-Cell/pathology , Lymphoma, Mantle-Cell/rehabilitation , Lymphoma, Mantle-Cell/therapy , Male , Middle Aged , Neoplasm Staging , Rituximab , Transplantation Immunology , Transplantation, Autologous
6.
Bone Marrow Transplant ; 43(6): 477-80, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18978825

ABSTRACT

Graft failure after allogeneic blood or marrow transplantation, although generally uncommon, can be a devastating complication. This report includes the outcome of nine patients who received a salvage transplant for failure to engraft after one (n=8) or 2 (n=1) prior transplants. Eight patients received allografts from the original donor. All received fludarabine 30 mg/m(2) i.v. and alemtuzumab 20 mg i.v. daily from days -6 to -2. Daily CYA was begun on day -2, and the allograft was infused on day 0. The therapy was well tolerated with low toxicity, and all nine patients engrafted, recovering neutrophils at a median of 12 days after transplant. Four patients died: two of relapse, one of a fungal infection in the setting of GVHD and one of multiple sclerosis. The combination of fludarabine and alemtuzumab is an effective and well-tolerated salvage conditioning regimen for patients who experience graft failure after blood or marrow transplants.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Neoplasm/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Bone Marrow Transplantation/methods , Transplantation Conditioning/methods , Vidarabine/analogs & derivatives , Adult , Alemtuzumab , Antibodies, Monoclonal, Humanized , Female , Graft Rejection , Humans , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Recurrence , Retrospective Studies , Salvage Therapy/methods , Treatment Outcome , Vidarabine/administration & dosage
7.
Bone Marrow Transplant ; 42(8): 523-7, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18622413

ABSTRACT

Allogeneic blood or marrow transplantation (BMT) is potentially curative for a variety of life-threatening nonmalignant hematologic diseases such as paroxysmal nocturnal hemoglobinuria (PNH) and hemoglobinopathies. The application of BMT to treat these disorders is limited by the lack of suitable donors and often end-organ damage from the underlying disease. We treated three patients with thrombotic PNH, one of whom also had sickle cell disease, with a nonmyeloablative, HLA-haploidentical BMT with post-transplant CY. Rapid engraftment without GVHD occurred in two of the patients, including the patient with sickle cell disease. Both patients are disease free with full donor chimerism and require no immunosuppressive therapy, with follow-up of 1 and 4 years, respectively. Nonmyeloablative, HLA-haploidentical BMT with post-transplant CY is a promising approach for patients with life-threatening nonmalignant hematologic disease who lack an HLA-matched sibling donor.


Subject(s)
Bone Marrow Transplantation , Budd-Chiari Syndrome/therapy , Cyclophosphamide/administration & dosage , Graft Survival/drug effects , Immunosuppressive Agents/administration & dosage , Adult , Anemia, Sickle Cell/complications , Anemia, Sickle Cell/therapy , Budd-Chiari Syndrome/complications , Female , Follow-Up Studies , Histocompatibility Testing , Humans , Male , Transplantation, Homologous
8.
Biol Blood Marrow Transplant ; 8(7): 377-86, 2002.
Article in English | MEDLINE | ID: mdl-12171484

ABSTRACT

Cyclophosphamide (Cy) is a potent immunosuppressive agent that is selectively toxic to lymphocytes proliferating in response to recent antigen stimulation. In animal models, both graft rejection and GVHD after histoincompatible BMT can be inhibited by the posttransplantation administration of high-dose Cy. Therefore, a phase I clinical trial was undertaken to determine the minimal conditioning, including posttransplantation Cy, that permits the stable engraftment of partially HLA-mismatched marrow (up to 3 HLA antigens) from first-degree relatives. Thirteen patients (median age, 53 years) with high-risk hematologic malignancies received conditioning with fludarabine, 30 mg/m2 per day from days -6 to -2, and TBI, 2 Gy on day -1. All patients received Cy, 50 mg/kg on day 3, mycophenolate mofetil from day 4 to day 35, and tacrolimus from day 4 to day > or = 50. Three patients in cohort 1 received no additional conditioning, and 2 experienced graft rejection. Ten patients in cohort 2 received identical conditioning with the addition of Cy 14.5 mg/kg on days -6 and -5. Sustained donor cell engraftment occurred in 8 of these patients, with a median time to absolute neutrophil count > 500/microL of 15 days (range, 13-16 days) and to unsupported platelet count > 20,000/microL of 14 days (range, 0-26 days). All patients with engraftment achieved > or = 95% donor chimerism within 60 days of transplantation. Two patients with myelodysplastic syndrome rejected their grafts but experienced autologous neutrophil recovery at 24 and 44 days. Histologic acute GVHD developed in 6 patients (grade II in 3 patients, grade III in 3 patients) at a median of 99 days (range, 38-143 days) after transplantation and was fatal in 1 patient. At a median follow-up of 191 days (range, 124-423 days), 6 of 10 patients in cohort 2 were alive, and 5 were in complete remission of their disease, including both patients with graft rejection. These data demonstrate that partially HLA-mismatched bone marrow can engraft rapidly and stably after nonmyeloablative conditioning that includes posttransplantation Cy. Clinically significant antitumor responses occur, even among patients who reject their donor grafts.


Subject(s)
Bone Marrow Transplantation/methods , Cyclophosphamide/administration & dosage , Histocompatibility , Immunosuppressive Agents/administration & dosage , Transplantation Conditioning/methods , Adolescent , Adult , Aged , Bone Marrow Transplantation/immunology , Child , Child, Preschool , Female , Graft Survival , Hematologic Neoplasms/mortality , Hematologic Neoplasms/therapy , Histocompatibility/genetics , Histocompatibility/immunology , Histocompatibility Testing/methods , Humans , Infant , Male , Middle Aged , Nuclear Family , Transplantation, Homologous/immunology , Transplantation, Homologous/methods , Treatment Outcome
9.
Blood ; 98(12): 3456-64, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11719388

ABSTRACT

Treatment of leukemia by myeloablative conditioning and transplantation of major histocompatibility complex (MHC)-mismatched stem cells is generally avoided because of the high risk of graft rejection or lethal graft-versus-host disease (GVHD). This study shows that MHC-incompatible cells can engraft stably after nonmyeloablative conditioning with immunosuppressive chemotherapy and low-dose total body irradiation (TBI). Long-term mixed hematopoietic chimerism, clonal deletion of donor-reactive T cells, and bidirectional cytotoxic T-cell tolerance were achieved by transplanting MHC-mismatched marrow cells into recipients conditioned with pretransplantation fludarabine or cyclophosphamide (Cy), 50 to 200 cGy TBI on day -1, and Cy 200 mg/kg intraperitoneally on day 3. In this model, long-term donor chimerism was proportional to the dose of TBI or donor marrow cells. Pretransplantation fludarabine and posttransplantation Cy were both required for alloengraftment, but the drugs had additional effects. For example, fludarabine sensitized host stem cells to the toxicity of TBI, because animals conditioned with both agents had higher chimerism than animals conditioned with TBI alone (P <.05). Also, posttransplantation Cy attenuated lethal and nonlethal GVH reactions, because F(1) recipients of host-reactive, parental spleen cells survived longer (P <.05) and had lower donor cell chimerism (P <.01) if they received posttransplantation Cy than if they did not. Finally, delayed infusions of donor lymphocytes into mixed chimeras prolonged survival after leukemia challenge (P <.0001) without causing lethal GVHD. These results indicate that stable engraftment of MHC-incompatible cells can be induced after fludarabine-based, nonmyeloablative conditioning and that it serves as a platform for adoptive immunotherapy with donor lymphocyte infusions.


Subject(s)
Cyclophosphamide/therapeutic use , Hematopoietic Stem Cell Transplantation , Immunosuppressive Agents/therapeutic use , Transplantation Conditioning , Vidarabine/analogs & derivatives , Vidarabine/administration & dosage , Whole-Body Irradiation , Animals , Graft Survival , Graft vs Host Disease/prevention & control , Graft vs Leukemia Effect , Histocompatibility , Mice , Mice, Inbred AKR , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , T-Lymphocytes, Cytotoxic/immunology , Transplantation Chimera
10.
Blood ; 97(12): 3960-5, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11389040

ABSTRACT

Sickle cell anemia (SCA) is an inherited disorder of beta-globin, resulting in red blood cell rigidity, anemia, painful crises, organ infarctions, and reduced life expectancy. Allogeneic blood or marrow transplantation (BMT) can cure SCA but is associated with an 8% to 10% mortality rate, primarily from complications of marrow-ablative conditioning. Transplantation of allogeneic marrow after less intensive conditioning reduces toxicity but may result in stable mixed hematopoietic chimerism. The few SCA patients who inadvertently developed mixed chimerism after BMT remain symptom free, suggesting that mixed chimerism can reduce disease-related morbidity. However, because the effects of various levels of mixed chimerism on organ pathology have not been characterized, this study examined the histologic effects of an increasing percentage of normal donor hematopoiesis in a mouse model of BMT for SCA. In lethally irradiated normal mice that were reconstituted with varying ratios of T-cell-depleted marrow from normal and transgenic "sickle cell" mice, normal myeloid chimerism in excess of 25% was associated with more than 90% normal hemoglobin (Hb). However, 70% normal myeloid chimerism was required to reverse the anemia. Organ pathology, including liver infarction, was present in mice with sickle Hb (HbS) levels as low as 16.8% (19.6% normal myeloid chimerism). Histologic abnormalities increased in severity up to 80% HbS, but were less severe in mice with more than 80% HbS than in those with 40% to 80% HbS. Therefore, stable mixed chimerism resulting from nonmyeloablative BMT may reduce the morbidity from SCA, but prevention of all disease complications may require minimizing the fraction of circulating sickle red cells. (Blood. 2001;97:3960-3965)


Subject(s)
Anemia, Sickle Cell/therapy , Bone Marrow Transplantation , Hematopoiesis , Transplantation Chimera , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/pathology , Animals , Female , Hemoglobin, Sickle/metabolism , Leukocyte Count , Linear Models , Liver/pathology , Male , Mice , Mice, Transgenic , Models, Animal , Reticulocyte Count , Spleen/pathology
11.
J Biomed Sci ; 3(5): 323-331, 1996.
Article in English | MEDLINE | ID: mdl-11725114

ABSTRACT

Both host cell and viral transcription factors regulate the long terminal repeat (LTR) of human immuno-deficiency virus (HIV) activity and viral replication. Using transient transfection, ligand-activated thyroid hormone and 9-cis-retinoic acid receptors (T(3)R and RXR) were found to stimulate HIV-1 and HIV-2 LTR activities. They also stimulated HIV-1 viral production. Drosophila SL2 cells that lack Sp1 and T(3)R were used to study HIV-1 and HIV-2 LTR activities. Both activities were stimulated by cotransfection of SP1 (120- and 180-fold, respectively); HIV LTR activities were also stimulated approximately 5-fold by ligand-activated T(3)R, approximately 10-fold by ligand-activated RXR and 20- to 30-fold by both receptors and their cognate ligands. T(3)R.RXR heterodimers bound to NF-kappaB and Sp1 response elements in both HIV LTRs having highest affinity for the HIV-1 NF-kappaB region. When U937 monocytic cells were cotransfected with HIV-1 viral DNA and T(3)R, RXR and retinoic acid receptor (RAR) expression plasmids, hormonal treatment increased viral replication up to 5-fold. Hormonal signals thus have the potential to regulate HIV transcription and viral production. Copyright 1996 S. Karger AG, Basel

12.
J Virol ; 70(3): 1596-601, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8627679

ABSTRACT

We have previously shown that hairpin ribozymes targeting the human immunodeficiency virus (HIV) genome can effectively inhibit virus replication in a variety of primary and cultured hematopoietic cells. To further increase antiviral potency and minimize the chance of viral resistance, we have now cloned the stem-loop II sequences of the HIV type 1 Rev response element into ribozyme transcription cassettes. Fusion RNA molecules were shown to function both as RNA decoys and ribozymes. Stable Molt-4/8 cell lines expressing fusion RNA of stem-loop II and a ribozyme directed at the HIV-type 1 U5 sequence (MSLMJT) or its disabled counterpart (MSLdMJT) were generated. The expression of fusion RNA was persistent for at least 6 months without apparent cytotoxicity. When virus inhibition was examined after the cocultivation of transduced cells with chronically infected Jurkat cells, much greater protection was observed in MSLMJT cells than in MSLdMJT or MMJT (expressing only the ribozyme) cells. Furthermore, to specifically compare the ribozyme activities in various transduced cells, we determined the quantitative levels of proviral DNA in the first round of virus replication (7 h after infection with HXB2). By competitive PCR, the proviral DNA levels in MSLMJT and MMJT cells were found to be reduced to 1/7 and 1/3, respectively, compared with those in MSLdMJT and MdMJT cells. These results suggest not only that the greater inhibition afforded by this fusion RNA was due to its function both as decoy and ribozyme but also that the ribozyme activity may be facilitated.


Subject(s)
HIV-1/physiology , RNA, Catalytic/pharmacology , RNA, Viral/metabolism , Base Sequence , Cell Line , Cloning, Molecular , DNA, Viral , Gene Products, rev/genetics , HIV-1/drug effects , HIV-1/genetics , Humans , Molecular Sequence Data , RNA, Catalytic/genetics , RNA, Viral/genetics , Virus Replication/drug effects , rev Gene Products, Human Immunodeficiency Virus
13.
AIDS Res Hum Retroviruses ; 11(7): 795-804, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7546906

ABSTRACT

The rev gene product of human immunodeficiency virus (HIV) is obligatory for viral replication. Rev interacts specifically with a structured RNA sequence within the viral genome termed the REV response element (RRE). Although the importance of Rev for the expression of viral proteins is well documented, its functional mechanism remains unresolved. Previous studies identified Rev in the absence of RRE to be a nuclear protein localized primarily within the nucleoli. To extend our understanding of the role of Rev in viral replication, immunolocalization studies of Rev and other nuclear components were carried out in transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV. In both types of cells, Rev-like immunoreactivity was distributed both in the nucleoplasm and cytoplasm. Within the nucleus, Rev immunoreactivity was not evenly distributed but was present within focal concentrations. In transfected cells that were double labeled for Rev and SC-35, which labels a known component of spliceosomes, the foci of Rev labeling were distinct from the "speckles" labeled by SC-35, although Rev foci and speckles were often juxtaposed. In addition, morphological changes in the three-dimensional network of speckles were observed in both transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV-1 and HIV-2. Our observations are consistent with the proposed dual role of Rev in mRNA transport and splicing.


Subject(s)
Gene Products, rev/analysis , HIV-2/physiology , Virus Replication , Base Sequence , Cell Nucleolus/virology , DNA Primers , Gene Expression , Gene Products, rev/biosynthesis , Genes, rev , Genome, Viral , HIV-2/genetics , HeLa Cells , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Restriction Mapping , Transfection , Viral Proteins/biosynthesis , rev Gene Products, Human Immunodeficiency Virus
14.
J Clin Invest ; 95(1): 328-32, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7814633

ABSTRACT

The replication of human immunodeficiency retroviruses involves a complex series of events that is regulated at both transcriptional and posttranscriptional levels. The tat gene product is a potent trans-activator of viral transcription and therefore an attractive target for the development of antiviral drugs. Tat-defective HIV-1 proviral DNA clones have been shown previously to be replication defective. In this study, we report that tat-defective HIV-1 and HIV-2 viral DNA transfected into U937 cells can direct efficient viral replication in the presence of transcriptional stimulators such as TNF-alpha and PMA. In MT-4 cells, tat-defective HIV-1 can replicate without any stimulation. The viruses recovered from MT-4 cells remained tat defective defined by their inability to infect T cell lines (e.g., Molt 4/8) although replication could be rescued with cytokines. Limited replication was observed in primary mononuclear cells. Furthermore, we showed that Ro 24-7429, a potent tat antagonist and antiviral compound, failed to suppress HIV-1 replication in TNF-alpha-stimulated T cells. These results have important implications for targeting tat as a therapeutic strategy for AIDS.


Subject(s)
Benzodiazepines/pharmacology , Gene Products, tat/metabolism , HIV/growth & development , Monocytes/virology , Pyrroles , Tumor Necrosis Factor-alpha/pharmacology , Cell Line , Defective Viruses/growth & development , Drug Antagonism , Gene Products, tat/antagonists & inhibitors , HIV-1/growth & development , HIV-2/growth & development , Humans , T-Lymphocytes/virology , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Virus Replication , tat Gene Products, Human Immunodeficiency Virus
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