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1.
Bone Marrow Transplant ; 38(9): 615-20, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16964267

ABSTRACT

Several recently published studies have suggested that patients who undergo ABO mismatched hematopoietic stem cell transplantation may be at increased risk for relapse, graft-versus-host disease, transplant-related mortality, and/or all-cause mortality. To investigate this issue further, we analyzed potential associations between the donor-recipient ABO mismatch pattern and the above outcome measures among 240 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation at our institution. Our analyses uncovered no significant associations between donor-recipient ABO mismatch pattern and overall survival, event-free survival, transplant-related mortality, incidence of acute graft-versus-host disease (GVHD), or incidence of chronic GVHD. Our data do not support recent assertions that donor-recipient ABO mismatching is a major risk factor for patients undergoing allogeneic transplant, nor do they support recent assertions that ABO matching should be an important consideration in selecting allogeneic hematopoietic stem cell donors.


Subject(s)
ABO Blood-Group System , Donor Selection , Hematopoietic Stem Cell Transplantation/mortality , Neoplasms/mortality , Tissue Donors , Blood Grouping and Crossmatching/methods , Disease-Free Survival , Donor Selection/methods , Female , Graft vs Host Disease/mortality , Humans , Male , Neoplasms/therapy , Recurrence , Remission Induction , Retrospective Studies , Time Factors , Transplantation, Homologous
2.
Bone Marrow Transplant ; 32 Suppl 1: S3-5, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12931231

ABSTRACT

Hematopoietic stem cell transplantation as a treatment for autoimmune disease began in 1996 and has subsequently spread worldwide. In Europe phase III trials have opened, while in America phase III trials are being designed and funded by the National Institutes of Health. On 6 June 2002, clinicians and scientists from around the world met at Snowbird, Utah to discuss the results and future directions of stem cell therapy for autoimmune diseases. What follows are general concepts from chairpersons of this meeting.


Subject(s)
Autoimmune Diseases/therapy , Hematopoietic Stem Cell Transplantation/methods , Humans , Regeneration , Transplantation Conditioning/methods
3.
Bone Marrow Transplant ; 31(6): 441-6, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12665838

ABSTRACT

Allogeneic stem cell transplantation (SCT) has been shown to be a curative therapy for some patients with non-Hodgkin's lymphoma (NHL). Total-body irradiation and high-dose cyclophosphamide combinations are the most established conditioning regimens used in this setting. We examined the efficacy and toxicity of cyclophosphamide, BCNU, and VP-16 (CBV) as a suitable chemotherapy-only regimen for NHL patients. In total, 18 patients, median age 42 years, with NHL were treated with CBV followed by allotransplant. Patients had received a median of two prior chemotherapy regimens. Median times to neutrophil and platelet recovery were 19 and 15 days, respectively. Interstitial pneumonitis occurred in one patient. There have been four relapses after a median follow-up of 39 months. Overall, there were four deaths, one because of relapse. The 2-year estimates of relapse-free and overall survival are 56 and 76%, respectively. CBV is a safe and an effective alternative to TBI-containing regimens before allogeneic SCT for NHL.


Subject(s)
Antineoplastic Agents, Alkylating/administration & dosage , Antineoplastic Agents, Phytogenic/administration & dosage , Carmustine/administration & dosage , Cyclophosphamide/administration & dosage , Etoposide/administration & dosage , Hematopoietic Stem Cell Transplantation , Lymphoma, Non-Hodgkin/drug therapy , Adolescent , Adult , Blood Platelets/cytology , Disease-Free Survival , Female , Graft vs Host Disease/mortality , Humans , Infections/mortality , Liver Diseases/mortality , Lung Diseases/mortality , Lymphoma, Non-Hodgkin/mortality , Male , Middle Aged , Neutrophils/cytology , Recurrence , Transplantation Conditioning/methods , Transplantation, Homologous , Treatment Outcome
4.
J Immunother ; 24(4): 384-8, 2001.
Article in English | MEDLINE | ID: mdl-11565840

ABSTRACT

Transforming growth factor (TGF)-beta3 has been hypothesized to prevent or alleviate oral mucositis (OM) in cancer patients receiving high-dose chemotherapy (CT). Two double-blind, placebo-controlled, multicenter, phase II studies of TGF-beta3 were initiated in the United States, Europe, and Argentina in patients with lymphomas or solid tumors who were receiving highly stomatotoxic CT regimens. Patients were to apply 10-mL mouthwash applications of TGF-beta3 (25 microg/mL) or placebo four times daily (or twice daily) 1 day before and all days during CT. The patients were subsequently evaluated for OM incidence, severity, and duration using National Institute of Cancer Common Toxicity Criteria (NCI-CTC) criteria and an objective scoring system (1). After the start of the trials, negative results from new preclinical studies suggesting suboptimal formulation and/or dosing led to an interim analysis of the ongoing clinical trials. One hundred fifty-two patients from the combined studies were included in the interim analysis, with 116 patients on the TGF-beta3 four times daily and placebo arms. Most (72%) patients had breast cancer, 22% had lymphomas, and 6% had other solid tumors. Although 98% (149 of 152) of patients experienced adverse events, only 14% (22 of 152) experienced events that were judged as possibly or probably related to the study drug (primarily gastrointestinal symptoms). No clinically relevant differences were seen between the treatment and placebo arms regarding safety, nor was there evidence for systemic absorption of TGF-beta3. Finally, there was no advantage of TGF-beta3 treatment regarding the incidence (TGF-beta3 four times daily versus placebo [46% versus 47%]), onset, or duration of NCI-CTC grade 3 or 4 OM. For this dose, formulation, regimen. and patient population, TGF-beta3 was not effective in the prevention or alleviation of CT-induced OM.


Subject(s)
Antineoplastic Agents/adverse effects , Mouth Mucosa/drug effects , Neoplasms/drug therapy , Transforming Growth Factor beta/therapeutic use , Adult , Aged , Double-Blind Method , Female , Humans , Inflammation/chemically induced , Inflammation/drug therapy , Lymphoma/drug therapy , Male , Middle Aged , Mouthwashes , Placebos , Pregnancy , Transforming Growth Factor beta/administration & dosage
5.
Ann N Y Acad Sci ; 938: 54-61; discussion 61-2, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11458526

ABSTRACT

The donor stem cell phenotype and host microenvironment determine the outcome of a stem cell transplant. In a series of transplant studies in syngeneic male to female or congenic Ly5.1/Ly5.2 models in which hosts have received no or minimal irradiation (100 cGy), evidence overwhelmingly supports the concept that syngeneic engraftment is determined by stem cell competition. These approaches can be extended to H-2 mismatched allogeneic mouse combination when antigen pre-exposure and CD40-CD40 ligand antibody blockage are employed. A human trial in patients with resistant neoplasia infusing pheresed blood with 10(8) CD3 cells/kg showed that tumor responses and complete chimerism occur with very low levels of CD34+ cells/kg and that the extent of previous treatment is a critical factor in determining chimerism. A major feature of transplants is the phenotype of the donor stem cell. This phenotype shows dramatic reversible plasticity involving differentiation, adhesion protein expression, and engraftment with cytokine-induced cell-cycle transit. Homing is probably also plastic. Marked fluctuations in engraftment capacity are also seen at different points in marrow circadian rhythm.


Subject(s)
Graft Survival , Hematopoietic Stem Cell Transplantation/methods , Transplantation Conditioning/methods , Adolescent , Adult , Aged , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Ly/immunology , Apoptosis/drug effects , CD40 Antigens/physiology , CD40 Ligand/drug effects , CD40 Ligand/physiology , Cell Lineage , Chimera , Circadian Rhythm , Clinical Trials as Topic , Dose-Response Relationship, Radiation , Female , Fluorouracil/pharmacology , Graft Enhancement, Immunologic/methods , Graft Survival/drug effects , Graft vs Host Disease , H-2 Antigens/immunology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/radiation effects , Histocompatibility , Humans , In Situ Hybridization, Fluorescence , Lymphocyte Transfusion , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle Aged , Neoplasms/therapy , Phenotype , Radiation Chimera , Spleen/cytology , Thalassemia/therapy , Transplantation Conditioning/adverse effects , Whole-Body Irradiation
6.
J Immunol ; 166(9): 5464-72, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11313384

ABSTRACT

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.


Subject(s)
CD5 Antigens/physiology , Receptors, Antigen, T-Cell/physiology , Signal Transduction/immunology , Animals , CD5 Antigens/biosynthesis , CD5 Antigens/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Membrane/genetics , Cell Membrane/immunology , Cell Membrane/metabolism , Cytoplasm/immunology , Down-Regulation/genetics , Down-Regulation/immunology , Female , Humans , Ligands , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Protein Binding/genetics , Protein Binding/immunology , Protein Structure, Tertiary/genetics , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/genetics , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism
7.
Psychol Bull ; 127(2): 249-66, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11316013

ABSTRACT

Gratitude is conceptualized as a moral affect that is analogous to other moral emotions such as empathy and guilt. Gratitude has 3 functions that can be conceptualized as morally relevant: (a) a moral barometer function (i.e., it is a response to the perception that one has been the beneficiary of another person's moral actions); (b) a moral motive function (i.e., it motivates the grateful person to behave prosocially toward the benefactor and other people); and (c) a moral reinforcer function (i.e., when expressed, it encourages benefactors to behave morally in the future). The personality and social factors that are associated with gratitude are also consistent with a conceptualization of gratitude as an affect that is relevant to people's cognitions and behaviors in the moral domain.


Subject(s)
Affect , Models, Psychological , Morals , Humans , Interpersonal Relations , Motivation , Social Behavior
8.
Bone Marrow Transplant ; 28(11): 1061-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11781617

ABSTRACT

Considerable progress has been made in improving the control of chemotherapy-induced emesis. The impact of available antiemetic options for patients receiving stem cell transplants is unclear, as few prospective data have been collected. We prospectively evaluated antiemetic outcome in patients receiving stem cell transplantation over a 7-day period following the initiation of chemotherapy. The primary endpoints were the number of emetic episodes and the extent of nausea measured on a four-point scale. Eighty-two patients were evaluated. Ninety-five percent of patients had nausea during the first week of treatment; 80% had at least one emetic episode. The percentage of patients with emesis was as follows: day 1: 13%, day 2: 21%, day 3: 30%, day 4: 38%, day 5: 44%, day 6: 39%, day 7: 18%. In multivariate analysis, gender, emesis with prior chemotherapy, history of morning or motion sickness, type of transplant (auto vs allo), use of total body irradiation, or use of dexamethasone did not effect emesis control. Most patients receiving high-dose chemotherapy experience incompletely controlled emesis. Control of nausea and emesis progressively worsened with each subsequent day following initiation of chemotherapy, reaching a nadir on day 5. New treatment approaches are needed to improve emesis control in this patient population.


Subject(s)
Antiemetics/therapeutic use , Antineoplastic Agents/adverse effects , Hematopoietic Stem Cell Transplantation , Adult , Aged , Female , Humans , Logistic Models , Male , Middle Aged , Nausea/chemically induced , Nausea/prevention & control , Prospective Studies , Transplantation Conditioning , Treatment Outcome , Vomiting/chemically induced , Vomiting/prevention & control
9.
Bone Marrow Transplant ; 26(2): 227-9, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10918437

ABSTRACT

We describe a successful autologous bone marrow transplant without the use of any blood products. The patient had relapsed large cell lymphoma. He was a Jehovah's Witness and would not accept transfusions of red blood cells or platelets. He enrolled in our Bloodless Medicine and Surgery Program and was maintained on a regimen of erythropoietin, iron, Amicar, and G-CSF throughout the transplant. He tolerated the transplant well and is alive with no evidence of disease 10 months after autografting.


Subject(s)
Bone Marrow Transplantation/methods , Adult , Anemia/drug therapy , Anemia/economics , Anemia/prevention & control , Blood Transfusion/economics , Blood Transfusion/psychology , Bone Marrow Transplantation/standards , Christianity/psychology , Humans , Lymphoma, Large B-Cell, Diffuse/therapy , Male , Thrombocytopenia/drug therapy , Thrombocytopenia/economics , Thrombocytopenia/prevention & control , Transplantation, Autologous/methods , Transplantation, Autologous/standards , Treatment Outcome
10.
Oncology ; 57(4): 287-92, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10575313

ABSTRACT

Hemorrhagic cystitis is a major cause of morbidity after bone marrow transplantation. Traditional methods of prevention have included mesna (2-mercaptoethane sodium sulfonate) and bladder irrigation. We report the use of hyperhydration as an alternative to these prophylactic measures. One hundred consecutive patients who underwent autologous or allogeneic bone marrow transplantation received high dose cyclophosphamide with hyperhydration using 5% dextrose normal saline at the rate of 250 ml/h and furosemide to maintain a urine output of >150 ml/h. Seventy-one of these patients also received high dose cyclophosphamide as mobilization chemotherapy. There were no episodes of hemorrhagic cystitis following mobilization chemotherapy. The incidence of hemorrhagic cystitis after transplant conditioning was 7% with 2 patients developing clinically significant hemorrhagic cystitis; one was a severe episode. The cost of hyperhydration was US$ 20 per course as opposed to US$ 1,500 per course for mesna, based on acquisition costs at our institution. We conclude that hyperhydration is a safe, inexpensive means of preventing hemorrhagic cystitis associated with high dose cyclophosphamide in bone marrow transplant recipients.


Subject(s)
Antineoplastic Agents, Alkylating/adverse effects , Bone Marrow Transplantation/adverse effects , Cyclophosphamide/adverse effects , Cystitis/prevention & control , Fluid Therapy , Hematuria/prevention & control , Adult , Antineoplastic Agents, Alkylating/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cyclophosphamide/therapeutic use , Cystitis/etiology , Female , Fluid Therapy/economics , Hematuria/etiology , Humans , Incidence , Male , Middle Aged , Treatment Outcome , United States
11.
Development ; 126(17): 3937-45, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10433921

ABSTRACT

The Drosophila spineless (ss) gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss(-) mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus. Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved.


Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins , Drosophila Proteins , Drosophila/growth & development , Drosophila/metabolism , Insect Proteins/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Transcription Factors , Alleles , Animals , Animals, Genetically Modified , Aryl Hydrocarbon Receptor Nuclear Translocator , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , DNA, Complementary/genetics , Dimerization , Drosophila/genetics , Enhancer Elements, Genetic , Extremities/growth & development , Gene Expression Regulation, Developmental , Genes, Insect , Insect Proteins/chemistry , Insect Proteins/genetics , Mutation , Phenotype , Protein Structure, Quaternary , Receptors, Aryl Hydrocarbon/chemistry , Receptors, Aryl Hydrocarbon/genetics , Sense Organs/growth & development
12.
Leuk Lymphoma ; 34(1-2): 105-9, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10350337

ABSTRACT

Some heavily pretreated cancer patients fail to mobilize enough peripheral blood stem cells (PBSC) after stimulation with chemotherapy and hematopoietic growth factors. For these patients the best way to obtain an adequate PBSC collection is unknown. Here we report 6 heavily pretreated cancer patients who failed to mobilize sufficient PBSC after stimulation with chemotherapy and G-CSF 5 microg/kg/day. In these cases, we used G-CSF 10 microg/kg/day alone for six days at least 3 weeks after the last chemotherapy. After three consecutive leukaphereses starting on day 5, five patients had adequate PBSC collections. With 6 days of G-CSF 10 microg/kg/day alone, 2.8 x 10(6) (+/- 1) CD34+ cells/kg were collected. This was significantly higher than the number of CD34+ cells/kg collected after chemotherapy and G-CSF 5 microg/kg 0.3 x 10(6) (+/- 0.1) [P = 0.05]. Four patients received high-dose chemotherapy with PBSC support. Hematologic recovery observed in these patients was as expected. In conclusion, G-CSF 10 microg/kg alone can mobilize progenitor cells into peripheral blood when previous mobilization with chemotherapy and G-CSF 5 microg/kg fails.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Granulocyte Colony-Stimulating Factor/administration & dosage , Hematopoietic Cell Growth Factors/therapeutic use , Hematopoietic Stem Cell Mobilization/methods , Hodgkin Disease/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Adult , Breast Neoplasms/blood , Combined Modality Therapy , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Granulocyte Colony-Stimulating Factor/adverse effects , Hematopoietic Stem Cell Transplantation , Hodgkin Disease/blood , Humans , Injections, Subcutaneous , Leukapheresis , Lymphoma, Non-Hodgkin/blood , Male , Middle Aged
13.
Ann N Y Acad Sci ; 872: 40-5; discussion 45-7, 1999 Apr 30.
Article in English | MEDLINE | ID: mdl-10372109

ABSTRACT

Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step; in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation. The stem cells that engraft in the nonmyeloablated are in G0, but are rapidly induced (by 12 hours) to enter the S phase after in vivo engraftment. Exposure of murine marrow to cytokines (IL-3, IL-6, IL-11 and steel factor) expands progenitor clones, induces stem cells into cell cycle, and causes a fluctuating engraftment phenotype tied to phase of cell cycle. These data indicate that the concepts of stem cell competition and fluctuation of stem cell phenotype with cell cycle transit should underlie any new stem cell engraftment strategy.


Subject(s)
Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/immunology , Lymphocytes/cytology , Transplantation, Homologous/immunology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Cell Differentiation , Cytokines/pharmacology , Graft Rejection/immunology , Graft vs Host Disease/immunology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Immunophenotyping , Lymphocytes/immunology , Mice , Transplantation Chimera
14.
Exp Hematol ; 27(5): 895-903, 1999 May.
Article in English | MEDLINE | ID: mdl-10340406

ABSTRACT

Several lines of evidence suggest that overexpression of interferon gamma (IFN-gamma) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-gamma by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-gamma. The effect of IFN-gamma on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-gamma vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-gamma-expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-gamma or control vector. Marrow cells transduced with either vector were transplanted into W/Wv recipient mice. The percentage of vector-containing cells in the mIFN-gamma mice was significantly lower than in the control mice, suggesting that mIFN-gamma-transduced primitive cells may not have survived culture, or that mIFN-gamma directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-gamma-transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-gamma group. These data indicate that ectopic or overexpression of mIFN-gamma, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo.


Subject(s)
Cell Division/genetics , Hematopoietic Stem Cells/cytology , Interferon-gamma/genetics , Stromal Cells/metabolism , Animals , Base Sequence , Coculture Techniques , DNA Primers , Female , Humans , Mice , Mice, Inbred C57BL , Recombinant Proteins/genetics , Retroviridae/genetics , Transduction, Genetic
15.
Hum Gene Ther ; 9(17): 2629-40, 1998 Nov 20.
Article in English | MEDLINE | ID: mdl-9853529

ABSTRACT

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.


Subject(s)
Bone Marrow Cells/metabolism , Gaucher Disease/therapy , Genetic Therapy , Glucosylceramidase/genetics , Retroviridae/genetics , Transduction, Genetic , Adult , Antigens, CD34/analysis , Base Sequence , Bone Marrow Cells/immunology , DNA Primers , Female , Gaucher Disease/enzymology , Gaucher Disease/genetics , Gene Transfer Techniques , Genetic Vectors , Glucosylceramidase/blood , Granulocyte Colony-Stimulating Factor/therapeutic use , Hematopoietic Stem Cell Mobilization , Humans , Male , Stromal Cells/cytology
16.
Proc Natl Acad Sci U S A ; 95(25): 14909-14, 1998 Dec 08.
Article in English | MEDLINE | ID: mdl-9843989

ABSTRACT

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3gamma, -delta, -epsilon, and zeta). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3gamma, -delta, or zeta results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3epsilon-/- mice, and thymocyte development is arrested at the early CD4(-)CD8(-) stage. Although these results suggest that CD3epsilon is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3gamma and CD3delta genes also is reduced in CD3epsilon-/- mice. Thus, it is unclear whether the phenotype of CD3epsilon-/- mice reflects the collective effects of CD3gamma, -delta, and -epsilon deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3epsilon gene via Cre/loxP-mediated recombination, we generated mice that lack CD3epsilon yet retain normal expression of the closely linked CD3gamma and CD3delta genes. These (CD3epsilonDelta/Delta) mice exhibited an early arrest in T cell development, similar to that of CD3epsilon-/- mice. Moreover, the developmental defect could be rescued by expression of a CD3epsilon transgene. These results identify an essential role for CD3epsilon in T cell development not shared by the CD3gamma, CD3delta, or zeta-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.


Subject(s)
CD3 Complex , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Animals , Cell Differentiation/immunology , Flow Cytometry , Immunophenotyping , Mice , Molecular Sequence Data , Mutation , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/cytology
18.
Hum Gene Ther ; 8(14): 1715-30, 1997 Sep 20.
Article in English | MEDLINE | ID: mdl-9322874

ABSTRACT

Fanconi Anemia (FA) is a rare genetic disorder characterized by progress pancytopenia, congenial abnormalities, and a predisposition to malignancy. Therapy is currently limited to allogeneic marrow transplantation; patients lacking a suitable donor usually die from aplasia or acute leukemia. Recently, mutation in a novel gene named FACC (Fanconi anemia C-complementing) has been identified as causing one type of FA. FACC mutations, which introduce splicing errors or stop codons, have been identified in approximately 15% of FA patients. We have recently been successful in functional complementation of four FA cell lines using retroviral vectors to transfer a copy of the normal FACC gene. We also analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. As for the lymphoid cell lines, these CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, the progenitors gave rise to increased numbers of colonies both in the absence and presence of up to 5nM MMC, whereas control cells were completely destroyed by 1nM MMC. In summary, we have demonstrated that: (1) retroviral vectors can be engineered to transfer a normal FACC gene to FA(C) lymphoid cell lines and primary hematopoietic cells; (2) introduction of a normal FACC gene into CD34+ progenitors markedly enhances their growth in the absence and presence of MMC. This study is designed to determine whether hematopoietic progenitors transduced with the normal FACC gene can be reinfused safely into FA(C) patients. CD34+ cells obtained from G-CSF mobilized peripheral blood will be transduced ex vivo over a 72-hour period in the presence of IL-3, IL-6, and Stem Cell Factor with the FACC retroviral vector. These transduced cells will be reinfused into FA(C) patients. Patients will be monitored for toxicities as well as evidence of successful gene transfer and expression. The procedure will be repeated up to a total of 4 times with each treatment 2-4 months apart. Theoretically, these rescued stem cells should have a selective growth advantage within the hypoplastic FA marrow environment in vivo.


Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins , Fanconi Anemia/therapy , Gene Transfer Techniques , Hematopoietic Stem Cells , Nuclear Proteins , Proteins/genetics , Cell Transplantation , Clinical Protocols , Fanconi Anemia Complementation Group C Protein , Fanconi Anemia Complementation Group Proteins , Genetic Therapy/methods , Genetic Vectors/genetics , Humans , Pilot Projects , Retroviridae/genetics
19.
Hum Gene Ther ; 8(11): 1313-9, 1997 Jul 20.
Article in English | MEDLINE | ID: mdl-9295126

ABSTRACT

Green fluorescent protein (GFP) has been used as a reporter molecule for gene expression because it fluoresces green after blue-light excitation. Inclusion of this gene in a vector could allow rapid, nontoxic selection of successfully transduced cells. However, many attempts by our laboratory to isolate stable retroviral producer cell clones secreting biologically active vectors containing either the highly fluorescent S65T-GFP mutant or humanized GFP have failed. Vector plasmids containing various forms of GFP and the neomycin resistance gene were transfected into three different packaging cell lines and fluorescence was observed for several days, but stable clones selected with G418 no longer fluoresced. Using confocal microscopy, the brightest cells were observed to contract and die within a matter of days. RNA slot-blot analysis of retroviral producer supernatants showed no viral production from the GFP plasmid-transfected clones, although all clones derived after transfection with an identical retroviral construct not containing GFP produced virus. Genomic Southern analysis of the GFP-transduced clones showed a much higher probability of rearrangement of the priviral sequences than in the control non-GFP clones. Overall, 18/34 S65T-GFP clones and 17/33 humanized-GFP clones had rearrangements, whereas 2/15 control non-GFP clones had rearrangements. Hence, producer cells expressing high levels of these GFP genes seem to be selected against, with stable clones undergoing major rearrangements or other mutations that both abrogate GFP expression and prevent vector production. These observations indicate that GFP may not be an appropriate reporter gene for gene transfer applications in our vector/packaging system.


Subject(s)
Genes, Reporter/genetics , Genetic Vectors/metabolism , Luminescent Proteins/genetics , Retroviridae/genetics , Animals , Blotting, Southern , Cell Line , Gene Transfer Techniques , Genes, Viral/genetics , Genetic Markers , Genetic Vectors/genetics , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Plasmids , RNA, Viral/analysis , Retroviridae/physiology , Virus Replication
20.
Blood ; 89(11): 4040-6, 1997 Jun 01.
Article in English | MEDLINE | ID: mdl-9166843

ABSTRACT

Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.


Subject(s)
Bone Marrow/pathology , Gene Transfer Techniques , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/pathology , Adult , Bone Marrow/drug effects , Drug Resistance, Microbial/genetics , Female , Genetic Vectors , Hematopoietic Stem Cells/drug effects , Humans , Male , Middle Aged , Neomycin/pharmacology , Retroviridae/genetics , Stromal Cells/drug effects , Stromal Cells/pathology , Stromal Cells/transplantation , Transplantation, Autologous
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