[show abstract][hide abstract] ABSTRACT: Recently, it has been reported that prophylactic administration of ciprofloxacin during cyclophosphamide (CY) conditioning was a high-risk factor for relapse in patients undergoing allogeneic BMT. In the present study, we investigated the possible mechanisms of this interaction in male Sprague-Dawley rats. The kinetics of CY and its active 4-OH-CY metabolite were determined, after 3 days pretreatment with ciprofloxacin (200 mg/kg) and compared to control rats without treatment. CY was administered as a high or low single intravenous dose (150 and 90 mg/kg, respectively). The expression of the CYP2B1, CYP2B2, CYP2C11, CYP3A1 and CYP3A2 genes was evaluated by SYBR Green I Dye real-time PCR for quantification of mRNA. The administration of ciprofloxacin resulted in a significant increase in the AUC (P=0.007) and a significant decrease in clearance (P=0.007) when CY was given as a high dose. In accordance, the metabolic ratio (AUC4-OH-CY/AUCCY) was significantly lower (P=0.007) compared to that found in the control group. Ciprofloxacin significantly suppressed gene expression of CYP2C11 (P=0.01) and CYP3A1 (P=0.04); however, no effect was observed on the gene expression of CYP3A2, CYP2B1 and CYP2B2. Our study revealed that ciprofloxacin interacts with CY and suppressed relevant cytochromes p450 at the transcriptional level. This study may have a great clinical impact when ciprofloxacin is used in therapy.
Bone Marrow Transplantation 03/2003; 31(3):197-203. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the setting of allogeneic hematopoietic stem cell transplantation, ex vivo culturing of donor T lymphocytes is a necessary step for processes such as gene modification. Often the aim is to enable control of undesired alloreactivity after in vivo administration of the cultured cells. However, it is not fully understood how T cell reactivity against donor and third-party targets is affected by the ex vivo cell culturing process. We have assessed how the activity of anti-Epstein Barr virus (EBV)-specific T lymphocytes from healthy EBV-seropositive donors is affected by in vitro cell culturing. Peripheral blood mononuclear cells (PBMCs) were expanded in X-VIVO 15 culture medium supplemented with 5% human serum. The cells were stimulated by either OKT3 (10 ng/ml) and interleukin (IL)-2 (500 U/ml) or by using anti-CD3/CD28-coated immunomagnetic beads and IL-2 (100 U/ml). Induction of polyclonal EBV-specific cytotoxic T lymphocyte cultures was attempted by stimulation of the in vitro-expanded cells at different time points during the cell expansion process, with pre-established autologous EBV-transformed lymphoblastoid cell lines (LCLs). While EBV-specific cytotoxic T lymphocytes (CTL) were generated from untreated PBMCs of 5 healthy donors, EBV-specific cytotoxicity was significantly decreased or absent in CTL cultures established from in vitro-expanded PBMCs. Our results indicate that the ex vivo cell expansion process itself significantly reduces the activity and/or the number of EBV-specific T cells. Additional stimulation with CD28 antibodies could not prevent this effect. Because T cell depleted bone marrow or stem cell grafts are known to contribute to the development of post transplant lymphoproliferative disorders, this should be taken into consideration if one considers expanding and administering PBMCs in conjunction with a T cell-depleted stem cell grafts.
Journal of Hematotherapy & Stem Cell Research 09/2002; 11(4):669-74.
[show abstract][hide abstract] ABSTRACT: Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-). However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells. The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.
Human Immunology 11/2001; 62(10):1092-8. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Between 1991 and 1999, 44 leukemic patients received donor lymphocyte infusions (DLIs) at our center (22 patients with chronic myelogenous leukemia [CML]; 10 with acute myelogenous leukemia; 11 with acute lymphatic leukemia; and 1 with myelodysplastic syndrome). Seventeen patients received graft-versus-host disease (GVHD) prophylaxis with methotrexate (MTX) at the time of DLI. In CML patients, 15 of 22 (68%) re-entered complete remission after DLI. At 3 years post-DLI, patients with cytogenetic (n = 10) or molecular (n = 3) relapse had a current leukemia-free survival (cLFS) rate of 85% compared with 0% for patients with hematologic relapse (P < .001). Among 15 CML patients who initially responded to DLI, 4 patients relapsed within the first 2 years. Four of 16 patients (25%) with acute leukemia had an initial response with complete remission after DLI. Two of them subsequently relapsed within 1 year. Patients with acute leukemia who relapsed within 1 year of hematopoietic stem cell transplantation (n = 9) had 0% cLFS at 18 months; patients with later relapse had 29% cLFS (P = .015). The overall probability of cLFS at 3 years for CML patients was 46%. For other diseases, cLFS was 13% at 18 months after DLI. Patients who developed chronic GVHD secondary to DLI showed a 3-year cLFS of 51% compared with 18% for patients without chronic GVHD (P = .022). This study emphasizes the importance of early disease stage and presence of chronic GVHD for effective DLI.
Biology of Blood and Marrow Transplantation 02/2001; 7(1):31-8. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable.
We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%). We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets. Buffy-coats from four healthy donors were expanded for 21 days. The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%). For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors.
In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117). For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance). When serum was added, this discrepancy was not seen. Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture. Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions. Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures. Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells.
Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells. These cells can easily be transduced and selected. There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days. The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.
[show abstract][hide abstract] ABSTRACT: Objective
In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable.
[show abstract][hide abstract] ABSTRACT: To minimize immunosuppression, allow a graft-versus-leukemia (GVL) effect, and reduce relapse incidence, 73 leukemic recipients of human leukocyte antigens-identical sibling marrow were given graft-versus-host disease (GVHD) prophylaxis based on the estimated risk of GVHD development. Methotrexate (MTX) monotherapy was given to patients with an estimated low risk of developing GVHD, whereas MTX + cyclosporine (CsA) was given to 'high-risk' patients. After engraftment, CsA was discontinued, and weekly MTX was reinstituted and given until 3 months post-bone marrow transplant. Conditioning consisted of busulfan (BU) + cyclophosphamide (CY) (n = 35) or CY + total body irradiation (TBI) (n = 38). Retrospective controls were given CY + TBI and MTX + CsA (n = 39). The median observation time was 5 yr 11 months. Chronic GVHD increased to 53% in the individual BU + CY group and 46% in the individual CY + TBI group, compared to 25% in the control group (p = 0.05). This increase was restricted to the limited form. The actuarial relapse incidence decreased to 20% in the individual BU + CY group, compared to 52% in the control group, p = 0.03. In the individual CY + TBI group, the relapse incidence was 44% (n.s. versus controls, p = 0.04 versus individual BU + CY). The 5-yr relapse-free survival (RFS) in the individual BU + CY group was 66%, in the control group, 41% (p = 0.07), and in the individual CY + TBI group, 45% (p = 0.1 versus individual BU + CY). Patients with early leukemia in the individual BU + CY group had a RFS of 83%, compared to 44% in the control group (p = 0.02) and 42% in the individual CY + TBI group (p = 0.01). In the multivariate analysis, advanced leukemia beyond first complete remission and first chronic phase and conditioning with CY + TBI were correlated to poor RFS. In summary, the individualized prophylaxis itself did not reduce the relapse incidence. However, in patients with early leukemia conditioning with BU + CY, our method of individualizing the GVHD prophylaxis might be of value, since this group had the best RFS in this study.
[show abstract][hide abstract] ABSTRACT: Between 1990 and 1996, three patients (1.1%), all with CML, among 272 patients with haematological malignancies, developed bilateral subdural haematomas (SDH) after treatment with i.t. MTX before HSCT in our unit. Since October 1996, we have given i.t. MTX only to patients at increased risk of CNS leukaemia such as ALL and AML M4 or M5. We suggest that intrathecal treatment before HSCT should only be given to patients at increased risk of CNS leukaemia.
Bone Marrow Transplantation 12/1999; 24(9):1033-5. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: A study was done to compare treatment with Filgrastim (r-metHuG-CSF) given at three different times after unrelated bone marrow transplantation (BMT). Sixty-nine patients grafted with HLA-A, -B and -DR-compatible unrelated bone marrow were randomized to Filgrastim (5 microg/kg/day) starting on day 0 (n = 23), day +5 (n = 23) or day +10 (n = 23) after BMT. No significant differences were detected in hematological recovery, days with fever, days on antibiotics, incidence of bacteremia or need for erythrocyte, platelet and granulocyte transfusions between the three groups. Patients given Filgrastim starting on day 0, day +5 or day +10, respectively, reached an absolute neutrophil count (ANC) >0.5 x 109/l on a median of 17, 16 and 16 days after BMT. Starting Filgrastim treatment on day +10, rather than on day 0, reduced the costs of Filgrastim by $1060, with no significant change in the median number of days-to-hospital discharge in the three Filgrastim-treated groups. The incidences of acute and chronic GVHD, transplantation-related mortality, relapse, leukemia-free survival and patient survival (PS) were similar in all groups.
Bone Marrow Transplantation 11/1999; 24(8):831-6. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Low-dose cyclosporine (CsA), starting at 1 mg/kg/day i.v. with early discontinuation, and four doses of methotrexate (MTX), was given to 82 consecutive leukaemic patients receiving HLA-identical sibling marrow transplants. Retrospective controls (n = 40) received CsA, starting at 5-7.5 mg/kg/day i.v., given for 1 year, and MTX. In the low-dose group, the risk of acute GVHD grades I-II was 78% as compared to 57% among the controls (P < 0.01). The risk of acute GVHD grades III-IV was 2% and 5%, respectively (NS). Chronic GVHD occurred in 60% in the low-dose group and 24% in the controls (P < 0. 001). Extensive chronic GVHD did not differ between the groups (3% vs6%). In multivariate analyses, low-dose CsA was the only factor associated with acute GVHD grades I-IV (P = 0.02). Significant risk factors for chronic GVHD included low-dose CsA (P = 0.002) and CML (P = 0.03). Transplant-related mortality at 3 years post-BMT was 22% and 19%, in the low-dose group and controls, respectively (NS). The probability of relapse was 26% in the low-dose group and 53% in the controls (P = 0.06). In multivariate analysis, high-dose CsA was the strongest risk factor for relapse (P = 0.03). The 3-year relapse-free survival was 58% in the low-dose group and 43% in the controls (P = 0.1).
Bone Marrow Transplantation 10/1999; 24(6):629-35. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Patients who do not respond to autologous stem cell transplantation (ASCT) have a poor prognosis. Concerns about toxicity limit the use of unrelated donor stem cell transplantation (UDSCT), but the knowledge about outcome after UDSCT post-ASCT is limited. We carried out a retrospective analysis of the outcome in seven consecutive patients with leukemia (n = 5), myeloma (n = 1) and graft failure (n = 1) who underwent UDSCT after ASCT. Donors were matched for HLA-A, -B and -DR (n = 6) or had one class I antigen mismatch (n = 1). Tissue typing was performed by a high-resolution genomic technique for class II. Median patient age was 34 (11-54) years and time from ASCT to UDSCT was 16 (3-22) months. Patients with malignant diseases were given TBI and a CY preparatory regimen. In addition, all patients received T cell antibodies prior to UDSCT. Grade I acute GVHD developed in all seven patients, but there was no sign of more severe acute GVHD. Two of four evaluable patients developed limited chronic GVHD. Three died of transplant-related toxicity, all due to pulmonary complications. Four patients are alive at 1.1, 1.5, 3.1 and 4.9 years post-UDSCT. A closely matched UDSCT could be considered for selected patients who are not cured by an ASCT.
Bone Marrow Transplantation 09/1999; 24(3):279-82. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several preventive strategies against cytomegalovirus (CMV) disease have been developed during the last decade. These have frequently been used in combination, and it has been difficult to identify each strategy's contribution.
Risk factors for CMV disease, death in CMV disease and transplant-related mortality were analyzed in 584 patients, who underwent a total of 594 allogeneic bone marrow transplants.
The overall probability of CMV disease was 8.9%. No seronegative patient who had a seronegative marrow donor developed CMV disease. The corresponding probabilities for seronegative patients with seropositive donors, seropositive patients with seronegative donors, and seropositive patients with seropositive donors were 5.4%, 13.7%, and 11.7%, respectively. In multivariate Cox models, the use of preemptive antiviral therapy and being CMV-seronegative reduced the risk for CMV disease, CMV-associated death, and transplant-related mortality (TRM). Patients who received unrelated or mismatched family donor transplants had increased risks for CMV disease, CMV-associated death, and TRM. Older age was a significant risk factor for CMV disease and TRM. A total of 258 patients who were monitored by polymerase chain reaction for CMV DNA were analyzed separately to assess whether addition of another CMV preventive strategy could give benefit. Patients who received mismatched or unrelated donor transplants had increased risk for CMV disease, death in CMV disease, and TRM. High-dose acyclovir prophylaxis or addition of intravenous immune globulin had no influence.
Preemptive therapy based on polymerase chain reaction for CMV DNA was associated with reduced risks for CMV disease, CMV-associated death, and TRM, whereas other prophylactic modalities did not give additional benefit.
[show abstract][hide abstract] ABSTRACT: Among 551 consecutive recipients of allogeneic bone marrow transplants, 451 survived more than 3 months and were evaluated for chronic graft-versus-host disease (GVHD). Most of the donors were HLA-identical siblings or parents (n = 334). Patients with HLA-mismatched donors (n = 30) and matched unrelated donors (MUD) (n = 87) were also included in the study. In the analysis of all patients, the 5-year cumulative incidence of chronic GVHD was 45%. We analysed 34 risk factors. High recipient age was the single most important risk factor (P < 0.001). Other significant risk factors in multivariate analysis were: acute GVHD grades I-IV (P < 0.001), immune female donor to male recipient (P = 0.006) and chronic myelogenous leukaemia (CML), compared with all other diagnoses (P = 0.014). The cumulative 5-year incidence of chronic GVHD, with no significant risk factors present, was 9%, 29% with one risk factor, 53% with two, 68% with three and 75% with all four risk factors present. In patients with HLA-identical sibling donors and GVHD prophylaxis consisting of a combination of methotrexate (MTX) and cyclosporin A (CsA) (n = 208), increasing recipient age (P < 0.001) and CML (P = 0.007), were found to be significant risk factors for chronic GVHD. Finally, a multivariate analysis in recipients of bone marrow from unrelated donors (n = 89) showed recipient age alone (P = 0.006) to be significantly associated with chronic GVHD.
Bone Marrow Transplantation 11/1998; 22(8):755-61. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using unrelated bone marrow, there is an increased risk of graft-versus-host disease (GVHD).
HLA-A-, HLA-B-, and HLA-DR-compatible unrelated bone marrow was given to 132 patients. The diagnoses included chronic myeloid leukemia (n=43), acute lymphoblastic leukemia (n=29), acute myeloid leukemia (n=27), myelodysplastic syndrome (n=4), lymphoma (n=3), myeloma (n=1), myelofibrosis (n=1), severe aplastic anemia (n=12), and metabolic disorders (n=12). The median age was 25 years (range 1-55 years). HLA class I was typed serologically, and class II was typed by polymerase chain reaction using sequence-specific primer pairs. Immunosuppression consisted of antithymocyte globulin or OKT3 for 5 days before transplantation and methotrexate combined with cyclosporine.
Engraftment was seen in 127 of 132 patients (96%). Bacteremia occurred in 47%, cytomegalovirus (CMV) infection in 49%, and CMV disease in 8%. The cumulative incidences of acute GVHD > or = grade II and of chronic GVHD were 23% and 50%, respectively. The 5-year transplant-related mortality rate was 39%. The overall 5-year patient survival rate was 49%; in patients with metabolic disorders and severe aplastic anemia, it was 61% and 48%, respectively. The disease-free survival rate was 47% in patients with hematological malignancies in first remission or first chronic phase and 38% in patients with more advanced disease (P=0.04). Acute GVHD was associated with early engraftment of white blood count (P=0.02). Poor outcome in multivariate analysis was associated with acute myeloid leukemia (P=0.01) and CMV disease (P=0.04).
Using HLA-A-, HLA-B-, and HLA-DR-compatible unrelated bone marrow and immunosuppression with antithymocyte globulin, methotrexate, and cyclosporine, the probability of GVHD was low and survival was favorable.
[show abstract][hide abstract] ABSTRACT: Three hundred and six patients with low- and intermediate-risk leukaemias undergoing allogeneic BMT between 1980 and March 1996 were studied regarding transplantation-related mortality (TRM), relapse, and leukaemia-free survival (LFS). Among the patients were 262 recipients of marrow from HLA-identical siblings and 44 patients receiving marrow from HLA-A, -B, and -DR identical unrelated donors. Between 1986 and 1993, 153 adult patients received ciprofloxacin continuously during Cy conditioning, but since November 1993 ciprofloxacin has not been given until after Cy treatment. TRM at 5 yr showed an incidence of 30%. Significant risk factors in Cox regression multivariate analysis comprised acute GVHD grades II-IV (p < 0.0001), seropositivity for 3-4 herpes viruses prior to BMT (p = 0.002), intermediate risk disease (p = 0.008), female donor to male recipient (p = 0.015), and a donor age over 17 yr (p = 0.025). The risk of relapse was studied from 90 d after BMT, and the overall 5-yr incidence was 32%. Significant risk factors comprised acute leukaemia, as compared to CML (p = 0.003), total body irradiation (TBI) compared to busulphan treatment (p = 0.011), gram-negative prophylaxis with ciprofloxacin during cyclophosphamide (Cy) conditioning (p = 0.024), GVHD prophylaxis using a combination of methotrexate (MTX) and cyclosporine (CSA), compared to monotherapy (p = 0.037) and absence of chronic GVHD (p = 0.050). The 5-yr probability of relapse in patients receiving ciprofloxacin prophylaxis during Cy conditioning was 40%, compared to 24% in patients not receiving this treatment (p = 0.01). Overall, LFS at 5 yr was 49%. LFS was evaluated from day 30 after BMT until relapse or death of the patient. We found no difference in TRM, relapse or LFS between recipients of HLA-identical sibling or unrelated bone marrow, risk factors significantly associated with an inferior LFS included acute GVHD grades II-IV (p = 0.0002), intermediate risk disease (p = 0.003), donor seropositivity for 3-4 herpes viruses (p = 0.046), and TBI conditioning (p = 0.048).
[show abstract][hide abstract] ABSTRACT: Twenty-seven patients above 40 years of age (range 40-55) with leukaemia underwent transplantation with haematopoietic stem cells from HLA-A, -B and -DR identical unrelated donors. They were compared to 69 younger patients, median age 23. In the older group, the diagnoses were acute myeloid leukaemia (AML) five, acute lymphoblastic leukaemia (ALL) three and chronic myeloid leukaemia (CML) 19. The corresponding figures in the younger patients were 21, 27 and 21, respectively. Conditioning consisted of cyclophosphamide (120 mg/kg) combined with 10 Gy total body irradiation. Immunosuppression was ATG or OKT3 for 5 days before transplantation and methotrexate combined with cyclosporin A. The probabilities of grades II-IV acute graft-versus-host disease (GVHD) were 23 and 21%, and the cumulative incidences of chronic GVHD were 64 and 50% in the older and younger patient cohorts, respectively. Overall, 3-year transplant-related mortality rates were 46% in patients > or =40 years of age and 32% in patients <40 years of age (P = 0.16). Three-year patient survival rates were 54 and 46% in the two groups, respectively. In patients with chronic phase CML, the corresponding figures were 67 and 68%, respectively. We conclude that patients above 40 years of age should be considered for transplantation with marrow from unrelated donors.
Bone Marrow Transplantation 01/1998; 21(1):43-9. · 3.54 Impact Factor