Durable treatment-free remission after high-dose cyclophosphamide therapy for previously untreated severe aplastic anemia.

BACKGROUND: Severe aplastic anemia is a life-threatening bone marrow failure disorder. High-dose cyclophosphamide therapy followed by allogeneic bone marrow transplantation cures the disease. However, it requires a suitable donor and carries the risk for graft-versus-host disease. A small pilot study demonstrated that high-dose cyclophosphamide therapy without bone marrow transplantation leads to durable, treatment-free complete remission. OBJECTIVE: To confirm the safety and efficacy of high-dose cyclophosphamide therapy alone in patients with severe aplastic anemia. DESIGN: Uncontrolled clinical trial. SETTING: Three tertiary care hospitals. PATIENTS: 19 patients with untreated severe aplastic anemia. INTERVENTION: Cyclophosphamide, 50 mg/kg of body weight per day for 4 consecutive days. MEASUREMENTS: Probability of response and overall survival were measured. Complete remission was defined as normal blood count for age and sex. Partial remission was defined as independence from transfusion and an absolute neutrophil count greater than 0.5 x 10(9) cells/L without growth factor support. Nonresponders were patients who remained transfusion dependent or died. Relapse was defined as no longer meeting criteria for partial or complete remission. RESULTS: The median time to an absolute neutrophil count of 0.5 x 10(9) cells/L was 49 days. The probability of survival was 84% (95% CI, 59% to 95%) at 24 months. The probability of achieving treatment-free remission was 73% (CI, 51% to 91%) at 24 months, and the probability of achieving complete remission was 65% (CI, 39% to 89%) at 50 months. No responding patients have had relapse or have developed secondary clonal disorders. CONCLUSIONS: High-dose cyclophosphamide therapy without bone marrow transplantation produces durable treatment-free remission in severe aplastic anemia. This approach deserves further study in patients with severe aplastic anemia who are not suitable candidates for allogeneic bone marrow transplantation.

Pulmonary T cell repertoire in patients with graft-versus-host disease following blood and marrow transplantation.

Pulmonary inflammation is one of the risk factors associated with blood and marrow transplantation (BMT). To determine the potential role of T cells in pulmonary complications after transplantation, we analyzed the T-cell repertoire expressed in bronchoalveolar lavage fluids from eleven patients with graft-versus-host disease following BMT. A reverse transcriptase-polymerase chain reaction was used to amplify rearranged TCR transcripts in unfractionated, CD4+, and CD8+ T cells from bronchoalveolar lavage fluids. The relative expression of TCR variable (V) gene families and the diversity of junctional region lengths associated with different AV and BV gene families were analyzed. Nearly all TCR AV and BV gene families were detected in bronchoalveolar lavage cells from BMT recipients. Oligoclonal patterns of TCR junctional region lengths were observed in unfractionated, CD4+, and CD8+ bronchoalveolar T cells. The oligoclonal expansion of bronchoalveolar T cells in patients was confirmed by DNA sequencing. TCRV gene expression is almost completely restored in the lungs of BMT recipients as early as two weeks after transplantation. Increased oligoclonality among TCR gene families suggests either an incomplete restoration of TCR diversity or an antigen-driven expansion of T cells in the lungs of BMT recipients with graft-versus-host disease, not necessarily related to pulmonary infection.

Plasma concentrations and pharmacokinetics of dimethylsulfoxide and its metabolites in patients undergoing peripheral-blood stem-cell transplants.

PURPOSE: Dimethylsulfoxide (DMSO) is used to cryopreserve hematopoietic stem cells and is obligatorily infused into patients who receive stem-cell transplants. This study characterized the plasma concentrations and pharmacokinetics of DMSO and its metabolites in patients who underwent peripheral-blood stem-cell transplants. MATERIALS AND METHODS: Plasma concentrations of DMSO, dimethylsulfone (DMSO2), and dimethylsulfide (DMSH2) were assessed in 10 patients who underwent autologous transplants with stem cells, cryopreserved in 10% DMSO (vol/vol). Blood was sampled at multiple times after the stem-cell infusion. Urine was pooled during the 24 hours postinfusion. DMSO, DMSO2, and DMSH2 were assayed simultaneously by gas chromatography. A one-compartment model with saturable elimination proved most suitable for fitting plasma DMSO concentration-versus-time data. RESULTS: Stem-cell volumes infused ranged between 180 and 585 mL (254 to 824 mmol DMSO). Infusions lasted between 20 and 120 minutes. Peak plasma DMSO concentrations were 19.1 +/- 6.3 mmol/L (mean +/- SD). Pharmacokinetic parameters for volume of the central compartment (Vc), maximum velocity (Vmax), and Michaels-Menten constant (Km) were 37.3 +/- 17 L, 0.99 +/- 0.57 mmol/L/h, and 5.2 +/- 5.0 mmol/L, respectively. Plasma DMSO2 concentrations increased during the first 24 hours, plateaued at 4.4 +/- 1.2 mmol/L, and remained there until 48 hours (the last sample). DMSH2 concentrations were at steady-state by 5 minutes and remained between 3 and 5 mmol/L for 48 hours. Urinary excretion of DMSO and DMSO2 accounted for 44% +/- 4% and 4% +/- 1%, respectively, of the administered DMSO dose. Renal clearance of DMSO was 14.1 +/- 3.4 mL/min. CONCLUSION: These data (1) document plasma concentrations of DMSO and metabolites in patients following peripheral-blood stem-cell transplants; (2) allow consideration of potential effects of these concentrations on stem-cell engraftment and drug-drug interactions; and (3) can facilitate a concentration-guided phase I trial of DMSO.

Transplantation in patients with multiple myeloma: a multicenter comparative analysis of peripheral blood stem cell and allogeneic transplant.

We performed a multicenter comparative analysis of autologous peripheral blood stem cell transplantation (PBSCT) and allogeneic bone marrow transplantation (alloBMT) in multiple myeloma. Forty-eight consecutive patients received either PBSCT (24 patients) or alloBMT (24 patients) at one of three institutions in the study group. Preparatory regimens consisted of melphalan and total body irradiation (TBI) or melphalan alone in the PBSCT group. The alloBMT group received one of four regimens: cyclophosphamide and TBI; cyclophosphamide, VP-16 and 1,3-bis(2-chloroethyl)-1-nitrosourea (CVB); busulfan and cyclophosphamide (BU/CY) and total marrow irradiation (TMI); or melphalan and TBI. Procedure-related mortality was 12.5% for the PBSCT group and 25% for the alloBMT group. With a median follow-up for survivors in the PBSCT and alloBMT groups of 11 months (range, 4-46) and 15 months (range, 2-84 months), respectively, there was no significant difference in median overall survival (33.5 versus 38.6 months, p = 0.7637) or event-free survival (16.7 versus 31 months, p = 0.8450). There was, however, a plateau in survival at 40% in the alloBMT group. No plateau in survival was seen in the PBSCT group. Clinical relapses occurred as late as 39 months posttransplant. Patients have survived up to 28 months postrelapse.

Complete remission in severe aplastic anemia after high-dose cyclophosphamide without bone marrow transplantation.

Severe aplastic anemia (SAA) can be successfully treated with allogeneic bone marrow transplantation (BMT) or immunosuppressive therapy. However, the majority of patients with SAA are not eligible for BMT because they lack an HLA-identical sibling. Conventional immunosuppressive therapy also has major limitations; many of its remissions are incomplete and relapse or secondary clonal disease is common. Cyclophosphamide is a potent immunosuppressive agent that is used in all BMT conditioning regimens for patients with SAA. Preliminary evidence suggested that high-dose cyclophosphamide, even without BMT, may be beneficial to patients with SAA. Therefore, 10 patients with SAA and lacking an HLA-identical sibling were treated with high-dose cyclophosphamide (45 mg/kg/d) for 4 consecutive days with or without cyclosporine. A complete response (hemoglobin level, > 13 g/dL; absolute neutrophil count, > 1.5 x 10(9)/L, and platelet count > 125 x 10(9)/L) was achieved in 7 of the 10 patients. One of the complete responders died from the acquired immunodeficiency syndrome 44 months after treatment with high-dose cyclophosphamide. The 6 remaining patients are alive and in continuous complete remission, with a median follow-up of 10.8 years (range, 7.3 to 17.8 years). The median time to last platelet transfusion and time to 0.5 x 10(9) neutrophils/L were 85 and 95 days, respectively. None of the complete responders has relapsed or developed a clonal disease. These results suggest that high-dose cyclophosphamide, even without BMT, may be more effective than conventional immunosuppressive therapy in restoring normal hematopoiesis and preventing relapse or secondary clonal disorders. Hence, further studies confirming the efficacy of this approach in SAA are indicated.

Sinus disease in the bone marrow transplant population: incidence, risk factors, and complications.

BACKGROUND: Fever associated with sinus disease in the immunocompromised bone marrow transplant recipient requires prompt evaluation and therapy. Very little is known about the incidence, risk factors, and sequelae of nonsurgically treated sinus disease in this population. METHODS: A retrospective review of 107 consecutive allogeneic and autologous bone marrow transplant recipients from August 1987 to July 1989 was performed to determine (1) the overall incidence of sinus disease; (2) factors that influence the development of sinus disease; and (3) the sequelae of sinus disease treated nonsurgically. RESULTS: Overall 33 (31%) of 107 bone marrow transplant recipients had sinus disease defined as a radiographic abnormality with clinical symptoms. Eleven (10%) of 107 recipients had preexisting sinus disease. Sinus disease developed in 22 (21%) of 107 recipients after bone marrow transplantation. Sinus abnormalities were significantly higher among allografted bone marrow transplant recipients than among autografted recipients (p = 0.027). The diagnosis, stage of disease, cytoreductive regimen, or graft-vs.-host disease were not different between recipients in whom sinus disease did and did not develop. There were no deaths as a result of sinus complications. CONCLUSIONS: Sinus disease developed in 21% of the studied population after bone marrow transplantation. Allogeneic recipients had a higher incidence of sinus disease than autologous recipients. There were no deaths attributed to sinus complications. All sinus disease in this bone marrow transplant population was treated medically. No patient required surgical intervention either before or after bone marrow transplantation.

Anti-CD3-activated splenocytes enhance survival in lethally irradiated mice after transplant of syngeneic hematopoietic stem cells.

Although cytokines produced by activated T cells may accelerate immunohematopoietic reconstitution after autologous bone marrow transplantation (ABMT), there is no direct evidence that infusion of anti-CD3 mAb-activated T cells can accelerate engraftment by hematopoietic stem cells. This study tests the ability of anti-CD3-activated murine splenocytes (ASC) to enhance the rescue of lethally irradiated (9 Gy) BDF1 mice by transplant of a limiting dose of fresh unmanipulated syngeneic splenocytes (SC). A minority (14.8%, 10-25%) of mice could be rescued with 5 x 10(5) SC after 9 Gy total-body irradiation (TBI). When 10(6) or 10(7) ASC were added to 5 x 10(5) SC, survival increased to 50% in those that received 5 x 10(5) SC + 10(6) ASC (not significant [NS]) and to 81.4% (77.7-88.0%) in those that received 5 x 10(5) SC + 10(7) ASC (p < 0.001). Furthermore, adding a fixed dose of 10(7) ASC to increasing doses of SC (10(5), 5 x 10(5), and 10(6)) enhanced survival at the different doses of SC. ASC alone did not rescue mice. CD3+ cells were the predominant population (77.6 +/- 6.7%) in the ASC inoculum, while NK cells remained low (1.2 +/- 0.9%). Colony-forming unit-spleen (CFU-S) yield after injection of SC showed dose dependence, whereas injection of 10 x 10(6) ASC alone failed to show any CFU-S yield in 23 of 25 recipient spleens. These results show that ASC enhanced survival of mice rescued with limiting doses of SC and that this effect was ASC dose-dependent but not dependent on the addition of extra stem cells.

Association between Lyn protein tyrosine kinase (p53/56lyn) and the beta subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human megakaryocytic leukemia cell line (M-07e).

The role of the lyn product (p53/p56lyn), a membrane-associated protein tyrosine kinase in the signaling pathway used by granulocyte macrophage-CSFR (GM-CSFR) was investigated by using the GM-CSF-dependent human megakaryoblastic leukemia cell line M-07e. M-07e cells express GM-CSFR and are dependent on GM-CSF for survival and proliferation in vitro. Treatment with anti-lyn Abs coimmunoprecipitated, along with lyn product, the beta subunit of GM-CSFR and a phosphoprotein with a molecular mass of 120 kDa (p120) in the lysates of M-07e cells but not in the lysates of human monocyte-derived macrophages (HMDM) or human lymphoid leukemia cells. That the 120-kDa phosphoprotein coimmunoprecipitated by anti-lyn Abs is the beta subunit of GM-CSFR was confirmed in the immunoprecipitates (IP) of M-07e cells with the use of an agarose-conjugated anti-p-tyr mAb. The formation of GM-CSF/GM-CSFR/lyn signaling complexes was verified in an autoradiographic study with anti-lyn IP of M-07e cells that had been bound with 125I-labeled recombinant human (rh)GM-CSF. The p120 protein (beta subunit) was not detected in the IP of M-07e cells with anti-fyn or anti-PI3 Abs. A direct association of Lyn kinase with the beta subunit of GM-CSFR was illustrated with a reversed approach showing the recovery of Lyn protein in anti-beta (CRS1) but not anti-alpha IP of M-07e cells that had been starved for a prolonged period. Finally, the interaction of Lyn kinase with the GM-CSFR complexes was further corroborated using anti-GM-CSF (G133) mAb, which coimmunoprecipitated both the p120 beta subunit and lyn product in the lysates of M-07e cells that had been bound with rhGM-CSF before cell lysis. Removal of rhGM-CSF from culture medium for 10 to 12 h resulted in a marked decrease in lyn-associated kinase activity but not the beta subunit/lyn kinase complex formation. Taken together, our results showed that, in M-07e cells, Lyn protein tyrosine kinase (p53/p56lyn) is stably associated with a constitutively phosphorylated beta subunit of the GM-CSFR in a manner that seems to be independent of lyn kinase activity.

Phase I trial of sequential cyclophosphamide, cyclosporin A, and interferon-alpha in patients with cancer: attempt to induce autologous graft-versus-host reaction to elicit an antitumor response.

Previous reports of autologous bone marrow transplant (auto-BMT) have demonstrated that myeloablative therapy followed by cyclosporin A (CsA), with and without interferon (IFN), can generate autoreactive cytotoxic T lymphocytes (auto-CTL) with potential therapeutic benefit. This is the first report of an attempt to generate auto-CTL using CsA and IFN after a non-myeloablative regimen. Cyclophosphamide (CTX) 1,200 mg/m2 i.v. day 1 was followed by CsA and IFN-alpha days 2-28, administered in a sequential three-step Phase I dose-escalation scheme. Patients were evaluated twice weekly for clinical evidence of graft-versus-host (GVH) reaction. Peripheral blood mononuclear cells (PBMCs) were obtained before treatment, at time of clinical GVH reaction, and days 21 and 28, and analyzed for auto-CTL, natural killer (NK) cell, and lymphokine-activated killer (LAK) cell activity. Patients also underwent punch skin biopsy at the time of clinical GVH reaction or day 21 to identify histologic evidence of GVH. Fourteen patients completed therapy and were evaluable for immunologic studies and anti-tumor response. No increase in auto-CTL, NK cell, or LAK cell activity was seen. Clinical or histologic evidence of GVH reaction did not occur. We conclude that this myelosuppressive dose of CTX combined with CsA and IFN is unable to generate clinical or immunologic evidence of an auto-GVH reaction. Further efforts are warranted to evaluate other therapeutic attempts to generate auto-CTL with anti-tumor activity based on preliminary results of clinical benefit in auto-BMT.

Prevention of hemorrhagic cystitis following allogeneic bone marrow transplant preparative regimens with cyclophosphamide and busulfan: role of continuous bladder irrigation.

High dose cyclophosphamide and/or busulfan conditioning treatment of recipients of bone marrow transplants proved to be highly effective but associated with substantial and sometimes life threatening hemorrhagic cystitis. To prevent this complication, a prophylactic continuous bladder irrigation program was instituted in patients receiving cyclophosphamide and/or busulfan in preparation for bone marrow transplantation. Retrospective analysis of 199 patients who underwent allogeneic bone marrow transplantation revealed that continuous bladder irrigation significantly decreased the frequency of hemorrhagic cystitis in patients receiving busulfan and cyclophosphamide (continuous bladder irrigation 23% versus no bladder irrigation 53%, p < 0.004). There was no difference in the frequency of hemorrhagic cystitis between the different preparative regimens in patients who underwent continuous bladder irrigation. There was no relationship between the incidence of hemorrhagic cystitis and the severity of graft-versus-host disease or the time to engraftment. The duration of hemorrhagic cystitis and overall survival rates were similar in both groups, and there was no increase in complications related to catheterization. In general, continuous bladder irrigation was well tolerated, decreased the incidence of hemorrhagic cystitis and may be useful in bone marrow transplant patients.

Allogeneic bone marrow transplantation in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent an acquired group of clonal disorders of the pleuripotent stem cells, resulting in progressive life-threatening cytopenias or transformation into acute leukemias. A major issue of using alloBMT in MDS is the criteria used for patient selection. Therapeutic trials of lesser intensity such as differentiating agents, and cytokines could be the preferable choice for patients with good prognostic features. On the other hand, patients with poor prognostic features may urgently need to establish a normal hematopoiesis through allogeneic bone marrow transplantation (alloBMT). Important prognostic indicators in MDS include FAB classification, presence of abnormal localization of immature precursors, degree of cytopenias and cytogenetic abnormalities. We used a novel preparative regimen--"BAC" consisting of the consecutive administration of 1 mg/kg of busulfan every 6 hours for 16 doses followed by 2 g/M2 of cytosine arabinoside (ara-C) given every 12 hours for four doses, and finally 60 mg/kg of cyclophosphamide daily for 2 days. Thirty two patients transplanted had a median age of 33 years. Nine of the patients had either RA or RARS, 21 had RAEB or RAEB-T and 2 were unclassified MDS. Twenty two of our patients had chromosomal abnormalities while 10 had a normal karyotype analysis. Nine of the 32 patients had documented leukemic transformation and received induction therapy prior to BMT. The median time from diagnosis to BMT was 5.6 months, ranging from 1.3 to 30.2 months. Nineteen out of 32 patients are alive without disease, with a median follow up of 24 months. The actuarial event-free survival for the entire group is 52%. Two patients have relapsed with an actuarial relapse rate of 12%. Only significant favorable prognostic indicator for the event-free survival was in the recipient of a genotypically matched graft (76%) compared to recipients of a non-genotypic graft (23%) (p = 0.02). "BAC" is a unique preparative regimen for alloBMT in MDS with excellent results.

Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin's lymphoma.

A prospective comparative trial of allogeneic versus autologous bone marrow transplant (BMT) was conducted. Sixty-six consecutive patients (median age, 41; range, 15 to 60; female:male ratio = 21:45) entered this clinical trial. Priority for allogeneic BMT was given to patients who were 55 or younger and had a major histocompatibility complex-matched or 1-antigen-disparate sibling donor. Autologous BMT was offered to all other patients whose age was 60 or younger. Patients who had no sibling donor and who had BM involvement at the time of evaluation were not eligible. Thirty-one patients received an allograft, and 35 patients received an autograft. Thirteen patients received a BM graft purged with 4-hydroperoxycyclophosphamide because of previous BM involvement. Patients who had previous radiation to the thoracic and/or abdominal areas of more than 20 Gy received a preparative regimen consisting of cyclophosphamide (1,800 mg/m2/d for 4 days), VP-16 (200 mg/m2 every 12 hours for 8 doses), and 1,3-bis(2-chloroethyl)-1-nitrosourea (600 mg/m2 as 1 dose). Other patients received cyclophosphamide 1,800 mg/m2/d for 4 days followed by total body irradiation of 12 Gy administered as a single daily fraction over 4 days. With a median follow-up of 14 months, the progression-free survival (PFS) for autograft and allograft recipients was 24% +/- 8% (+/- SE) and 47% +/- 9%, respectively, (P = .21). However, the probability of disease progression was significantly higher in the autologous group (69% +/- 9%) than in the allogeneic group (20% +/- 10%; P = .001). When other confounding prognostic factors were adjusted in the multivariate analysis, chemosensitive disease and allograft were found to have a significant favorable influence on probability of disease progression (P = .03 and .003), but only chemosensitive disease had a significant influence on the PFS (P < .002). Our results suggest the existence of graft-versus-lymphoma effect and also support the rationale of using immunotherapy after autologous BMT. Allogeneic BMT should be preferable to autologous BMT in younger patients with lymphoma.

Graft failure in children receiving HLA-mismatched marrow transplants with busulfan-containing regimens.

Identifying risk factors that lead to graft failure may reduce morbidity and mortality after bone marrow transplantation (BMT) for hematologic malignancies. We evaluated engraftment of all patients with acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and myelodysplastic syndrome (MDS) receiving an unmanipulated marrow allogeneic BMT at the Detroit Medical Center from 1987 to 1992 using a busulfan, cyclophosphamide +/- cytarabine preparative regimen. Three of 118 patients had graft failure (2.5%; (95% confidence interval (CI) 0.7%, 6.4%). Graft failure was high in patients < or = 15 years with 3 of 12 patients with failure (25.0%) compared with 0 of 106 patients > 15 years (p = 0.002). Failure to engraft was not seen in HLA-identical (related or unrelated) donor transplants (0 of 103) whereas 3 of 15 HLA-mismatched donors failed (p = 0.003). Patient diagnosis, locus of HLA-mismatch, cytarabine in the preparative regimen, marrow cell dose and the relative reactive index (RRI) were not significant factors. Altered busulfan kinetics secondary to young age was probably not a major factor since 8 of 8 HLA-identical donor transplants engrafted in children. These findings demonstrate that patients receiving an unmanipulated marrow graft using busulfan-containing regimens were at a high risk for graft failure only if they were < or = 15 years of age and had an HLA-mismatched donor. More immunosuppressive preparative regimens, possibly including total body irradiation, should be considered to prevent potential graft failure in children.

Phase I study of alpha-interferon augmentation of cyclosporine-induced graft versus host disease in recipients of autologous bone marrow transplantation.

To explore the augmentation of cyclosporin-induced graft-versus-host disease (GVHD) in autologous bone marrow transplantation (BMT), we conducted a phase I dose escalation trial of interferon (IFN)-alpha 2a. A dose of either 1 or 3 x 10(6) units of IFN-alpha 2a was given by daily sc injection starting on day 0 of BMT and continuing for 28 days. Cyclosporine (CYA) was also started on day 0 of BMT at a dose of 1 mg/kg/day for 28 days. We enrolled 22 patients (median age 43 years, range 19-55 years, male/female ratio = 9/13) which included 11 patients with lymphoma, 5 patients with Hodgkin's disease, 4 patients with AML and 1 patient each with acute lymphoblastic leukemia (ALL) and myeloma. Patients were divided into four groups: two control groups received either CYA or IFN-alpha 2a alone and the other two groups received IFN-alpha 2a at a dose of either 1 x 10(6) or 3 x 10(6) units/day sc concomitantly with CYA for 28 days. IFN-alpha 2a treatment was terminated early in 5 patients: 2 patients receiving IFN-alpha 2a at a dose of 3 x 10(6) units/day developed intractable fatigue, nausea and vomiting and 3 other patients had life-threatening transplant-related complications not related to IFN-alpha 2a (1 patient receiving 3 x 10(6) units/day, and 2 receiving 1 x 10(6) units/day). These patients were considered not evaluable. Of the 17 evaluable patients, all 13 who received IFN-alpha 2a developed GVHD regardless of whether they received CYA whereas only 2 of the 4 patients who received CYA alone developed detectable GVHD.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical studies using immobilized OKT3 to activate human T cells for adoptive immunotherapy: optimal conditions for the proliferation and induction of non-MHC-restricted cytotoxicity.

In order to obtain large numbers of T cells for adoptive immunotherapy after bone marrow transplantation (BMT), we optimized conditions for long-term proliferation of T cells that exhibit non-MHC-restricted cytotoxicity using immobilized anti-CD3 (OKT3) activation and culture in IL-2. Proliferation and cytotoxicity directed at Daudi, K562, and B cell lines were used to determine (1) the optimal concentration of IL-2 and the optimal time of exposure to immobilized OKT3 for maintaining growth and cytotoxicity, (2) the starting populations that can be used, (3) the T cell subsets that mediate cytotoxicity, and (4) the optimal medium and concentration of serum for maintaining growth and cytotoxicity. Peripheral blood lymphocytes (PBL) activated with OKT3 would proliferate and mediate cytotoxicity at IL-2 doses as low as 30 IU/ml. Increasing the IL-2 concentrations beyond 600 IU/ml did not augment the proliferative or cytotoxic responses of PBL. A 24-hr incubation on OKT3 was sufficient to activate PBL. Increasing the incubation time on OKT3 from 24 to 72 hr did not significantly enhance cytotoxicity. Comparisons between PBL and purified T cells (E-rosette) indicated that either cell population could be activated with OKT3 in the presence of IL-2 to proliferate and mediate non-MHC-restricted cytotoxicity. Purified populations of CD4+ or CD8+ T cells demonstrated equivalent proliferation and cytotoxicity when activated using IL-2 and OKT3. With equal concentrations of human or fetal bovine serum, RPMI 1640 and X-Vivo 10 were comparable for supporting proliferation and cytotoxicity. These conditions are being used to activate and expand T cells for clinical trials that involve infusing activated T cells into recipients after autologous BMT.

Constitutive and mitogen-stimulated cytokine mRNA expression by peripheral blood mononuclear cells from most autologous and allogeneic bone marrow transplant recipients is intact.

The immunodeficiency that occurs after human bone marrow transplantation (BMT) leaves BMT recipients susceptible to fatal infections. Although cytokines are critical for coordinating immune responses and immune reconstitution after BMT, there are still gaps in our knowledge about the expression of mRNA for cytokines in peripheral blood mononuclear cells (PBMC) after BMT. Therefore, we systematically studied cytokine gene expression by PBMC from 11 allogeneic and four autologous BMT recipients from 111 to 837 days after BMT and compared the results with PBMC from seven normal controls tested in parallel. PBMC were examined for mRNA expression for IL-2r alpha, IL-2, IL-3, IL-4, IL-6, and IL-7 using reverse transcription polymerase chain reaction (RT/PCR). PBMC from 11 allogeneic recipients constitutively expressed mRNA for IL-2r alpha in 2 of 11 and IL-2 in 1 of 9 samples tested whereas the same PBMC constitutively expressed mRNA for IL-3 in 8 of 11, IL-4 in 3 of 7, IL-6 in 6 of 7 and IL-7 in 3 of 6 samples tested. After PHA/PMA stimulation, PBMC from the same recipients frequently expressed mRNA for IL-2r alpha in 9 of 11, IL-2 in 8 of 9, IL-4 in 3 of 7 and IL-6 in 7 of 7. PBMC from four autologous recipients (two short-term and two long-term) frequently constitutively expressed mRNA for IL-2r alpha (3 of 4) IL-2 (3 of 4), and IL-3 (4 of 4). Stimulation of PBMC from the autologous recipients did not alter cytokine expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Successful treatment of human Waldenström's macroglobulinemia with combination biological and chemotherapy agents.

The immunomodulating effects and antitumor activity of two biological agents, bryostatin 1 (Bryo1) and alpha-interferon, were tested in vitro and in vivo either alone or prior to chemotherapy agents, against a Waldenström's macroglobulinemia tumor line (WSU-WM). Bryol caused a decrease in the expression of CD10, CD19, IgM, Leu10, and CD22 and a temporary growth inhibition as measured by cell cycle analysis. alpha-Interferon did not show any major effects. In vivo, severe combined immunodeficient mice were used to test the activity of the agents against WSU-WM. Bryo1 (i.p.) was given either alone or sequentially with doxorubicin (i.v.), vincristine (i.v.), melphalan (i.v.), and alpha-interferon (i.v.). Bryo1 given 24 h before vincristine or melphalan resulted in the highest tumor growth inhibition, tumor growth delay, and tumor cell kill. Two of five mice receiving Bryo1/vincristine combination were free of tumors > 200 days after treatment and were considered cured. In light of our findings, we recommend that Bryo1 be considered for clinical investigation in human B-cell tumors and might best be given combined with other chemotherapy agents used in the treatment of that disease. Whether Bryo1 is acting as a differentiating agent or as a direct anti-Waldenström's macroglobulinemia tumor agent, remains unclear.

Coactivation with anti-CD28 monoclonal antibody enhances anti-CD3 monoclonal antibody-induced proliferation and IL-2 synthesis in T cells from autologous bone marrow transplant recipients.

Recent studies show that costimulation of T cells with anti-CD28 Mab (anti-CD28) enhances anti-CD3 Mab (anti-CD3)-induced proliferative responses and cytokine production. This study determines if coactivation with anti-CD3 and anti-CD28 corrects defects in proliferation and IL-2 secretion in peripheral blood lymphocytes (PBL) from bone marrow transplant (BMT) recipients. PBL or T cells from 5 of 16 autologous and 5 of 22 allogeneic recipients increased their anti-CD3-induced proliferation responses by > 50% after coactivation. In short-term (< 180 days after BMT) autologous recipients, the group mean response increased after anti-CD3 activation from 62,900 to 97,800 cpm after coactivation. In long-term (> 180 day after BMT) autologous recipients, the group mean response after anti-CD3 activation increased from 62,600 to 78,400 cpm after coactivation. The long-term autologous recipient group had costimulated responses from PBL that were significantly higher than the paired anti-CD3-induced responses (p < 0.01); in contrast, such differences were not seen in allogeneic recipient groups. After anti-CD3 stimulation, the mean response of 88,000 cpm for PBL from short-term allogeneic recipients and the mean response of 83,600 cpm for PBL from long-term allogeneic recipients were higher than those in PBL from autologous recipients were higher than those in PBL from autologous recipient groups. The amount of IL-2 secreted by T cells from three autologous and three allogeneic recipients was enhanced 0.9-25-fold by coactivation. Coactivation of PBL from selected recipients increased proliferation into the normal range and increased IL-2 secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of murine peritoneal macrophage colony-forming cells by peritoneal administration of macrophage inflammatory protein-1 alpha.

Peritoneal injection of thioglycollate medium (TM) to mice results in a dramatic increase in total number of peritoneal macrophages within 48 to 72 hours. Unlike resident macrophages, a fraction (10 to 20%) of these newly arrived young macrophages, designated as macrophage colony-forming cells (M-CFC), are highly proliferative and formed macrophage colonies in vitro in the presence of either macrophage or granulocyte-macrophage colony-stimulating factor (M-CSF or GM-CSF). Using a reverse transcriptase polymerase chain reaction (RT-PCR) technique, peritoneal exudate macrophages (PEM) obtained 2 to 5 days after a single TM injection actively expressed mRNA for recombinant murine macrophage inflammatory protein-1 alpha (rmMIP-1 alpha). Yet none or only a trace amount of mRNA for MIP-1 alpha was detected in normal resident macrophages or PEM obtained 7 days after TM treatment. The effect of rmMIP-1 alpha on the induction of exudate M-CFC was investigated. Multiple intraperitoneal (IP) administration of rmMIP-1 alpha caused a marked increase in the total number of peritoneal M-CFC and macrophages similar to but weaker than the increase in TM-injected mice. The total number of neutrophils, mast cells, and eosinophils also increased, but with different kinetics, following multiple injections of rmMIP-1 alpha. rmMIP-1 alpha alone did not stimulate the proliferation of M-CFC, nor did it potentiate their responsiveness to either rmGM-CSF or recombinant human (rh) M-CSF in vitro. Taken together, our results suggest that MIP-1 alpha released by exudate macrophages is a major chemoattractant responsible for the migration of M-CFC from the circulation to the peritoneal cavity during a TM-induced inflammatory response.