High dose chemotherapy without hematopoietic cell support for the treatment of refractory lymphoma.

Conventional dose combination chemotherapy for patients with relapsed or refractory lymphoma is rarely curative. High dose chemotherapy followed by hematopoietic progenitor cell transplant (HPCT) has a clearly defined role in patients who have first relapsed after standard CHOP chemotherapy for lymphoma. However, the role of HPCT is less well defined for patients with chemo-resistant, or chemo-refractory disease. Sixteen patients (15 Non-Hodgkin's, 1 Hodgkin's Disease) were entered into a phase II study to determine if a dose intensive induction regimen in heavily pre-treated refractory lymphoma patients could permit further consolidation with HPCT. The primary endpoints were survival, response, toxicity, and resource utilization. The regimen consisted of continuous infusion etoposide 1 or 2 gm/m2/72 hours, idarubicin 12 mg/m2/d for 3 days followed by cytarabine 2 gm/m2/72 hours on days 8, 9, and 10 (VIC). Fifteen patients were evaluable for objective response. The overall response rate was 53% with 7 patients achieving a partial response and 1 patient achieving a complete response. Of the 8 responders, 6 patients subsequently received high dose chemotherapy followed by HPCT (4 autologous, 2 allogeneic). The median survival was 176 days for the non-responders contrasted with 722 days for the responders. The average duration of hospitalization was 38 days. Toxicity was mainfest primarily as mucositis with a median grade of 3 among the first 13 patients, and a median grade of 2 in three subsequent patients who received an etoposide dose of 1 gm/m2/72 hours. All patients had an episode of neutropenic fever and 5 patients developed clinically significant pneumonitis during therapy. The VIC regimen is active in the treatment of chemo-refractory lymphoma with clinically significant differences in survival for patients who respond to therapy. Further modifications to the regimen could include the addition of a topoisomerase I inhibitor for synergy with etoposide, and using VIC as part of a tandem transplant regimen where response to VIC would allow further therapy with a myeloablative induction followed by HPCT.

Cytokine upregulation of the antigen presenting function of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) cells are malignant counterparts of normal myeloid pathway progenitors. Myeloid progenitors differentiate into professional antigen presenting cells (APC) under the essential influence of GM-CSF along with additional cytokines. Twelve cases of human AML were tested for ability to be differentiated toward a professional APC phenotype in short-term culture with addition of GM-CSF and the following recombinant proteins: TNFalpha, IL-4, CD40 ligand, Flt3 ligand and SCF. Significant upregulation of CD80 (B7-1) and enhancement of alloantigen presentation was seen with the addition of GM-CSF and TNFalpha alone or with additional cytokines. The combination of GM-CSF and TNFalpha, either alone or in combination with an additional cytokine, resulted in enhancing alloantigen presentation by at least two-fold over the media control group in 10/12 patients studied, and resulted in CD80 expression of greater than 15% in 11/12 patients studied. In AML cultures with GM-CSF and TNFalpha, coexpression of CD80 and either CD34 or an aberrant surface marker (CD56) was seen. In one case, sorted CD80, cells retained a characteristic cytogenetic marker and CD34 expression, proving their derivation from an AML precursor. These studies verify other reports of in vitro differentiation of human AML precursors into enhanced APC, suggesting that this phenomenon could be utilized for immunotherapy strategies aimed at enhancing presentation of leukemia antigens to T cells.

Hematologic and virologic effects of lineage-specific and non-lineage-specific recombinant human and rhesus cytokines in a cohort of SIVmac239-infected macaques.

The hematologic abnormalities of SIV and HIV are well described, although the mechanisms that lead to hematopoietic dysfunction are yet to be fully defined. A number of growth factors and cytokines have been used to induce the differentiation, maturation, and proliferation of appropriate lineages, with the aim that such therapy will lead to functional hematopoietic reconstitution. Within this context, some cytokines have been shown to influence HIV and SIV replication in vitro and, in selected cases, in vivo. However, few studies detail the effects of hematopoietic cytokines such as IL-3, Flt-3 ligand, G-CSF, Tpo, and Epo or correlate the effects on virus replication. In an effort to address this issue, we infected 12 rhesus macaques with 500 TCID50 of SIVmac239 and intensively evaluated hematologic, virologic, and immunologic parameters during administration of cytokines. When all animals had lymphadenopathy, hepatosplenomegaly, and CD4+ cell counts > or =1000/microl, subgroups of three rhesus macaques were administered either rhFlt-3; rrIL-3a; combination of rhG-CSF, rhTpo, and rhEpo (rhGET); or rrIL-12. Fourteen days of rhFlt-3 administration induced expansion of the bone marrow CD34+ cells and granulocyte-macrophage colony-forming units (GM-CFUs) and increased absolute peripheral blood CD34+ cells and total CFUs. Following rrIL-3 and rhGET administration absolute peripheral blood CD34+ cells and total CFUs increased. rhGET also increased granulocyte, platelet, and reticulocyte counts by day 14 of administration. Branched DNA and coculture assays did not demonstrate any significant change in viral load with any of the cytokines administered. These data suggest that SIV-infected rhesus macaques have the hematopoietic capability to expand and mobilize CD34+ and GM-CFU progenitors and formed elements at 6-8 months postinfection in response to various cytokines, without increasing viral load.

Allogeneic bone marrow transplantation with matched unrelated donors for patients with hematologic malignancies using a preparative regimen of high-dose cyclophosphamide and fractionated total body irradiation.

Allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling donor is effective therapy for patients with bone marrow failure states and those with hematologic malignancies. However, only a minority of them will have an HLA-identical sibling donor; unrelated donors, matched or partially mismatched, have been used successfully for patients lacking a related donor. Even though results with allogeneic transplants using unrelated donors are encouraging, the incidence of complications including graft-versus-host disease (GVHD) and graft rejection or late graft failure is increased compared to identical sibling transplants. The combination of cyclophosphamide and total body irradiation (TBI) has been used as an effective preparative regimen for allogeneic transplants, however, the total dosage and dosing schedule of both the cyclophosphamide and TBI has varied significantly among studies. To decrease the rate of graft rejection and late graft failure with volunteer donors, we evaluated a preparative regimen of high-dose cyclophosphamide (200 mg/kg over 4 consecutive days, days -8, -7, -6, -5) followed by fractionated TBI (1400 cGy administered in eight fractions over 4 days, days -4, -3, -2, -1). GVHD prophylaxis included FK506 and methotrexate. From July 1993 to January 1996, 43 adult patients, median age 38 years (range 18-58 years), were treated with this preparative regimen. Seventeen patients had low-risk disease and 26 had high-risk disease. Thirty-one donor/recipient pairs were matched for HLA-A, -B, and -DR by serology and molecular typing. Seven additional pairs were minor mismatched at the HLA-A or HLA-B loci. Four other donor/recipient pairs were HLA-A,-B, and -DR identical by serology but allele mismatched at either DRB1 or DQB. Forty patients were evaluable for myeloid engraftment. Engraftment occurred in all 40 patients at a median of 19 days. There were no cases of graft rejection or late graft failure. Nephrotoxicity was the primary adverse event with 26 patients (60%) experiencing a doubling of their creatinine. Hepatic veno-occlusive disease occurred in seven patients, six of whom had high-risk disease. All patients who had relapsed or refractory disease prior to BMT achieved a complete remission following BMT. Six patients transplanted for high-risk disease relapsed a median of 377 days post-BMT. None of the patients with low-risk disease have relapsed following transplant; the Kaplan-Meier survival for those patients with low-risk disease is 62% and 37% for those patients transplanted with high-risk disease (P = 0.0129). The median Karnofsky performance status is 100% (range 70-100%). Therefore, a preparative regimen of high-dose cyclophosphamide and fractionated TBI is an acceptable regimen for patients receiving an allograft from unrelated donors.

The outcome of unrelated donor bone marrow transplantation in patients with hematologic malignancies using tacrolimus (FK506) and low dose methotrexate for graft-versus-host disease prophylaxis.

Initial studies of FK506 combined with methotrexate (MTX) in patients receiving unrelated donor BMT have demonstrated a possible-decrease in the incidence of severe GVHD but high rates of severe stomatitis and nephrotoxicity. With this background, we undertook a pilot study evaluating FK506 in combination with a lower than usual dose of MTX in an attempt to improve the tolerability of this immunoprophylaxis regimen. Between July 1993 and October 1994, 26 consecutive adults receiving unrelated donor BMT at Emory University Hospital were enrolled on this study. All patients received FK506 intravenously at an initial dose of 0.03 mg/kg/day beginning day -1 and continuing until oral FK506 was tolerated. Patients also received MTX intravenously at 5 mg/m2 on days 1, 3, 6, and 11. The preparative regimen administered to all but one patient included cyclophosphamide at 200 mg/kg over 4 days followed by total body irradiation (TBI) at 1400 cGy in twice daily fractions over 4 days. The median age of patients was 31 years (range: 19 to 52). Sixteen donor/recipient pairs were matched for HLA-A, -B, and -DR by serology and molecular typing. Ten paris were minor mismatches at either class I or class II. Twenty-two of 26 patients (85%) completed four doses of MTX on schedule. Nephrotoxicity was the most common adverse event associated with the administration of FK506: 88% of patients experienced a doubling of their serum creatinine. One patient died of central nervous system hemorrhage prior to engraftment. Twenty-four of the remaining 25 patients (96%) engrafted. Fourteen of 24 patients (50%) evaluable developed grades 2-4 acute GVHD. The rate of severe (grades 3-4) acute GVHD was 25%. Chronic GVHD developed in 11 of 20 (55%) evaluable patients. At a median follow-up of 461 days, 14 patients (54%) are alive. All are relapse-free with a median Karnofsky performance status of 90% (range: 70-100%). The cumulative probability of 2-year disease-free survival is 50% (95% confidence interval [CI]: 0.33 to 0.77); for low risk patients 67% (95% CI: 0.47 to 0.95) and for high risk patients 23% (95% CI: 0.049 to 1.00). These preliminary data indicate that FK506-based immunosuppression following unrelated donor BMT is effective in preventing severe acute GVHD and warrants comparison to CSA-based regimens.

Vancomycin-resistant Aureobacterium species cellulitis and bacteremia in a patient with acute myelogenous leukemia.

A 39-year-old male with acute myelogenous leukemia and concomitant porphyria cutanea tarda was admitted to the hospital for consolidation chemotherapy of his leukemia. During his hospitalization, he developed cellulitis of the left hand and persistent bacteremia with a yellow-pigmented, nonfermenting coryneform bacterium that was identified as Aureobacterium sp. The portal of entry for the Aureobacterium infection was probably through the skin lesions due to porphyria cutanea tarda. The infection developed while the patient was receiving vancomycin prophylaxis, and the vancomycin MIC for the isolate was 32 micrograms/ml.

Profile of cell cycle in hematopoietic malignancy by DNA/RNA quantitation using 7AAD/PY.

Using 7-amino-actinomycin-D/pyronin Y (7AAD/PY), we analyzed the surface phenotypes and cell cycle of 22 hematopoietic cell lines based on their cellular DNA/RNA content. Populations of G1a, G1b, S, and G2M, the DNA index (DI), and the RNA index of S phase (SRI) were calculated by means of DNA/RNA dot plots. Two new parameters were extracted from the cell-cycle profiles: the nucleic acid index of S phase (NI) and the coefficient of variations in the RNA at S phase (SVC). DNA/RNA dot plots of cell lines revealed four characteristic profiles of the cell cycle, defined with the calculated NI and SCV. These were type 0 (small NI, large SCV), type I (small NI, small SCV), type II (large NI, small SCV), and type III (large NI, large SCV). Type O included four stem cell lines: one t(1;19) leukemia, two Ph1+ acute lymphocytic leukemia (ALL), and one biphenotypic crisis of chronic granulocytic leukemia (CGL). Type I included five ALL cell lines: three T-ALL and two common B-ALL. Type II contained 10 myeloid cell lines: five AML and five myeloid crisis of CGL. Type III contained three relatively immature lymphoma cell lines: two Burkitt's lymphoma and one follicular center lymphoma. Calculated NI/SCV (%) were as follows: type 0, 2.27 +/- 0.19/16.7 +/- 3.7; type I, 2.20 +/- 0.30/11.1 +/- 0.7; type II, 3.64 +/- 0.52/11.8 +/- 1.0; and type III, 3.60 +/- 0.53/17.5 +/- 1.9. Cell-cycle analysis of blasts using 7AAD/PY combined with surface phenotyping may yield important information for classifying hematopoietic malignancy within 2 hours of patient admission.

Reverse transcriptase-polymerase chain reaction for bcr/abl fusion in chronic myelogenous leukemia.

Reverse transcriptase-polymerase chain reaction (RT-PCR) has shown promise as a means of detecting low levels of cells bearing the Philadelphia chromosome (Ph1) and for detecting cytogenetically inapparent ("masked") Ph1 in patients with chronic myelogenous leukemia (CML). For detection by karyotyping, dividing cells must be used, precluding use of peripheral blood samples in cases with low peripheral blood blast counts. Reverse transcriptase-polymerase chain reaction was performed in 83 bone marrow and 30 peripheral blood samples from patients with CML to compare results with karyotyping and to evaluate utility of this test on peripheral blood samples. Using isolated total cellular RNA and a single primer pair, cDNA was transcribed, amplified, electrophoresed, and probed for bcr/abl fusion involving M-bcr exons 2 and 3 of the bcr gene. Fifty-three samples were from untreated or conventionally treated patients (pre-BMT), and 60 were from patients who had undergone bone marrow transplantation (post-BMT). Fifty of 53 pre-BMT samples were positive by RT-PCR. Two samples, negative by RT-PCR, had complex translocations, t(9;16;22) and t(4;14;22). One case was indeterminate by RT-PCR, but positive on retesting. Forty-five of 53 had Ph1 by karyotyping; 8 were negative, including 5 peripheral blood samples, 2 bone marrow samples with "masked" Ph1, and 1 bone marrow sample with poor growth. Thirty-five of 60 post-BMT samples were positive by RT-PCR. Fourteen of 60 post-BMT samples had Ph1 by karyotyping. Of the RT-PCR+/Ph1- cases, most showed a weak but definite band by RT-PCR, suggesting a low level of the bcr/abl fusion gene. Nineteen patients had concurrent peripheral blood and bone marrow samples analyzed by RT-PCR and karyotyping. Of 16 patients with satisfactory RNA extraction, 15 had concordant results by RT-PCR. Five patients had adequate metaphase cells for karyotypic analysis. All had Ph1 in bone marrow, but were negative in peripheral blood. Our results indicate that RT-PCR for detection of bcr/abl fusion is more sensitive than karyotyping in pre- and post-BMT samples. Furthermore, RT-PCR can be successfully performed on peripheral blood, yielding excellent correlation with bone marrow samples.

Minimal toxicity and mortality in high-risk breast cancer patients receiving high-dose cyclophosphamide, thiotepa, and carboplatin plus autologous marrow/stem-cell transplantation and comprehensive supportive care.

PURPOSE: To assess the clinical toxicity and outcome associated with a comprehensive supportive care approach in poor-risk breast cancer (BrCA) patients with high-dose chemotherapy (HDC). PATIENTS AND METHODS: One hundred twenty-five consecutive patients with stages II, III or metastatic breast cancer received HDC between February 1992 and June 1994. Recipients received 4 days of continuous infusion of cyclophosphamide 1.5 g/m2/d, thiotepa 125 mg/m2/d, and carboplatin 200 mg/m2/d followed by infusion of bone marrow or peripheral-blood stem cells (PBSC) and recombinant human growth factor (rhu-GF) support. Patients received similar supportive care that included administration of prophylactic antibiotics, management of neutropenic fevers, and transfusion support. RESULTS: There were 38 women with stage II or III (27 patients with > or = 10 lymph nodes), four with stage IIIB, and 83 with metastatic breast cancer. The median age was 44 years (range, 27 to 61). Grade II or greater nonhematologic toxicities included diarrhea (66%), stomatitis (33%), hepatic venoocclusive disease (VOD) (5%), and pulmonary toxicity (4%). Myeloid and platelet engraftment was comparable between bone marrow and PBSC recipients (P > .1). Infectious complications were rare and consisted of gram-negative bacteremia (1.6%), gram-positive bacteremia (1.6%), fungemia (1.6%), and documented or suspected aspergillosis infection (3%). There was one treatment-related death secondary to severe VOD. CONCLUSION: A comprehensive supportive care approach was associated with a low treatment-related mortality rate of less than 1%. With the observed reduction in treatment-related mortality, it is reasonable to evaluate the efficacy of HDC in women with less than 10 positive nodes and stage II disease in well-designed clinical trials.

Association of busulfan area under the curve with veno-occlusive disease following BMT.

Busulfan pharmacokinetics, specifically area under the concentration curve (AUC), have been correlated with the occurrence of veno-occlusive disease (VOD) following BMT. To evaluate the risk of VOD, we studied 66 patients who received pharmacotherapeutically monitored busulfan regimens in combination with CY, etoposide (VP16) and/or Ara-C in preparation for BMT. These patients received a total of 16 doses of busulfan dosed as 1 mg/kg/dose q 6 h beginning at 09.00 (n = 39), 18.00 (n = 2), 21.00 (n = 1) or 24.00 (n = 24) h. With the first dose, blood samples were obtained at baseline, every 15-30 min for 2 h, then every 1-2 h for 4 h. Blood was analyzed for busulfan concentration by high performance liquid chromatography and AUC calculated by the trapezoidal rule. Seventeen patients (25.8%) were not evaluable for AUC calculation due to slow absorption and/or elimination: 13 of 27 (48.1%) received the first dose between 18.00-24.00 vs four of 39 (10.2%) patients who received the first dose at 09.00 (P < 0.001). Eighteen of 51 (35.3%) evaluable patients had an AUC > 1500 mumol x min/l; 10 of whom received doses reduced proportionally to achieve an AUC = 1200 mumol x min/l starting with the 10th to 15th dose. Six of 18 (33.3%) patients with an initial AUC > 1500 mumol x min/l developed VOD vs one of 33 (3.0%) patients with an initial AUC < 1500 mumol x min/l (relative risk = 11.1; P = 0.0056). Other pharmacokinetic parameters, age, gender, type of BMT, previous therapy or pre-transplant liver function tests were not predictive of VOD. A higher incidence of VOD occurred in patients receiving BUCY (4 of 10) compared to those receiving BUCYAra-C (1 of 18) or BUCYVP16 (7 of 38), which could not be attributed to increased busulfan exposure in the BUCY patients. Routine pharmacotherapeutic monitoring of busulfan is recommended with further study to evaluate the impact of earlier and greater overall dose reduction in patients with high initial busulfan exposures.

High dose cyclophosphamide, fractionated total body irradiation with involved field irradiation and autologous bone marrow transplantation in malignant lymphoma.

The objective of this clinical trial was to determine if radiation to areas of recurrence or bulky disease prior to total body irradiation (TBI) and chemotherapy followed by autologous bone marrow transplantation (ABMT) altered the site of relapse and/or prolonged survival. Forty-eight patients with recurrent or refractory malignant lymphoma were treated with high-dose cyclophosphamide and fractionated TBI followed by ABMT. Thirty-four patients were eligible to receive involved field radiation therapy (IF-RT) to sites of recurrence or bulky disease. The overall response rate in 46 evaluable patients was 89% with 33 complete remissions (CR) and 8 partial remissions (PR). In a multivariant analysis increasing LDH, decreased serum albumin, older age, and lack of sensitivity to prior chemotherapy were associated with poorer survival. There were 10 deaths due to treatment related complications, 8 died of pulmonary complications of whom 6 were in CR. Of 11 who had received IF-RT and subsequently relapsed, 4 recurred in or adjacent to the involved field. We conclude that intensive chemo-radiotherapy proved to be an effective salvage therapy for patients with recurrent malignant lymphoma, resulting in a projected actuarial 33% DFS at 5 years, but was associated with a high transplant-related mortality.

Treatment of chronic neutropenia associated with large granular lymphocytosis with cyclosporine A and filgrastim.

A patient with neutropenia and life-threatening infections secondary to T-gamma lymphoproliferative disease, who did not respond to treatment with recombinant human G-CSF (filgrastim), was treated with filgrastin plus cyclosporine A (CyA). The patient achieved a good response in the absolute neutrophil count and subsequently required a dose reduction in the filgrastim. The patient was eventually discontinued from the CyA but continues on filgrastim alone. While on therapy, the large granular lymphocytes disappeared from the circulation and the beta-TCR rearrangement, which was present prior to beginning therapy, became undetectable. The patient had no significant toxicity to the CyA or the filgrastim and he has not experienced any serious infections or required hospitalization. Filgrastim has proven to be relatively nontoxic and of some benefit to patients with this disease and should probably be utilized first when treatment is necessary. However, if improvement is not observed, these findings suggest that a trial of the combination of CyA plus filgrastim may be beneficial.

Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y.

We developed an improved technique that permits simultaneous DNA and RNA quantitation by a flow cytofluorometry using 7-amino-actinomycin D (7AAD) and pyronin Y (PY), respectively. Detailed cell cycle analyses based upon the cellular DNA/RNA levels were performed using cells suspended in a buffer containing 0.004% saponin. This method preserved the light scattering properties of human peripheral blood cells, thus lymphocyte, monocyte and granulocyte populations could be evaluated. In addition, since 7AAD and PY exhibit red (> 650 nm) and orange fluorescence (570 nm) respectively, the green fluorescence channel of the flow cytometer was reserved for surface phenotyping using FITC-conjugated antibodies. The 7AAD/PY method is applicable to the simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation when combined with FITC-conjugated surface markers in heterogeneous samples. To demonstrate the three-color analysis, PHA-activated human peripheral blood lymphocytes were stained for cell surface markers with monoclonal antibodies. The cells were suspended in buffer containing 0.004% saponin, then stained with 7AAD and PY. The DNA and RNA were analyzed in indivisual CD4+, CD8+ and CD20+ cells, and the characteristic cell cycle status was found. Cell activation was further analyzed using antibodies against interleukin-2 (IL-2) receptors (CD25), transferrin receptors (CD71) or HLA-DR molecules. Transferrin receptors were expressed in late G1 phase (G1B) just before the initiation of DNA synthesis, whereas IL-2 receptors and HLA-DR were expressed very early in the G1 phase (G1T). Since this technique preserves both light scatter properties as well as cell surface proteins, it is ideally suited for detailed cell cycle analyses of heterogeneous samples such as peripheral blood or bone marrow cells.

Phase I multicenter trial of interleukin 6 therapy after autologous bone marrow transplantation in advanced breast cancer.

Our purpose was to determine the maximum tolerated dosage of rhIL-6 after high-dose cytotoxic chemotherapy and autologous BMT in patients with advanced breast cancer. Twenty patients (median age 43.5 years) received either CY and thiotepa (n = 3) or CY, thiotepa and carboplatin (n = 17) for 4 days. Unpurged autologous BM was reinfused 72 h later. Daily rhIL-6 therapy began the day of marrow infusion and continued until recovery of neutrophils (> or = 1.5 x 10(9)/l) and platelets (> or = 50 x 10(9)/l) or for a maximum of 28 days at a dosage of 0.3 microgram/kg/day (n = 7), 1 microgram/kg/day (n = 6) or 3 micrograms/kg/day (n = 7). Two of the initial 4 patients given rhIL-6 at 0.3 mu/kg i.v. experienced grade 4 hyperbilirubinemia, so subsequent patients received s.c. rhIL-6. Most toxicities attributable to rhIL-6 were reversible or mild constitutional symptoms, but dose-limiting grade 4 hyperbilirubinemia also occurred in 3 of the 7 patients receiving the 3 micrograms/kg dose. At the 0.3 and 1 microgram/kg/day doses, 8 of 13 patients completed the study vs. only 2 of 7 at the 3 micrograms/kg/day dose. During rhIL-6 treatment, neutrophil recovery (> or = 500 x 10(6)/l) occurred in 12 patients and platelet recovery (> or = 20 x 10(9)/l) occurred in 6 patients, 5 of whom received the 0.3 or 1 microgram/kg/day s.c. dose. The maximal tolerated dose of rhIL-6 after autologous BMT appeared to be 1 microgram/kg/day s.c., a dose appreciably lower than the maximal tolerated dose after conventional cytotoxic therapy.

Chemotherapy-induced hemolytic uremic syndrome: description of a potential animal model.

Hemolytic uremic syndrome (HUS) is an uncommon complication of chemotherapy that contributes to the morbidity of oncology and bone marrow transplant patients. The pathogenesis is not well understood and no established clinical animal model exists. We studied four rhesus monkeys (RM) that developed fatal HUS following high-dose chemotherapy. Microangiopathic hemolytic anemia (pre-Hct 40% and day 5-8 Hct 31% (P < .05), increased BUN (168 mg/dl), creatinine (8.2 mg/dl), and lactate dehydrogenase (1458 IU/L) (mean day 5-8 measurements) were observed. Platelets counts decreased to 39 +/- 15 x 10(9)/l from a mean of 397 +/- 31 x 10(9)/L (P < .0001). vWF, ATIII, thrombin:anti-thrombin complex (T:AT) and prothrombin fragment F1.2 levels were not different from a control group (N = 2). The data presented describe chemotherapy-induced HUS with typical clinical and laboratory features which may provide an animal model for the study of this important syndrome.

Effect of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on hematopoietic regeneration as demonstrated in a nonhuman primate chemotherapy model.

Using a recently developed hepsulfam-induced pancytopenia model in rhesus macaques, we have studied the effects of recombinant human interleukin-6 (rhIL-6) and rhIL-3 on marrow regeneration. Control animals were given hepsulfam (1.5 g/m2 by a single 30-minute intravenous [i.v.] injection, n = 4), while study animals received hepsulfam followed by rhIL-6, rhIL-3, or a combination of rhIL-6 and rhIL-3 (n = 3 per study group). Each cytokine was administered by once-daily subcutaneous (SC) injection (15 micrograms/kg/d) for 3 weeks beginning the day after chemotherapy (days 2 through 22). Mean platelet counts in control animals were < 100,000/microL on days 15 through 24, with 50% of the counts < 50,000/microL and two of four animals requiring platelet transfusion. In the rhIL-6- and rhIL-6/rhIL-3-treated groups, the nadir mean platelet counts were 164,000 +/- 58,700/microL and 162,300 +/- 23,800/microL, respectively, and occurred on day 15. Platelet counts in the rhIL-3-treated group were similar to those in controls. Mean absolute neutrophil counts (ANCs) < 1,000/microL occurred on days 10 through 29 in control animals, days 8 through 15 in rhIL-6-treated animals, and days 6 through 8 and 13 in rhIL-6/rhIL-3-treated animals. The frequency of ANCs < 500/microL was significantly less in the rhIL-6- and rhIL-6/rhIL-3-treated groups versus control groups (2.7 +/- 0.6 and 2.0 +/- 1.0 vs 7.0 +/- 1.4 occurrences, respectively; P < .05). rhIL-3-treated animals had ANCs similar to those in controls; one animal died with septicemia on day 21. Monkeys receiving rhIL-6 were significantly more anemic during the cytokine administration period; however, the anemia resolved by day 24. Coadministration of rhIL-3 and rhIL-6 partially corrected the anemia. The data indicate that rhIL-6 prevents significant thrombocytopenia and shortens the neutropenic period in this chemotherapy model.

T-cell receptor gene rearrangement in T-cell large granular leukocyte leukemia: preferential V alpha but diverse J alpha usage in one of five patients.

T-gamma lymphoproliferative disease (T-gamma LPD) is a chronic disorder of mature T cells that is associated with neutropenia and autoimmune phenomena. Although the progression of the lymphoproliferation is indolent, it is often associated with a monoclonal proliferation of T-cell-type large granular lymphocytes (LGL) that manifest multiple in vitro suppressor and cytotoxic activities. We considered the possibility that the granulocytopenia or anemia might represent an autoimmune disorder mediated by the monoclonal LGL via T-cell receptor (TCR) recognition of an antigen involved in hematopoiesis. Therefore, in an effort to characterize the usage of the TCR alpha- and beta-chain genes in patients with T-gamma LPD, we cloned and sequenced TCR alpha- and beta-chain mRNAs derived from the T-cell type LGL of five patients. The five patients studied did not use a common V alpha nor a common J alpha segment. However, an unusual finding was observed in one of the patients where the occurrence of a single variable-diversity-junctional (VDJ) rearrangement of the beta chain confirmed the monoclonal origin of the LGL proliferation. In accord with this evidence for monoclonality, many of the cells studied used a common V alpha (V alpha 19.1). In contrast to this common V alpha usage, there was a marked diversity of the J alpha segments and N-region addition that were associated with the V alpha 19.1 segment. This pattern of common V alpha usage associated with different N and J alpha segments suggests an immune-mediated selection process affecting the TCR alpha chain occurring after the transformation event that established the clone. We suggest that the T-cell-type LGL malignant clone might have developed autoreactivity conferred by the selected TCR alpha chain and that this autoreactivity might be implicated in this patient's anemia.

Different mechanisms of inhibition by alkyl-lysophospholipid and phorbol ester of granulocyte-macrophage colony-stimulating factor binding to human leukemic cell lines.

We investigated the effects of rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on granulocyte-macrophage colony-stimulating factor (GM-CSF) binding to human leukemic cell lines HL60, U937, KG-1, KG-1a, and K562. HL60, U937, and KG-1 exhibited the high-affinity receptors, but KG-1a and K562 revealed no demonstrable receptors. ET-18-OCH3 inhibited GM-CSF binding to HL60, U937, and KG-1 cells with a half-maximal inhibitory concentration of 16, 10, and 78 microM, respectively. ET-18-OCH3 at 10 microM reduced GM-CSF binding sites on HL60, U937, and KG-1, but had little effect on the dissociation constant (Kd). ET-18-OCH3 at 10 and 30 microM significantly (p < 0.01) decreased, in a dose-dependent manner, the total uptake, surface binding, and internalization of GM-CSF. The internalization of GM-CSF was more profoundly inhibited than its surface binding. TPA at 1 and 10 nM inhibited GM-CSF binding. Inhibition of GM-CSF binding by a combination of ET-18-OCH3 (10 microM) and TPA (1 or 10 nM) was less than additive, and ET-18-OCH3 partially inhibited TPA-induced protein kinase C (PKC) depletion in the cytosol and translocation to the particulate fractions. It is suggested that the inhibition of GM-CSF binding by ET-18-OCH3 is due in part to disruption of the plasma membrane and that the inhibition of GM-CSF binding by TPA is due to activation of PKC.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on platelet survival and activation using a nonhuman primate model.

In humans and nonhuman primates, the in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) consistently results in marked increase of megakaryocyte ploidy and size similar to that observed with interleukin-6 (IL-6). However, whereas the administration of IL-6 also results in an increase in circulating platelets, there is no predictable corresponding increase in peripheral blood platelets following treatment with rhGM-CSF. To determine whether the failure of rhGM-CSF to produce thrombocytosis is secondary to cytokine-related increased platelet activation and consumption in vivo, we quantified autologous platelet survival time and in vivo platelet activation before and during 5 days of administration of rhGM-CSF to two rhesus monkeys. Platelet survival was measured using autologous platelets labeled with 111Indium-oxine. Platelet activation was assessed by flow cytometric determination of the expression of the major platelet membrane glycoprotein (GP) IIb/IIIa complex, and an activation-dependent epitope on GPIIb/IIIa (recognized by monoclonal antibodies [MABs] LJ-P4 and PAC1, respectively). Platelet activation was also assessed by dose-response aggregometry using adenosine diphosphate (ADP). While megakaryocyte ploidy increased during rhGM-CSF administration, peripheral platelet counts were 418 x 10(9)/L and 525 x 10(9)/L before and 402 x 10(9)/L and 508 x 10(9)/L during cytokine treatment in animals 1 and 2, respectively. No changes were observed in the mean platelet volume. 111Indium-labeled platelet recovery in circulation was similar before (94.7%, 91.8%) and during (92.9%, 92.8%) rhGM-CSF administration, which indicates that cytokine-related in vivo sequestration of platelets does not occur. Autologous platelet survival was 5.6 and 6.2 days before and 5.0 and 5.4 days during the rhGM-CSF treatment (p = 0.07), without significant change in the corresponding platelet turnover rate (derived from the platelet count and survival time). The flow cytometric analysis showed no increase in the binding of either LJ-P4 or PAC1 MABs to the platelet membrane during rhGM-CSF administration. The aggregometry studies demonstrated similar concentrations of ADP inducing half-maximal aggregation (ED50). Overall, the above data indicate that treatment with rhGM-CSF is not associated with in vivo activation, sequestration, or increased consumption of platelets. The data suggest that the failure of rhGM-CSF-stimulated megakaryocytes to increase peripheral platelet count is a manifestation of ineffective megakaryocytopoiesis resulting from inability to increase platelet delivery to the circulation.

Peripheral blood stem cell acquisition by large-volume leukapheresis in growth factor-stimulated and unstimulated rhesus monkeys: development of an animal model.

Twelve (eight unstimulated [UNS], four growth factor-stimulated [ST]) anesthetized adult male rhesus monkeys underwent a single large-volume leukapheresis (> 3 blood volumes processed) in an attempt to define an animal model for use in future peripheral blood stem cell (PBSC) transplantation studies. The cell separator was primed with autologous blood and saline and set according to the manufacturer's mononuclear cell (MNC) protocol using the granulocyte separation and small volume collection chambers with additional modifications. Stimulated animals received 5 micrograms/kg recombinant human interleukin-3 (rhIL-3) on days -12 to -5, 5 micrograms/kg granulocyte-macrophage colony-stimulating factor (GM-CSF) on days -4 to -1, and large-volume leukapheresis on day 0. UNS animals did not receive growth factors. Vascular access was via a triple lumen intra-aortic catheter; blood pressure was monitored via the third lumen. Pre- and post-apheresis blood counts were determined and product hematocrit (Hct), MNC, colony-forming units-granulocyte/macrophage (CFU-GM), CD34+, and lymphocyte subsets were studied. During the 100-minute large-volume leukapheresis with mean flow rate 26.4 +/- 3.9 mL/min, the pre- and post-Hct were 36.6 +/- 2.6 and 32.3 +/- 7.0%, platelets 447 +/- 305 and 154 +/- 77 x 10(9)/L, and MNC 2.7 +/- 0.9 and 1.9 +/- 1.4 x 10(9)/L (all p < .05) in UNS animals. In ST animals, the pre- and post-Hct were 39.0 +/- 5.6 and 34.9 +/- 3.7%, platelets 507 +/- 100 and 150 +/- 9 x 10(9)/L (p < .05), and MNC 4.9 +/- 1.6 and 2.8 +/- 0.7 x 10(9)/L (p < .05). The product contained 98.5 +/- 1.4% MNC, 4.1 +/- 4.1% Hct, 1.9 +/- 0.6 x 10(9) MNC, 9.2 +/- 7.3 x 10(4) CFU-GM, and 3.5 +/- 2.1 x 10(6) CD34+ cells in UNS animals. In ST monkeys, the product contained 49.5 +/- 32% MNC, 6.4 +/- 4.4% Hct, 3.6 +/- 1.8 x 10(9) MNC, 49.3 +/- 39 x 10(4) CFU-GM, and 9.1 +/- 5.5 x 10(6) CD34+ cells. Greater than 75% of the product MNC were CD2+ T cells in ST and UNS animals. Large-volume leukapheresis in rhesus monkeys was tolerated well. Herein we characterize an animal model for large-volume leukapheresis in UNS monkeys that is similar to that of human PBSC leukapheresis. In ST animals there is more than a five-fold increase in CFU-GM collected and an increase in circulating CFU-GM/MNC.(ABSTRACT TRUNCATED AT 400 WORDS)