Paclitaxel vs cyclophosphamide in peripheral blood stem cell mobilization: comparative studies in a murine model.

Paclitaxel is a promising drug for the treatment of breast and ovarian cancer. It also may play a role in mobilization of peripheral blood stem cells (PBSC), as an alternative to cyclophosphamide (Cy). We investigated the PBSC-mobilizing potential of paclitaxel compared to Cy in a murine model. C57B1/6 mice were primed with intraperitoneal injections of Cy (200 mg/kg) or paclitaxel (60 mg/kg) and were sacrificed 4, 6, 8, or 10 days later. Spleens were harvested and processed to obtain low-density mononuclear cells that were used as PBSC. The number of hematopoietic progenitors (CFU-C) on day 4 was significantly higher in the paclitaxel group when compared to mice receiving Cy (72.0 +/- 1.8 vs 9.8 +/- 2.8, p < 0.001). By day 6, CFU-C became significantly higher in the Cy-treated group compared to the paclitaxel-treated group (195.6 +/- 31.9 vs 95.8 +/- 20.7, p < 0.05) and this trend was maintained. However, the total number of CFU-C recovered per spleen was greater in the paclitaxel-treated group (1.27 x 10(5) +/- 0.53 x 10(5) vs 1.06 x 10(5) +/- 0.36 x 10(5), NS). In contrast to paclitaxel, mobilization with Cy was associated with marked perturbation in the proportion of lymphoid cell subsets in the PBSC population along with functional impairment of lymphocytes. After 24 hours of in vitro IL-2 activation, the cytotoxic effector cell function of the Cy-mobilized PBSC population was lower than that of paclitaxel-mobilized cells when tested against three tumor cell lines (B16, melanoma; C1498, AML; and Yak-1, lymphoma). These results indicate that paclitaxel is an efficient mobilizer of PBSC, leading to early (day 4 to 6) mobilization of PBSC when compared to Cy (day 6 to 8). In addition, paclitaxel was associated with less perturbation of phenotypic and functional characteristics of cells contained within the mobilized PBSC population.

Suppression of progenitor cell growth by vancomycin following autologous stem cell transplantation.

The occurrence of hematologic side-effects resulting from the use of vancomycin is rare. Prior to this report, vancomycin-induced neutropenia was believed to be due to a hypersensitivity reaction since antibodies directed against circulating neutrophils have been discovered in the serum of some patients. We demonstrate suppression of hematopoietic bone marrow progenitor cells in a patient experiencing vancomycin-induced neutropenia after an autologous hematopoietic stem cell transplantation for multiple myeloma. A bone marrow (BM) specimen obtained at the time of neutropenia demonstrated direct suppression of progenitor cell growth in vitro when vancomycin was added at increasing concentrations (1, 10 and 50 microg/ml). No such trend was noted in a BM sample from the same patient obtained 11 months prior to transplantation and a normal control BM. The decrease in the total number of colony-forming units (CFU) was statistically significant at all the dose levels of vancomycin when compared to the number of CFU in the baseline BM sample (P < 0.05). The myeloid maturation arrest observed in the bone marrow sample obtained during the period of neutropenia and the dose dependent growth inhibition by vancomycin observed in vitro suggest a novel nonimmune mechanism of hematologic effects due to suppression of bone marrow progenitor cell growth.

Effect of the in vivo priming regimen for peripheral blood stem cells (PBSC) mobilization on in vitro generation of cytotoxic effectors by IL-2 activation of PBSC in a murine model.

Priming of patients with different PBSC mobilizing regimens leads to an increase by several fold in circulating hematopoietic progenitors in peripheral blood. However, the effect of these mobilizing regimens on lymphoid cells contained within the harvested PBSC population is not well understood. We have studied the effect of CY and/or G-CSF +/- IL-2 containing regimens on lymphoid cells, and their capacity to give rise to cytotoxic effectors on subsequent in vitro IL-2 activation in a murine model of PBSC mobilization. C57B1/6 mice were given CY 100 or 200 mg/kg on day 0 followed 48 h later by G-CSF 125 micrograms/kg twice a day and/or IL-2 60000 i.u. twice a day in different schedules. Mice were sacrificed on day 4, 6, 8 and 10 following CY and the number of hematopoietic progenitors mobilized to the spleens of these mice was assessed by CFC assay and cytotoxicity was evaluated by 4 h 51Cr release assay against both NK-sensitive (Yak-1), and NK-resistant (B16, C1498) cell lines after 24 h in vitro IL-2 activation in the presence of 6000 i.u./ml of IL-2. Peak numbers of CFC in the splenic PBSC population were seen on day 6 following CY. Administration of CY 200 mg/kg + GCSF, the most potent regimen for CFC mobilization, led to a marked decrease in proportion of CD3+ cells in day 6 PBSC as compared to controls (17.7% vs 3.9%) and was associated with a significant decrease in generation of cytotoxic cells after IL-2 activation. Combining IL-2 to CY + G-CSF prevented the marked loss in cytotoxicity associated with this regimen without any decrease in number of CFC mobilized. When IL-2 was combined with CY without G-CSF, the number of CFC mobilized was comparable to that seen with CY + G-CSF and these CY + IL-2 mobilized PBSC generated potent cytotoxic effectors after in vitro IL-2 activation. Thus our results indicate that combining IL-2 with a PBSC mobilizing regimen can avert a decrease in the cytotoxic potential of mobilized cells without compromising the number of hematopoietic progenitors.

Interleukin-2-activated hematopoietic stem cell transplantation for breast cancer: investigation of dose level with clinical correlates.

Incubating hematopoietic stem cells with IL-2 in vitro for 24 h generates cytotoxic T cells. When infused into patients, these cells may stimulate a graft-versus-tumor (GVT) effect. This clinical trial was designed to assess the ability of IL-2 activated peripheral blood stem cells (PBSC) to reconstitute hematopoiesis, to investigate dose levels and dose-limiting toxicities of IL-2, and to evaluate clinical results and preliminary laboratory effects using a combination of IL-2-activated autologous PBSC followed by IL-2 after transplantation. Sixty-one women with stage II-IV breast cancer were treated. After the administration of carboplatin (200 mg/m2/day for 3 days) and cyclophosphamide (2 g/m2/day for 3 days), patients received autologous PBSC that were cultured in IL-2 for 24 h followed by parenteral administration of IL-2 beginning the day of transplantation. Three escalating doses of IL-2 were evaluated with increasing duration up to 4 weeks. Of the 57 patients receiving IL-2 after tranplantation, 19 patients (33.3%) were unable to complete the planned course of IL-2 therapy due to persistent fevers (n = 9), diarrhea (n = 2), pulmonary capillary leak syndrome (n = 3), development of a rash (n = 1), atrial fibrillation (n = 1), or patient's request (n = 3). One death occurred during hospitalization. Engraftment of neutrophils occurred on day 11.5 (mean; range 8-21 days) and platelets on day 11.7 (mean; range 7-33 days). The maximal tolerated dose of IL-2 was 6 x 10(5) IU/m2/day for 4 weeks. Disease-free survival rates for all stages were comparable to current reports in the literature. Preliminary laboratory evaluations include FACScan analysis of the IL-2 activated PBSC demonstrating an increased percentage of CD3+, CD25+, HLA-DR+ T cells. Phenotypically similar cells were present in peripheral blood samples of patients when tested 15 days after transplantation. This study demonstrates successful engraftment with IL-2-activated PBSC after high-dose chemotherapy for women with stage II-IV breast cancer. The regimen is feasible and, although toxicities are common, they are manageable and correlate with increasing dose and duration of IL-2.

Comparative study of hemopoietic precursors from fetal and adult bone marrow: utilization of stem cells derived from miscarriages.

Hemopoietic and immune capacities of fetal bone marrow (FBM) obtained from 2nd-trimester lost pregnancies and adult bone marrow (ABM) were compared. Progenitor cell assays for both sources were also enumerated. Out data showed ontogeny-related functional differences between hemopoietic cells, particularly in the ability to produce CD34+ cells (24.6% in FBM, 3.1% in ABM). The phenotypic composition of FBM and ABM were quite different. The clonogenic/proliferative potentials, as measured by CFU-C assays, were significantly higher in FBM when compared to ABM (202.5 vs. 73.5/10(5) cells). Moreover, FBM had a lower percentage of CD3+ T lymphocytes as compared to ABM (1.47 vs. 7.58), and there was a significantly decreased proliferative responsiveness in mixed lymphocyte reactions of FBM as compared to ABM. Thus, our data clearly showed distinct advantages of FBM over ABM, which include a higher number of stem cells, lower immunological reactivity, and higher clonogenic/proliferative potential. These characteristics provide optimal conditions for successful engraftment without graft-versus-host disease. These data support the possible advantages of FBM from these sources for hemopoietic stem cell reconstitution and gene therapy.

Hematopoietic potential of IL-2-cultured peripheral blood stem cells from breast cancer patients.

Patients receiving autologous transplants for various malignancies generally experience an increased incidence of relapse compared with patients receiving unmanipulated allogeneic transplants. We initiated a protocol for IL-2 activation of peripheral blood stem cells (PBSC) for induction of in vitro and in vivo autologous graft-versus-tumor (GVT) activity in patients with breast cancer. In this study we analyzed the effects of 24 h of IL-2 incubation on the hematopoietic potential of PBSC from these patients. Cells collected by leukapheresis were first cryopreserved and stored in liquid nitrogen, then thawed rapidly and incubated with IL-2 in a serum-free system for 24 h, with samples analyzed before and after incubation. Although there was a significant drop in mononuclear cells (MNC) (from 4.5 to 3.7 x 10(8)/kg) and CD34+ cells (from 12.3 to 7.5 x 10(6)/kg) after 24 h in culture, there was no significant change in colony-forming units (CFU) (from 12.5 to 11.5 x 10(5)/kg). Time to engraftment (neutrophils: < 0.5 x 10(9)/l; platelets: > 20 x 10(9)/l) was comparable to a cohort of similar patients receiving non-cultured PBSC transplants. These results indicate that mobilized frozen/thawed PBSC which have been cultured in IL-2 for 24 h retain adequate potential for hematopoietic reconsistution in this group of patients.

Towards endothelial-cell-directed cancer immunotherapy: in vitro expression of human recombinant cytokine genes by human and mouse primary endothelial cells.

Recent studies have demonstrated the feasibility of cytokine gene transfer to enhance the antitumor activities of host immune cells. Endothelial cells forming the vascular supply of tumors may be useful vehicles for the delivery of cytokine molecules in order to effect tumor immunotherapy. In order to determine whether primary endothelial cells can express cytokine transgenes efficiently, we constructed two retroviral vectors containing a cDNA encoding either recombinant human interleukin-1 alpha (rhIL-1 alpha) or recombinant human interleukin-2 (rhIL-2), called LNCIL-1 alpha and LNCIL-2 respectively, and studied the expression of the two cytokines in vitro in non-immortalized endothelial cells. Human umbilical vein endothelial cells (HUVEC) transduced with LNCIL-1 alpha or LNCIL-2 secreted 1.8-33 ng/10(6) cells/24 h and 40-246.7 ng/10(6) cells/24 h of biological active rhIL-1 alpha and rhIL-2 respectively. Mouse microvascular endothelial cells (MMEC) transduced with LNCIL-1 alpha and LNCIL-2 secreted 1.5 ng/10(6) cells/24 h and 5.8-24.7 ng/10(6) of biologically active rhIL-1 alpha and rhIL-2 proteins respectively. Cocultivation of HUVEC/IL-2 and MMEC/IL-2 with normal human bone marrow cells generated potent cytotoxic activity against K562, Daudi and other cell targets in a 51Cr-release assay. While IL-2 transgene-expressing HUVEC and MMEC retained their normal morphology, rhIL-1 alpha transgene expression inhibited the growth and altered the morphology of both HUVEC and MMEC in culture. The cytokine-gene-transduced endothelial cells retained other endothelial cell features, including uptake of acetylated low-density lipoprotein (Ac-LDL) and expression of von Willebrand factor, and were euploid as shown by flow cytometry. These results demonstrate that endothelial cells, by sustaining the production of biologically active rhIL-2 at levels that are sufficient for the activation of potent cytotoxic lymphocyte activity, may be useful agents for cancer gene therapy.

Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2.

Earlier evidence suggests that transforming growth factor beta (TGF beta) plays a significant role in tumor progression and metastasis. The most likely mechanism of the action of TGF beta is induction of immunosuppression in the host, allowing for unchecked tumor growth and metastasis. We attempted to test that hypothesis and to compare antitumor effects of anti-TGF beta antibody alone and in combination with interleukin-2 (IL-2). Six- to 8-week-old female C57B1-6 mice were induced with murine B16 melanoma by tail vein injection. Therapy was started 48 h after tumor injections. Monoclonal anti-TGF beta antibody (2G7) was administered intraperitoneally (i.p.) at 500 micrograms every other day, and IL-2 at 10,000 U i.p. twice daily, for 21 days. A threefold decrease in the number of lesions in the anti-TGF beta/IL-2 treatment group compared with the control group was observed, a highly significant statistical difference (p = 0.002). No statistically significant differences were seen between the control group and other studied groups (IL-2 alone, anti-TGF beta alone). Analysis of TGF beta levels in plasma by the TGF beta-1 Quantikine assay indicated normal levels in the control and IL-2 groups, and significantly diminished levels in the two groups that received TGF beta antibody. However, acid-ethanol extraction of plasma (to reverse antibody binding before assay) showed normal plasma TGF beta levels in all groups, suggesting that the antibody may alter the availability of TGF beta in vivo. Microscopic analysis of metastases revealed a decrease in the average size of lesions in the groups treated with IL-2. Thus, combination therapy using anti-TGF beta antibody and IL-2 may be a novel, less toxic approach to tumor immunotherapy.

Stem cell transplantation with chemoradiotherapy myeloablation and interleukin-2.

Interleukin 2 (IL-2) stimulates the proliferation of T-cells both in vitro and in vivo. When murine or human peripheral blood (PB) or bone marrow (BM) mononuclear cells are incubated with IL-2 in vitro for 24 hours, cytotoxic T-cells are generated. If these activated cells are infused into mice, the enhanced cytotoxicity continues if low dose IL-2 is administered. This combination of administering activated cells with the subsequent low dose IL-2 infusion results in enhanced tumor cell destruction and improved survival rates in mice with acute myeloid leukemia. The encouraging results of these laboratory experiments prompted the initiation of phase I clinical trials in patients with refractory/relapsed hematologic malignancies and patients with breast cancer (Stages II-IV). Results from these trials demonstrate that stem cell transplantation with IL-2 activated stem cells (either PB or BM) with or without parenteral administration of IL-2 results in hematopoietic reconstitution with mild-to-moderate toxicities. This regimen also generates cutaneous and visceral autologous graft versus host disease (AuGVHD). The majority of our patients with relapsed/refractory hematologic malignancies or breast cancer developed either clinical and/or histological evidence of AuGVHD. Further studies are being conducted to determine if patients who develop AuGVHD experience improved disease-free survival from a possible autologous graft versus tumor (GVT) effect. Current laboratory evaluations include the elucidation of the pathogenesis of AuGVHD and molecular evaluation of the purging efficacy of IL-2.

A syndrome of fibrosing pleuritis, pericarditis, and synovitis with infantile contractures of fingers and toes in 2 sisters: "familial fibrosing serositis".

We describe a family in which 2 sisters born to consanguineous parents developed childhood onset fibrosing pleuritis in association with constrictive pericarditis and bilateral deforming arthropathy of large and small joints of upper and lower extremities including flexion contractures of several fingers (camptodactyly) and toes. One patient also had mitral value prolapse. Histopathological examination of the synovium and pericardium revealed fibrosis, and ultrastructural study of synovium showed abundant inter and intracellular mesh of 9 nm microfibrils. We describe this distinct clinicopathological entity with pleiotropic manifestations, the common features of which appears to be the fibrosis of serous membranes. Therefore, the term "familial fibrosing serositis" is proposed for this entity.

Interleukin-12 (IL-12) alone or in synergistic combination with IL-2 for in vitro activation of human bone marrow: differential effects at different time points.

IL-12 augments many effector functions associated with T and NK cells including induction of cytotoxic activity and secretion of various cytokines. In addition, synergistic responses with IL-2 in most of the functions attributed to that cytokine make IL-12 an attractive candidate to have a potential role as an immunotherapeutic agent. We have evaluated the effect of rhIL-12 either alone or in combination with IL-2 on the generation of cytotoxic effectors from normal human bone marrow. IL-12 induced cytotoxic effectors against NK sensitive targets within 24 h of stimulation and this activity was maintained in cultures with IL-12 supplementation for up to 2 weeks tested. The decline in NK activity seen in control cultures over a period of 2 weeks was thus not observed in IL-12 stimulated cultures. In addition, a small increase was also observed in cytotoxic activity against NK resistant Daudi cell targets. IL-12, when combined with a low to intermediate dose of IL-2, led to enhancement in IL-2 induced cytotoxicity against both NK sensitive and resistant targets. However an increase in cytotoxicity against NK resistant targets was evident only after 1 week of stimulation. Cellular yield and number of hematopoietic precursors (LTC-IC, CFU-GM, BFU-e) in IL-12 stimulated cultures was comparable to or higher than control cultures at all the three time points (days 1, 7, 14) tested. In contrast, cultures stimulated with a combination of IL-12/IL-2 showed an increased number of hematopoietic precursors at day 1 followed by a marked decrease in the number of these precursors at day 7 and day 14.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 activation of chemotherapy and growth factor-mobilized peripheral blood stem cells for generation of cytotoxic effectors.

Based on our previous studies demonstrating marked anti-tumor activity of interleukin-2 (IL-2)-activated bone marrow in vitro and in vivo, we studied the generation of anti-tumor cytotoxic effectors from chemotherapy- and growth factor-mobilized PBSC from 12 patients with different solid tumors. As chemotherapy and growth factor priming could lead to important qualitative and quantitative alterations of lymphoid cells, we also looked at the distribution of lymphoid cells contained in primed PBSC. In addition, different variables were defined for successful application of the technique to clinical protocols. The cells were placed in culture at varying cell densities in either serum-containing or serum-free culture medium, supplemented with IL-2, 100 or 1000 Cetus units/ml at 37 degrees C for 24 or 72 h, in flasks or in culture bags. Anti-tumor cytotoxicity was tested against A375 (melanoma), K562 (CML) and Daudi (Burkitt's lymphoma) cell lines in standard 4 h 51Cr release assay. Marked cytotoxicity was seen against all cell lines tested (A375: 32.7% +/- 5.2; K562: 52.8% +/- 4.8; Daudi: 50.5% +/- 7.2). Cytotoxicity was comparable in serum-containing and serum-free culture conditions and in tissue culture flasks and bags. Cell density up to 10 x 10(6)/ml was not associated with any significant decline in cytotoxicity. IL-2 activation of PBSC after thawing led to the generation of cytotoxicity comparable to that obtained with fresh PBSC. On flow cytometric analysis, the proportion of CD8+ T cells and NK cells (CD56+) was found to be higher in primed PBSC than in control peripheral blood mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 activation of human bone marrow in long-term cultures: an effective strategy for purging and generation of anti-tumor cytotoxic effectors.

Our previous studies have shown that IL-2 activated bone marrow cells develop potent tumoricidal activity in vitro and in vivo. With the dual aim of in vitro purging and generation of effectors which could mediate graft-versus-leukemia effects in vivo, IL-2 activation of human bone marrow in long-term cultures (LTC) was tested. Marked cytotoxicity was seen against A375 (melanoma), K562 (CML) and Daudi (lymphoma) cell lines in IL-2 (1000 U/ml) stimulated cultures. Hematopoietic progenitor cell number in these cultures was assessed by day 14 clonogenic assays. In 1-week-old IL-2 stimulated cultures a higher number of clonogenic cells was seen than control LTCs without IL-2. However, thereafter accelerated decrease in the number of clonogenic cells was seen in IL-2 cultures. In vitro purging efficacy was tested by elimination of A375 and K562 cells mixed with normal marrow at 1:10 and 1:100 ratios and co-cultured for 10 days. In IL-2 stimulated cultures, A375 cells capable of proliferation were not detectable at both mixing ratios. K562 elimination was complete only at 1:100 ratio. After 10 days in culture, no Ph1-positive metaphases were seen in IL-2 stimulated BM cultures of 4 patients with CML. These results indicate that IL-2 activation of BM in 1-2 week cultures can lead to generation of marked anti-tumor cytotoxicity and effective in vitro purging in a variety of tumor types.

Scleromyxoedema with features of systemic sclerosis.

The case is described of a patient with scleromyxoedema with features typical of systemic sclerosis. The features were so characteristic that the disease was misdiagnosed as systemic sclerosis. A brief review of the association of the two diseases is given.