Mizolastine therapy also has an effect on nasal blockade in perennial allergic rhinoconjunctivitis. RIPERAN Study Group

High-dose therapy with autografting of peripheral blood stem cells (PBSCs) has become an accepted treatment modality. However, gene-marking studies in patients with acute myeloid leukemia and neuroblastoma have revealed that malignant cells reinfused along with leukapheresis products (LPs) contribute to relapse. Thus, a reduction in the number of malignant cells in autografts is desirable. We analyzed the percentage of malignant cells and the number of CD34+ PBSCs in LPs mobilized by granulocyte colony-stimulating factor (G-CSF) alone (LP-S) compared with high-dose cyclophosphamide plus G-CSF (LP-CY) in patients with multiple myeloma (MM). A quantitative polymerase chain reaction assay involving CDR3-specific primers based on the method of limiting dilutions was used to determine the tumor loads of LPs. Sixteen LPs from eight patients with MM were analyzed intraindividually in matched pairs. The percentage of malignant cells was lower in LP-CY (p = 0.017; median 0.0067 vs. 0.009%), whereas the number of CD34+ cells was higher (p = 0.012; median 0.3 vs. 0.095%). The calculated number of malignant cells per CD34+ cell was significantly lower in LP-CY as well (p = 0.017). We conclude that mobilization by cyclophosphamide plus G-CSF leads to a lower number of malignant cells per CD34+ cell in LPs compared with G-CSF alone.     

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.     

Mobilization of peripheral blood progenitor cells with high-dose cyclophosphamide (4 or 7 g/m2) and granulocyte colony-stimulating factor in patients with multiple myeloma

High-dose cyclophosphamide (HD-CY) has been shown to decrease the tumor mass in multiple myeloma (MM) patients and to be effective in the mobilization of PBPC. By administering hematopoietic growth factor the quantity of progenitor cells in the peripheral blood increased and the hematological toxicity of CY could be reduced. Thirty-two patients with stage II and stage III MM were treated to mobilize and harvest a sufficient amount of PBPC for autologous transplantation. Sixteen patients received 4 g/m2 CY and 16 patients 7 g/m2 CY in divided doses of 1 g/m2 every 2 h. Both patient groups were comparable for disease stages as well as previous therapies. Twenty-four hours after chemotherapy 300 micrograms GCSF were administered subcutaneously once daily until the last day of leukapheresis. Administration of 7 g/m2 HD-CY resulted in statistically significantly higher peak values for CD34+ progenitor cells (47.86/microliters vs 18.75/microliters, P = 0.0198) in the peripheral blood. PBPC autografts containing > 2.5 x 10(6) CD34+ cells/kg BW could be obtained at the first attempt from 14 of 16 patients treated with 7 g/m2 CY as compared to 10 of 16 patients treated with 4 g/m2 CY (P = 0.11). The analysis of potentially malignant CD19+ B cells showed a highly significant lower mean CD19+ cell content/kg BW per leukapheresis in the 7 g/m2 compared to the 4 g/m2 CY group (0.75 vs 1.81 x 10(6), P = 0.001). WHO grade IV treatment-related non-hematologic toxicity was not observed. We prefer the 7 g/m2 CY dosage followed by cytokine administration for the mobilization of PBPC in advanced state MM patients pretreated with alkylating agents.     

In vitro expansion of CD34+ cells from peripheral blood of myeloma and lymphoma patients

We studied the feasibility of in vitro expansion of CD34+ cells from patients with multiple myeloma (MM) or follicular non Hodgkin lymphoma (NHL). CD34+ cells were selected from peripheral blood (PB) using avidinbiotin immunoadsorption columns: purified CD34+ cells from three MM and five NHL patients were expanded. First, CD34+ cells (2 MM, 4 NHL) were grown for 14 days in 5 ml of IMDM plus 12.5% horse serum (HS), 12.5% fetal calf serum (FCS) and a commonly used combination of cytokines: IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (10 ng/ml each) and EP (4 UI/ml). In these conditions, at day 14, average increase in CD34+, CFU-GM and total cell numbers were, respectively: x 6.0 x 23 and x 2,113 fold with 20 to 35% of granulocytic cells. In terms of CD34+ cell, CFU-GM and total cell outputs, MM cultures were comparable to NHL cultures, but MM cultures seemed to produce less granulocytic cells than NHL cultures. Next, in vitro expansion of PB CD34+ cells was tested in culture media suitable for clinical use. Two cultures (1 MM, 1 NHL) were carried out for 14 days in 20 ml of X-Vivo 10 medium, 2% human serum, IL1alpha, IL3, IL6, SCF, GM-CSF, G-CSF (6 ng/ml each) and EP (2 UI/ml). Increase in CD34+, CFU-GM and total cell numbers in these conditions were, respectively: x 5.7 and x 19.7, x 11.9 and x 40.9, x 424 and x 408 fold, with at least 75% of granulocytic cells in both cultures. We conclude that, although further improvements are necessary, in vitro expansion of PB CD34+ cells can presumably be carried out successfully for MM patients as well as for NHL patients, including in conditions suitable for clinical use.     

Dendritic cell-based vaccines in the setting of peripheral blood stem cell transplantation: CD34+ cell-depleted mobilized peripheral blood can serve as a source of potent dendritic cells

We are investigating the use of tumor-pulsed dendritic cell (DC)-based vaccines in the treatment of patients with advanced cancer. In the current study, we evaluated the feasibility of obtaining both CD34+ hematopoietic stem/ progenitor cells (HSCs) and functional DCs from the same leukapheresis collection in adequate numbers for both peripheral blood stem cell transplantation (PBSCT) and immunization purposes, respectively. Leukapheresis collections of mobilized peripheral blood mononuclear cells (PBMCs) were obtained from normal donors receiving granulocyte colony-stimulating factor (G-CSF) (for allogeneic PBSCT) and from intermediate grade non-Hodgkin's lymphoma or multiple myeloma patients receiving cyclophosphamide plus G-CSF (for autologous PBSCT). High enrichment of CD34+ HSCs was obtained using an immunomagnetic bead cell separation device. After separation, the negative fraction of mobilized PBMCs from normal donors and cancer patients contained undetectable levels of CD34+ HSCs by flow cytometry. This fraction of cells was then subjected to plastic adherence, and the adherent cells were cultured for 7 days in GM-CSF (100 ng/ml) and interleukin 4 (50 ng/ml) followed by an additional 7 days in GM-CSF, interleukin 4, and tumor necrosis factor alpha (10 ng/ml) to generate DCs. Harvested DCs represented yields of 4.1+/-1.4 and 5.8+/-5.4% of the initial cells plated from the CD34+ cell-depleted mobilized PBMCs of normal donors and cancer patients, respectively, and displayed a high level expression of CD80, CD86, HLA-DR, and CD11c but not CD14. This phenotypic profile was similar to that of DCs derived from non-CD34+ cell-depleted mobilized PBMCs. DCs generated from CD34+ cell-depleted mobilized PBMCs elicited potent antitetanus as well as primary allogeneic T-cell proliferative responses in vitro, which were equivalent to DCs derived from non-CD34+ cell-depleted mobilized PBMCs. Collectively, these results demonstrate the feasibility of obtaining both DCs and CD34+ HSCs from the same leukapheresis collection from G-CSF-primed normal donors and cancer patients in sufficient numbers for the purpose of combined PBSCT and immunization strategies.     

Mifepristone modulation of ACTH and CRH regulation of bovine adrenocorticosteroidogenesis in vitro

In this article, we review neoplastic contamination in the peripheral blood (PB) of patients with multiple myeloma (MM) upon stem cell mobilization. We first evaluated PB samples from pretreated MM patients following administration of high-dose cyclophosphamide (Cy, 7 g/m2 or 4 g/m2) and granulocyte colony-stimulating factor (G-CSF) for the presence of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic immunoglobulin (cIg) were counted by immunofluorescence microscopy after incubation with appropriate antisera against light and heavy chain Ig. Flow cytometry studies were performed to determine the presence of malignant B lineage elements, using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Prior to PBSC mobilization, circulating plasma cells were detected in all MM patients at 0.1%-1.8% of the mononuclear cell (MNC) fraction (mean value 0.7 +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after administration of chemotherapy and G-CSF. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors, with the peak coinciding with the optimal period for the collection of PBSC. The absolute number of plasma cells showed a 10-50-fold increase over the baseline value. Apheresis products contained 0.7 +/- 0.2% SD myeloma cells (range 0.2%-2.7%), which demonstrated the capacity of plasma cells to proliferate, differentiate, and mature in response to c-kit ligand (SCF), IL-3, IL-6, and a combination of IL-3 and IL-6. Subsequently, in an attempt to reduce tumor cell contamination prior to autologous transplantation, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute bone marrow (BM) function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51%-94%), with a 75-fold enrichment compared with the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33%-95%) and 45% (range 7%-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log depletion of plasma cells and CD 19+ B lineage cells as determined by immunofluorescence studies, although DNA analysis of the CDR III region of the IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high-dose melphalan (140 mg/m2) or melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis. The median time to 0.5 x 10(9) neutrophils, 20 x 10(9) and 50 x 10(9) platelets/L of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. Our data demonstrate the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring normal hematopoiesis following a TBI-containing conditioning regimen.     

Autologous transplantation of mobilized peripheral blood CD34+ cells selected by immunomagnetic procedures in patients with multiple myeloma

In the use of autologous PBPC transplantation in patients with multiple myeloma, contamination of PBPC with myeloma cells is commonly observed. Enrichment for CD34+ cells has been employed as a method of reducing this contamination. In this study the reduction of myeloma cells in PBPC was accomplished by the positive selection of CD34+ cells using immunomagnetic bead separation (Isolex 300 system). PBPC were mobilized from 18 patients using cyclophosphamide (4.5 g/m2) and G-CSF (10 microg/kg/day). A median of two leukaphereses and one selection was performed per patient. The median number of mononuclear cells processed was 3.50 x 10(10) with a recovery of 1.11 x 10(8) cells after selection. The median recovery of CD34+ cells was 48% (range 17-78) and purity was 90% (29-99). The median log depletion of CD19+ cells was 3.0. IgH rearrangement, assessed by PCR, was undetectable in 13 of 24 evaluable CD34+ enriched products. Patients received 200 mg/m2 of melphalan followed by the infusion of a median of 2.91 x 10(6)/kg CD34+ cells (1.00-16.30). The median time to absolute neutrophil count >0.5 x 10(9)/l was 11 days, and sustained platelet recovery of >20 x 10(9)/l was 14 days. We conclude that immunomagnetic-based enrichment of CD34+ cells results in a marked reduction in myeloma cells without affecting engraftment kinetics.     

In vitro expansion of CD34+/CD41+ cells from human peripheral blood CD34+/CD41- cells: role of cytokines for in vitro proliferation and differentiation of megakaryocytic progenitors

The aim of this study is to clarify the transitional change of the proliferation and differentiation of human peripheral blood CD34+ cells to megakaryocytic lineage, focusing on its clinical application. We developed a rapid system to purify human peripheral blood CD34+ cells from healthy volunteers, which produced CD34+ cells with a 90% purity. The purified CD34+ cells predominantly consisted of CD41- cells, and the rate of coexpression of CD41 was 0.6% +/- 0.5%. When the purified cells were cultured in liquid phase for 10 days in the presence of recombinant human stem cell factor (rSCF: a ligand for c-kit), interleukin-3 (rIL-3), and thrombopoietin (rTPO: a ligand for Mpl), the number of CD34+/CD41+ cells increased to 19% +/- 7% of total expanded cells on day 4 (4 days of liquid culture) and then gradually decreased to 2.2% +/- 0.6% on day 10. The absolute number of CD34+/CD41+ cells increased and reached a plateau on day 6, and 1.7 +/- 0.6 x 10(5) CD34+/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The CD34-/CD41+ cells appeared on day 6, continuously increased in number until day 10, and constituted the main population of expanded cells on day 10, with a value of 38% +/- 18%. On day 10, 19.5 +/- 10.6 x 10(5) of CD34-/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The deletion of rTPO from this cytokine combination decreased the number of CD34+/CD41+ and CD34-/CD41+ cells, after days 6 and 8, respectively. Day 0 cells required rIL-3 for promoting colonies containing megakaryocytes, whereas rTPO alone promoted almost no megakaryocytic colonies from day 0 cells. Thus, a combination of IL-3 and SCF expands CD34+/CD41+ cells from CD34+/CD41- cells, and TPO mainly acts to increase CD34-/CD41+ cells. This study suggests that if the expansion of CD34+/CD41+ is performed in vitro, the 6 days' culture of peripheral blood CD34+/CD41- cells with a combination of IL-3 and SCF with TPO provides the most rapid and stable products of CD34+/CD41+ cells for the rapid recovery of platelets in patients with peripheral blood stem cell transplantation.     

Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- +/- 2-fold, myeloid colony-forming cells increased 140- +/- 36-fold, and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.     

Analysis of tumor specific immunoglobulin gene rearrangement in peripheral blood B-cells of multiple myeloma patients by a PCR-SSCP method

OBJECTIVE: To analyze the clonal relationship between lymphocytes in peripheral blood (PB) and myeloma cell in bone marrow (BM) for proving the existence of circulating tumor cells in multiple myeloma (MM) patients. METHODS: Eighteen patients with MM who have no cytomorphologic plasma cells and CyIg+ cells in PB demonstrated by anti-kappa and anti delta MoAbs using ABC method were involved in the present study, including 3 cases in phases I-II and 15 cases in phase III. The complementary determining region 3 (CDR3) of immunoglobulin heavy chain (IgH) gene was amplified by polymerase chain reaction (PCR). We further analysed the single strand conformation of the PCR products by single strand conformation polymorphism (SSCP) analysis to detect the mononuclear cells in PB and BM of the patients simultaneously. RESULTS: The same PCR products of IgH-CDR3 gene with BM samples were found in PB of 11 MM patients. The same PCR products and single strand conformation in both PB and BM were found in 9 cases. CONCLUSIONS: This study has proved the presence of identical clonal malignant cells in PB and BM of MM patients. B cells are involved in the pathogenesis of MM.     

The potential role of FLT3 ligand in progenitor and primitive hematopoietic cell expansion

We have previously defined the experimental conditions for hematopoietic cell expansion. CD34+ human marrow cells were maintained in a serum-free, stroma-free liquid culture system, at a concentration of 10(3) cells/ml, for 10 days at 37 degrees C, in the presence of various cytokine combinations. The basic combination of early cytokines SCF (100 ng/ml), IL3 (5 ng/ml), IL6 (10 ng/ml), has a modest stimulating effect on all compartments: the number of total cells increased 56-fold and CD34+ cells 1-fold; CFU-GM, BFU-E and CFU-MK, increased 6-fold, 5-fold and 3-fold respectively. As far as CD34+ cells are concerned, the subpopulation CD34+/CD38- was only maintained. Interestingly, the addition of 100 ng/ml of Flt3 ligand (FL) significantly enhanced the amplification of total cells (276-fold), CFU-GM (54-fold) and BFU-E (15-fold). The number of CD34+ cells and the subpopulation CD34+/38- increased to 7-fold and 22-fold respectively. Moreover, long term culture-initiating cells (LTC-ICs) in limiting dilution assay (LDA) were found to increase 3-fold. Further addition of MGDF (10 ng/ml), G-CSF (10 ng/ml) and Epo (0.5 U/ml), in various combinations, acted synergically with the previous cytokine combination to support the formation of multiple types of hematopoietic colonies. As expected, the addition of MGDF increased the number of CFU-MK up to 5-fold expansion. Interestingly, MGDF addition was synergistic also for BFU-E and CFU-GM expansion. In the combination of SCF+ IL3+ IL6+ FL + MGDF, CFU-GM expanded to 73-fold and BFU-E to 17-fold. G-CSF in SCF + IL3 + IL6 + FL conditions stressed the expansion of the granulopoietic compartment doubling the number of CFU-GM and CD33+ cells, with no consequence on LTC-IC or BFU-E. Surprisingly, G-CSF induced the expansion of the megakaryocytic lineage up to 6-fold, in a similar way as MGDF. Epo in presence of SCF+ IL3+ IL6+/-FL dramatically increased total cell expansion (2300-2800-fold), mainly erythroblastic (70% glycoA) without exhaustion of all other compartments. The simultaneous use of these three cytokines (MGDF + G-CSF + Epo) in presence of four early cytokines (SCF + IL3 + IL6 + FL) clearly allows a significant expansion of all hematopoietic compartments, precursors, progenitors, and primitive stem cells. In conclusion, these data show the ability of a stroma-free, serum-free liquid system to expand all myeloid lineages, including CFU-MK and LTC-IC which are critical for clinical application of ex vivo expanded cells.     

Dendritic cells cultured from mononuclear cells and CD34 cells in myeloma do not harbour human herpesvirus 8

Dendritic cells (DC) are antigen-presenting cells with the potential to be a powerful adjuvant in the immunotherapy of haematological malignancy, including myeloma. Recently, human herpesvirus 8 (HHV-8) infection of dendritic cells in the long-term bone marrow stromal cultures of patients with myeloma has been reported. This finding is of great potential importance regarding oncogenesis in myeloma in addition to having significant implications for the use of DC in the immunotherapy of this disease. Therefore DC generated from mobilized blood mononuclear cells (MO-DC) and purified CD34+ cells (CD34-DC) of myeloma patients were examined for the presence of HHV-8 using a sensitive PCR technique. HHV-8 was not demonstrated in MO-DC or CD34-DC and we conclude that these cells remain a suitable vehicle for investigation in the immunotherapy of myeloma.     

Rapid generation of antiplasma cell activity in the bone marrow of myeloma patients by CD3-activated T cells

We have recently shown that peripheral blood T cells of multiple myeloma (MM) patients are very susceptible to stimulation of the T-cell receptor/CD3 complex with anti-CD3 monoclonal antibodies (MoAbs). CD3 stimulation is currently under clinical investigation as a nonspecific approach to boost antitumor effector mechanisms. The aim of this study was to determine whether the hyperreactivity of MM T cells to CD3 stimulation could be exploited to generate antitumor activity. Bone marrow mononuclear cells (BMMCs) from 65 MM patients were stimulated with the anti-CD3 MoAb OKT3 and the effect of this stimulation on autologous T cells and plasma cells was evaluated. The number of CD3+ CD25+ cells on day 6 was significantly higher in MM than the controls (30 normal individuals) (P = .001). Kinetic studies showed that 3H-thymidine incorporation peaked on day 3 and that the T-cell expansion peaked on days 5 and 6. In MM, T-cell activation markedly affected the survival of autologous plasma cells; their number in OKT3-treated cultures was significantly lower than in unstimulated cultures (P < .0001). T-cell activation and plasma cell decrease were not observed when T cells were removed from BMMC preparations. MM produced significantly higher levels of interferon-gamma (P = .005) and tumor necrosis factor-beta (P = .001), but lower levels of tumor necrosis factor-alpha (P < .001) than normal individuals. Interferon-gamma only was partially involved in CD3-induced plasma cell killing. Transwell cultures showed that the main mechanism by which CD3+ CD25+ cells affected plasma cells was direct cell-to-cell contact rather than cytokines. In conclusion, T cells in MM BMMCs possess distinct features in terms of susceptibility to CD3 stimulation and cytokine production compared with normal bone marrow T cells that can be exploited to generate antiplasma cell activity.     

Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.     

Release of cytokines and soluble cell surface molecules by PBMC after activation with the bispecific antibody CD3 x CD19

Bispecific antibodies (BsAb) consist of two different heavy and light chains and may bind to two different antigens present on different cell types. With their dual specificity BsAb may recognize effector cells (e.g. T cells) on one hand and tumour cells (e.g. malignant B cells) on the other hand. The authors analysed whether T cell activation and subsequent killing of malignant B cells mediated by the bispecific antibody CD3 x CD19 was reflected by the release of cytokines. In addition, the authors investigated whether the in vitro cytokine release was similar to that observed in vivo in the patients treated with BsAb. The in vitro release of cytokines into the supernatant of cell cultures of peripheral blood mononuclear cells (PBMC) and malignant B cells was measured after incubation with either the bispecific antibody CD3 x CD19 or the monospecific anti-CD3 (aCD3) antibody in the presence or absence of interleukin (IL)-2. Release of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-8, IL-10, soluble (s) CD4, sCD8 and sCD25 by PBMC was equal under both conditions and could be used as an indicator for T cell activation. However, the cytokine pattern and level did not correlate with the cytotoxic capacity, which was 4 logs higher with BsAb + IL-2 compared to aCD3 + IL-2. The in vitro pattern of cytokine release was similar to that observed in vivo in the serum of patients treated with BsAb and IL-2, indicating the possibility of predicting cytokine release in future patients with other therapeutic regimens.     

An elevation of stem cell factor in patients with hyperthyroid Graves'' disease

The dose of cells expressing the surface antigen CD34 (CD34+) has been shown to be a reliable predictor of the time to engraftment following transplantation of PBPC to support high-dose chemotherapy. However, evaluation of rare cells is complicated by a number of factors, including the variability in operator and technical procedures. Recently, Becton Dickinson Immunocytometry Systems introduced a new CD34+ cell analysis system, the ProCOUNT cell enumeration kit, which automates the analysis of CD34+ cells and minimizes the variabilities of this procedure. We have evaluated the ProCOUNT system in comparison to a standard CD34 cell analysis (based on the Milan approach) using leukapheresis products from patients and normal donors mobilized with chemotherapy plus recombinant human G-CSF (rhG-CSF) or with rhG-CSF alone. In addition, we compared these analyses using CD34+ cell-selected mobilized leukapheresis products with purities of 75% or greater. The standard CD34 cell analysis methodology quantitated the frequency of cells identified as CD45+, low side scatter, and CD34+. A high correlation coefficient was obtained between the ProCOUNT methodology and the standard CD34 cell analysis methodology for cells obtained from leukapheresis products mobilized with chemotherapy plus rhG-CSF (r = 0.98), rhG-CSF alone (r = 0.96), and CD34+-selected mobilized leukapheresis products (r = 0.83). A comparison was also made between technicians using both analysis methods. Whereas the correlation coefficient between two technicians using the standard methodology was r = 0.77, the correlation coefficient was much higher when using ProCOUNT (r = 0.99). These data demonstrate that the use of ProCOUNT is associated with less variability between data analyzed by different operators. Also, ProCOUNT is consistent with existing CD34+ cellular analysis methodologies. An additional advantage is the ability to determine the absolute concentration of CD34+ cells, thereby allowing calculation of total CD34+ cell numbers without using WBC counts, which also have inherent errors. The ProCOUNT system provides an automated analysis procedure that minimizes the variables in CD34+ cell analysis and may be useful for standardization of methodology between laboratories.     

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 10(3)-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface IgM (sIgM) after 5 weeks of co-culture. CD34+CD19- cells also showed a similar development of CD19+ cells and CD19+sigM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19-CD13- CD33- cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19-CD13-CD33- progenitors require the cell-cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.     

Dental nurse training in Cambodia--a new approach

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.     

The absence of CD56 (NCAM) on malignant plasma cells is a hallmark of plasma cell leukemia and of a special subset of multiple myeloma

In this study, we show that malignant plasma cells from patients with either primary (n=12) or secondary (n=15) plasma cell leukemia (PCL) do not express CD56 at all, neither in the bone marrow nor the peripheral blood in 81% of cases. On the other hand, multiple myeloma (MM) at diagnosis overexpress it in 63 of 94 (67%) cases (P=0.0001). In three secondary PCL evaluated serially, CD56 was also lacking at diagnosis showing that CD56 is not downregulated at the end stage of the disease but rather not upregulated in this subset of patients. This last concept is strengthened by the observation that 29% of MM patients lacking CD56 or weakly expressing it at diagnosis present a detectable leukemic phase vs 11% only in CD561 MM (P=0.06). Forty percent of all the CD56(-/weak) malignant plasma cell disorders present or develop a leukemic phase vs only 15% of CD56+ cases (P < 0.008). CD56(-/weak) MM subset is also associated with a significantly less aggressive osteolytic potential (P=0.012). We conclude that the lack or weak expression of CD56 is a characteristic feature of PCL but also delineates a special subset of MM at diagnosis mainly characterized by a lower osteolytic potential and a trend for malignant plasma cells to circulate in the peripheral blood more overtly.     

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells

Ex vivo expanded bone marrow CD34+DR- cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1alpha (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR- cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61 + megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR- cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR- expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR- cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1alpha had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR- cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.