Biomedical subjects

S Fruehauf

Publications and source records attributed to S Fruehauf.

45 records · Page 3Linked to original sources

PubMed1996

Granulocytes harvested following G-CSF-enhanced leukocyte recovery retain their functional capacity during in vitro culture for 72 hours.

The purpose of this study was to compare two different in vitro culture conditions for the preservation of human granulocytes. These cells could be used in patients with severe neutropenia following cytotoxic chemotherapy if the functional capacity was retained, and autologous transfusions of granulocytes would circumvent the risk of alloimmunization. Granulocytes were obtained from the peripheral blood of healthy donors and patients with hematologic malignancies who received cytotoxic chemotherapy supported by recombinant human granulocyte colony-stimulating factor (R-metHuG-CSF, 300 micrograms/day, s.c.). Granulocytes were either cultured for 72 h at 4 degrees C in the presence of 100 ng/ml G-CSF or cryopreserved at -196 degrees C. The viability, surface antigen expression, and function of the granulocytes were assessed. Since effective microbial killing involves the attachment of granulocytes to blood vessel walls, transmigration into tissues, chemotaxis, and phagocytosis, the surface expression of the adhesion molecules LFA-1 (CD11a/CD18) and gp 150,95 (CD11c/CD18) was measured. In addition, the IgG receptors Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), as well as the complement receptor CR3 (CD11b/CD18), were assessed. Dynamic superoxide anion release served as a measure of the metabolic pathway of the oxidative burst after f-Met-Leu-Phe (fMLP) and phorbol-12-myristate-13-acetate (PMA) stimulation. Substantial differences in the preservation of granulocyte integrity and function were observed between the two storage conditions. Cryopreservation abolished reactivity to extracellular stimuli and severely affected the cell phenotype. On the other hand, functional activity could be maintained for up to 72 h when in vivo primed granulocytes of patients were incubated at 4 degrees C in the presence of G-CSF. This storage modality may permit the use of granulocyte autotransfusion to reduce the risk of neutropenic fever.

Antigens, Surface↗

PubMed1996

Human-mouse xenografts in stem cell research.

New progenitor cell transplantation strategies that change the composition of the graft, such as CD34+ cell selection, ex vivo expansion, and gene marking, are budding. The efficiency and safety of most techniques are evaluated by in vitro assays using human progenitor cells and murine intraspecies transplantation studies before clinical introduction. However, proliferation potential in culture and engraftment capability can be discrepant. Furthermore, some CD34 epitopes and cytokines are unique to humans, thus rendering clinical inferences from experimental results difficult. Therapeutic studies with malignant human hematopoietic cells also require appropriate models that take into account pharmacokinetics. Human-mouse interspecies progenitor cell grafts may allow us to bridge this gap. For engraftment of human cells, recipients need to be immunodeficient. The highest long-term engraftment rate of up to 96% was obtained following transplantation of peripheral blood progenitor cells into non-obese diabetic/severe combined immunodeficiency mice. Data obtained from several human-mouse xenograft transplantation models are presented and discussed.

Animals↗

PubMed1995

Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support.

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non-Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty-four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst-forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.

Adolescent↗

PubMed1995

Peripheral blood progenitor cell (PBPC) counts during steady-state hematopoiesis allow to estimate the yield of mobilized PBPC after filgrastim (R-metHuG-CSF)-supported cytotoxic chemotherapy.

Peripheral blood progenitor cells (PBPC) can be mobilized using cytotoxic chemotherapy and cytokines. There is a substantial variability in the yield of hematopoietic progenitor cells between patients. We were looking for predictive parameters indicating a patient's response to a given mobilization regimen. Multiparameter flow-cytometry analysis and clonogenic assays were used to examine the hematopoietic progenitor cells in bone marrow (BM) and peripheral blood (PB) before filgrastim (R-metHuG-CSF; Amgen, Thousand Oaks, CA)-supported chemotherapy and in PB and leukapheresis products (LPs) in the recovery phase. Fifteen patients (four with high-grade non-Hodgkin's lymphoma [NHL], two with low-grade NHL, two with Hodgkin's disease, two with multiple myeloma, three with breast cancer, one with ovarian cancer, and one with germ cell tumor) were included in this study. The comparison of immunofluorescence plots showed a homogenous population of strongly CD34+ cells in steady-state and mobilized PB whereas in steady-state BM, the CD34+ cells ranged from strongly positive with continuous transition to the CD34- population. Consistent with the similarity in CD34 antigen expression, a correlation analysis showed steady-state PB CD34+ cells (r = .81, P < .001) and colony-forming cells (CFCs; r = .69, P < .01) to be a measure of a patient's mobilizable CD34+ cell pool. Individual estimates of progenitor cell yields could be calculated. With a probability of 95%, eg, 0.4 steady-state PB CD34+ cells x 10(6)/L allowed to collect in six LPs 2.5 x 10(6) CD34+ cells/kg, the reported threshold-dose of progenitor cells required for rapid and sustained engraftment after high-dose therapy. For the total steady-state BM CD34+ cell population, a weak correlation (r = .57, P < .05) with the mobilized CD34+ cells only became apparent when an outlier was removed from the analysis. Neither the CD34+ immunologic subgroups defined by the coexpression of the myeloid lineage-associated antigens CD33 or CD45-RA or the phenotypically primitive CD34+/HLA-DR- subset nor the BM CFC count had a predictive value for the mobilization outcome. This may be caused by the additional presence of maturing progenitor cells in BM, which express lower levels of the CD34 antigen and do not circulate. Our results permit us to recognize patients who are at risk to collect low numbers of progenitor cells and those who are likely to achieve sufficient or high progenitor cell yields even before mobilization chemotherapy is administered.

Adult↗

PubMed1995

Blood-derived autografts collected during granulocyte colony-stimulating factor-enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells.

It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood-derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA-DR- cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony-forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM-derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy-related cytotoxic damage.

Adult↗

PubMed1995

Retroviral transfer of the multidrug resistance-1 gene into lineage-committed and primitive hemopoietic cells.

Transfer of the multidrug resistance-1 (MDR1) gene to hemopoietic cells for myeloprotection against cytostatic agents is a new and rapidly developing field in "cancer gene therapy." Before clinical application, safety and efficacy criteria need to be met. The retroviral producer cell lines and the retroviral supernatant need to be tested for replication-competent retrovirus and contamination with adventitious agents. The cell source needs to contain sufficient hemopoietic cells with repopulating ability. We used CD34(+)-selected mobilized peripheral blood progenitor cells (PBPC) for MDR1 transductions in order to obtain a favorable vector to target cell ratio. An analysis of 249 patients who had undergone PBPC harvesting revealed that primarily solid tumor and non-Hodgkin's lymphoma patients are eligible for CD34+ selection. They can be expected to retain sufficient CD34+ cells for rapid and sustained engraftment after myeloablative therapy if the CD34+ cell loss (approximately 50%) during the procedure is taken into account. Clinical MDR1 gene therapy protocols focus on these two patient groups. Next we characterized MDR1 gene transfer into lineage-committed and primitive hemopoietic cells. Provirus-specific polymerase chain reactions showed a high efficiency gene transfer into colony-forming-units granulocyte-macrophage and long-term culture cells. The level of the conferred P-glycoprotein expression was estimated by fluorescence-activated cell sorting analysis to be up to 3 log above mock-transduced controls. The cobblestone area forming cell assay, which is a stroma-dependent long-term culture assay measuring frequencies of stem cell subsets in a limiting-dilution set-up, allowed demonstration of sustained expression of the MDR1 gene in the progeny of primitive hemopoietic cells. This is a favorable basis for a clinical MDR1 gene therapy trial.

Drug Resistance, Multiple↗

PubMed1994

CD34 selection for purging in multiple myeloma and analysis of CD34+ B cell precursors.

Selection of CD34+ hematopoietic progenitor cells from autografts may be performed in multiple myeloma (MM) to minimize contamination with tumor cells. This approach is based on the assumption that the malignant cells do not express the CD34 antigen. Therefore, we first compared the CD34+/CD10+ and CD34+/CD19+ subpopulations from bone marrow (BM) and peripheral blood (PB) of fourteen MM patients and five normal controls. No difference between the respective early B cell subsets of both groups could be observed. Using tricolor flow cytometry, the CD19 expression on CD34+/CD10+ cells in BM was found to increase continuously from CD19- to CD19dim. In contrast, circulating CD34+/CD10+ cells did not coexpress the CD19 antigen. This population may contain myeloid progenitor cells or bipotential progenitor cells of the myeloid and lymphoid lineage as suggested by data obtained with fetal liver cells. Further functional studies are required. Enrichment of CD34+ cells with immunomagnetic beads was performed from BM of three MM patients and four normal donors. The CD34+ cells were selected with the HPCA-1 antibody and detached from the beads with chymopapain. Compared with the starting cell preparation, a 3.97 +/- 0.48 log (mean +/- SE) reduction of plasma cells could be achieved after CD34 selection. On morphological examination, 84% +/- 4% of the cells in the CD34+ fraction (MM) were immature blasts. The plating efficiency for hematopoietic colony forming cells was 9.7% +/- 2.8% in the CD34 selected fraction of the MM group, reflecting a 51-fold increase as compared with the starting population.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗

PubMed1994

Filgrastim post-chemotherapy mobilizes more CD34+ cells with a different antigenic profile compared with use during steady-state hematopoiesis.

For the mobilization of CD34+ peripheral blood progenitor cells (PBPC) filgrastim (R-metHuG-CSF) can be administered either during steady-state hematopoiesis or following cytotoxic chemotherapy. We compared both mobilization modalities intra-individually in seven patients with breast cancer. The number of circulating CD34+ cells was increased after filgrastim-supported chemotherapy compared with filgrastim administration during steady-state (on average, 129 vs. 19/microliters), resulting in a sevenfold higher yield of CD34+ cells per leukapheresis (5.73 vs. 0.79 x 10(6)/kg bodyweight). CD34+ PBPC harvested post-chemotherapy comprised a smaller proportion of early progenitor cells (CD34+/HLA-DR- or CD34+/CD38-) compared with filgrastim treatment alone. However, the absolute number of these early progenitor cells harvested was fivefold higher. The filgrastim-supported rebound after chemotherapy was characterized by a greater proportion of CD34+/CD33+ cells. Correspondingly, CD34+ PBPC mobilized post-chemotherapy contained a higher proportion of CFU-GM compared with filgrastim treatment during steady-state, while the cloning efficiency of CD34+ cells for BFU-E tended to be lower. Of note, the proportion of CD34+/CD19+ lymphoid progenitor and B cells was reduced after chemotherapy. In cancer patients, filgrastim mobilizes higher numbers of CD34+ cells when administered post-chemotherapy, compensating for the smaller proportion of early hematopoietic progenitors.

Adult↗

PubMed1993

Sustained remission of idiopathic hypereosinophilic syndrome following alpha-interferon therapy.

Two patients with idiopathic hypereosinophilic syndrome (HES) refractory to treatment with corticosteroids and hydroxyurea received alpha-interferon (alpha-IFN) for 3 and 1 years, respectively. Eosinophil counts dropped below 1.5 x 10(9)/l at weekly doses of 2-7 x 3 million units alpha-IFN. More than a year after discontinuation of alpha-IFN, 1 patient remains in a stable remission. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plasma levels were found to be normal in this patient, although GM-CSF is known to stimulate eosinophil proliferation in vitro. Based on the favorable clinical results of alpha-IFN, further studies are necessary to define its role in the treatment of HES.

Adult↗