Biomedical subjects

H J Bühring

Publications and source records attributed to H J Bühring.

At least 19 recordsLinked to original sources

PubMed2006

The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis.

BACKGROUND: The chemokine stromal derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 have been demonstrated to be crucial for the homing of stem cells and prostate cancers to the marrow. While screening prostate cancers for CXCL12-responsive adhesion molecules, we identified CD164 (MGC-24) as a potential regulator of homing. CD164 is known to function as a receptor that regulates stem cell localization to the bone marrow. RESULTS: Using prostate cancer cell lines, it was demonstrated that CXCL12 induced both the expression of CD164 mRNA and protein. Functional studies demonstrated that blocking CD164 on prostate cancer cell lines reduced the ability of these cells to adhere to human bone marrow endothelial cells, and invade into extracellular matrices. Human tissue microarrays stained for CD164 demonstrated a positive correlation with prostate-specific antigen levels, while its expression was negatively correlated with the expression of androgen receptor. CONCLUSION: Our findings suggest that CD164 may participate in the localization of prostate cancer cells to the marrow and is further evidence that tumor metastasis and hematopoietic stem cell trafficking may involve similar processes.

Bone Marrow Neoplasms↗

PubMed2006

Upregulation of frizzled 9 in astrocytomas.

Wnt/frizzled (FZD) cascades play important roles in controlling cell fate, proliferation, migration, tissue architecture and organogenesis during embryonic development and in adult organisms. The potential involvement of this pathway in tumorigenesis has been established in several types of cancers. Frizzled 9 (FZD9) is expressed in brain and its aberrant expression in gastric cancer was observed. However, its association with astrocytomas remains unknown therefore we studied FZD9 expression in astrocytomas of different malignancy. In the present study, FZD9 expression in 25 astrocytomas was investigated using immunohistochemistry with specific antibodies. Further FZD9 expression in native human brain tissue and glioblastoma cell line were analysed using real-time reverse transcription polymerase chain reaction (RT-PCR). In human astrocytomas, FZD9 immunoreactivity (IR) was observed in both microvessels and neoplastic cells. The percentage of FZD9+ microvessels in relation to FZD9+ vessels was significantly higher in malignant astrocytomas than in low-grade astrocytomas and positively correlated with the astrocytoma World Health Organization (WHO) grading (r = 1, P = 0.04). Furthermore, the FZD9 IR scores positively correlated with astrocytoma WHO grading (r = 1, P = 0.04) and proliferating activity (r = 0.77, P < 0.001). Real-time RT-PCR data showed that FZD9 expression in human glioblastoma was significant higher than in normal brain (P < 0.05) but FZD9 expression was only slightly induced in cobalt chloride-treated human glioblastoma T98G cells compared with untreated cells (P > 0.05). FZD9 is upregulated in astrocytomas, suggesting that FZD9 could be important in the tumorigenesis of human astrocytomas.

Adult↗

PubMed2001

The epithelial tumor antigen MUC1 is expressed in hematological malignancies and is recognized by MUC1-specific cytotoxic T-lymphocytes.

The epithelial mucin MUC1 is overexpressed on the cell surface of many epithelial malignancies as well as on some B-cell lymphomas and multiple myelomas. Recently, we identified two HLA-A2-restricted T-cell epitopes derived from the MUC1 protein. To further extend the potential application of these peptides, we analyzed the expression of MUC1 on blast cells from patients with acute myelogenous leukemia (AML; n = 43) and several other hematological malignancies including acute lymphoblastic leukemia (n = 24), chronic lymphocytic leukemia (n = 36), hairy cell leukemia (n = 9), follicular lymphoma (n = 7), and multiple myeloma (n = 12). Using reverse transcription-PCR and MUC1-specific monoclonal antibodies, MUC1 expression was found in 67% of AML samples and 92% of myeloma samples. To analyze the presentation of MUC1 peptides by primary AML blasts, we induced MUC1-specific CTLs in vitro using peptide-pulsed dendritic cells from HLA-A2+ healthy donors as antigen-presenting cells. These CTLs efficiently lysed in an antigen-specific and HLA-A2-restricted manner not only target cells pulsed with the antigenic peptide but also tumor cell lines including multiple myeloma cells and primary AML blasts that constitutively expressed both MUC1 and HLA-A2. The specificity of the CTLs was confirmed in a cold target inhibition assay. Our data demonstrate that MUC1-derived peptides are tumor antigens in AML and several other hematological malignancies that could potentially be used for immunotherapeutic approaches.

Antibodies, Monoclonal↗

PubMed2001

The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3.

It has recently been shown that monoclonal antibody (MoAb) 97A6 detects a surface antigen expressed on basophils and their CD34(+) precursor cells, as well as the basophil cell line KU-812. In this report the partial amino acid sequence of affinity chromatography- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated 97A6 antigen(s) from KU-812 lysates was determined. Sequence alignment of high-performance liquid chromatography-selected tryptic peptides from the resulting 130- and 150-kd bands revealed a 100% identity with amino acids 393 to 405 of ectonucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3; CD203c) but not of the related ectoenzyme PC-1 (E-NPP1). Moreover, MoAb 97A6 selectively recognized 293 cells transfected with human E-NPP3, but did not react with cells transfected with PC-1 or parental 293 cells. In addition, E-NPP3 messenger RNA expression was detected in basophils but not other peripheral blood cells. Finally, MoAb 97A6 immunoprecipitated phosphodiesterase activity from KU-812 cells and peripheral blood basophils, but not from other cell populations. These data demonstrate that MoAb 97A6 recognizes the functionally active type II transmembrane ectoenzyme E-NPP3.

Amino Acid Sequence↗

PubMed2001

Signal-regulatory protein alpha (SIRPalpha) but not SIRPbeta is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34(+)CD38(-) hematopoietic cells.

Signal-regulatory proteins (SIRPs) represent a new family of inhibitory/activating receptor pairs. They consist of 3 highly homologous immunoglobulin (Ig)-like domains in their extracellular regions, but differ in their cytoplasmic regions by the presence (SIRPalpha) or absence (SIRPbeta) of immunoreceptor tyrosine-based inhibitory motifs (ITIMs). To analyze the differential expression on hematopoietic cells, function and ligand binding capacity of SIRPalpha and SIRPbeta molecules, soluble fusion proteins consisting of the extracellular domains of SIRPalpha1, SIRPalpha2, and SIRPbeta1, as well as SIRPalpha/beta-specific and SIRPbeta-specific monoclonal antibodies (MoAbs) were generated. In contrast to SIRPalpha1 and SIRPalpha2, no adhesion of SIRPbeta1 to CD47 could be detected by cell attachment assays and flow cytometry. Using deletion constructs of SIRPalpha1, the epitope responsible for SIRPalpha1 binding to CD47 could be confined to the N-terminal Ig-like loop. Flow cytometry analysis with SIRPalpha/beta- and SIRPbeta-specific MoAbs revealed that SIRPalpha but not SIRPbeta is expressed on CD34(+)CD38(-) hematopoietic cells. In addition, a strong SIRPalpha expression was also observed on primary myeloid dendritic cells (DCs) from peripheral blood as well as on in vitro generated DCs. Analysis of the T-cell stimulatory capacity of in vitro generated DCs in the presence of soluble SIRPalpha1 fusion proteins as well as SIRPalpha/beta-specific and CD47-specific MoAbs revealed a significant reduction of T-cell proliferation in mixed lymphocyte reaction and inhibition of induction of primary T-cell responses under these conditions. In contrast, soluble SIRPalpha or SIRPbeta-specific antibodies had no effect. The data suggest that the interaction of SIRPalpha with CD47 plays an important role during T-cell activation and induction of antigen-specific cytotoxic T-lymphocyte responses by DCs.

ADP-ribosyl Cyclase↗

PubMed2001

Cutting edge: characterization of allorestricted and peptide-selective alloreactive T cells using HLA-tetramer selection.

The vast majority of alloreactive T cells recognize foreign MHC molecules in a peptide-dependent manner. A subpopulation of these peptide-dependent alloreactive T cells is peptide-specific and contains T cells that are of interest for tumor immunotherapy. Allorestricted T cells (i.e., peptide-specific and alloreactive) specific for tumor-associated Ags can be raised in vitro. However, it is technically difficult to distinguish between peptide-specific and peptide-nonspecific alloreactive T cells by functional assays in vitro. Here we show for the first time that allorestricted T cells specifically bind HLA-peptide tetrameric complexes, as nominal Ag-specific T cells would do. In consequence, fluorescent HLA-peptide tetrameric complexes can be used for sorting and cloning of allorestricted CTLs specific for a peptide of interest. We also show by the mean of HLA-peptide tetramers the existence of peptide-selective alloreactive T cells that recognize a conformation on the foreign-MHC brought about by some but not all peptides bound.

Cell Line↗

PubMed2001

Variable expression of activation-linked surface antigens on human mast cells in health and disease.

Mast cells (MC) are multipotent effector cells of the immune system. They contain an array of biologically active mediator substances in their granules. MC also express a number of functionally important cell surface antigens, including stem cell factor receptor (SCFR=kit=CD117), high affinity IgER (FcepsilonRI), or CSaR (CD88). Respective ligands can induce or promote degranulation, migration, or cytokine production. Other integral surface molecules can mediate adhesion or cell aggregation. Recent data suggest that a number of critical molecules are variably expressed on the surface of human MC. In fact, depending on the environment (organ), stage of cell maturation, type of disease, and other factors, MC express variable amounts of activation-linked antigens (CD25, CD63, CD69, CD88), cell recognition molecules (CD2, CD11, CD18, CD50, CD54), or cytokine receptors. At present, however, little is known about the mechanisms and regulation of expression of such antigens. The present article gives an overview of MC phenotypes in health and disease, and attempts to provide explanations for the phenotypic variability of MC.

Animals↗

PubMed2001

Quantitative, phenotypic, and functional evaluation of basophils in myelodysplastic syndromes.

BACKGROUND: The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders characterized by cytopenia(s), reduced differentiation-capacity of myeloid cells, and impaired leukocyte function. However, little is known so far about basophil granulocytes in MDS. DESIGN: We have compared the numbers, phenotype and function of basophils in MDS patients with those in healthy subjects. A total numer of 23 patients with MDS (refractory anaemia, n = 8; refractory anaemia with ringsideroblasts, n = 7; refractory anaemia with excess of blasts/refractory anaemia with excess of blasts in transformation, n = 8) and 20 healthy donors were included. RESULTS: The numbers of blood basophils in MDS patients (34.6 +/- 62.9 microL-1) was lower compared to healthy controls (58.6 +/- 64.9 microL-1). Correspondingly, whole blood histamine levels were lower in MDS patients (MDS 34.1 +/- 29.1 ng mL-1 vs. normal donors 72.0 +/- 36.9 ng mL-1). Like "normal" basophils, basophils in MDS expressed interleukin-3 receptor alpha (CD123), E-NPP3 (CD203c), CR1 (CD35), CR3 (CD11b), CR4 (CD11c), membrane co-factor protein (CD46), decay-accelerating factor (CD55) and membrane attack complex inhibitory factor (CD59), as well as receptors for C3a, C5a (CD88), and IgE. Recombinant human (rh) C5a and anti-IgE induced significant release of histamine from basophils in both groups of donors without significant differences between MDS and healthy controls. CONCLUSIONS: The absolute numbers of basophils in MDS patients are lower than in normal donors. However, basophils in MDS do not differ from their "normal counterparts" in terms of complement receptor expression, IgE-receptor expression, or functional responses to respective ligands.

Aged↗

PubMed2001

Immunohistochemical properties of bone marrow mast cells in systemic mastocytosis: evidence for expression of CD2, CD117/Kit, and bcl-x(L).

In an attempt to identify novel diagnostic markers for mast cell (MC)-proliferative disorders, serial bone marrow (bm) sections of 22 patients with mastocytosis (systemic indolent mastocytosis, n = 19; mast cell leukemia [MCL], n = 1; isolated bm mastocytosis, n = 2) were analyzed by immunohistochemistry using antibodies against CD2, CD15, CD29, CD30, CD31, CD34, CD45, CD51, CD56, CD68R, CD117, HLA-DR, bcl-2, bcl-x(L), myeloperoxidase (MPO), and tryptase. Staining results revealed expression of bcl-x(L), CD68R, and tryptase in neoplastic MCs (focal dense infiltrates) in all patients. Mastocytosis infiltrates were also immunoreactive for CD45, CD117 (Kit), and HLA-DR. In most cases, the CD2 antibody produced reactivity with bm MCs in mastocytosis, whereas in control cases (reactive bm, immunocytoma, myelodysplastic syndrome), MCs were consistently CD2 negative. Expression of bcl-2 was detectable in a subset of MCs in the patient with MCL, whereas no reactivity was seen in patients with SIM or bm mastocytosis. Mastocytosis infiltrates did not react with antibodies against CD15, CD30, CD31, CD34, or MPO. In summary, our data confirm the diagnostic value of staining for tryptase, Kit, and CD68R in mastocytosis. Apart from these, CD2 may be a novel useful marker because MCs in mastocytosis frequently express this antigen, whereas MCs in other pathologic conditions are CD2 negative.

Adolescent↗

PubMed2001

The monoclonal antibody W7C5 defines a novel surface antigen on hematopoietic stem cells.

We recently raised a monoclonal antibody, termed W7C5, against a surface antigen that is expressed at low levels on bone marrow and peripheral blood CD34+ stem/progenitor cells but at high levels on fetal liver CD34+ cells. A reasonable staining intensity was achieved using magnetofluorescent liposome conjugates to analyze expression of W7C5 antigen on CD34+CD38- bone marrow (BM) cells. Flow cytometric analyses revealed that W7C5 detects about 50% of immature CD34+CD38- BM cells that coexpressed the differentiation antigens CD164, CD133, and CD172a (SIRP alpha). In addition, W7C5 also recognized a CD34- BM fraction. These cells were negative for CD117 and CD133, but expressed CD45 and moderate levels of CD164. Injection of selected CD34+W7C5+ and CD34-W7C5+ cells into 55-60-day-old fetal sheep resulted in an engraftment of both fractions. Partial amino acid sequence analysis of affinity-purified lysates of KU-812 cells revealed that W7C5 detects a novel membrane protein. Together, W7C5 defines a novel molecule that is expressed on CD34+ as well as on CD34- stem cell subsets.

Animals↗

PubMed2001

Dendritic cell-based vaccines in patients with hematological malignancies.

The epithelial mucin MUC1 is overexpressed on many epithelial malignancies as well as on some B-cell lymphomas and multiple myelomas. In the present study, we described MUC1 expression also on primary AML blasts. To analyze the presentation of MUC1-derived HLA-A2 restricted peptides by primary AML blasts, we induced MUC1-specific cytotoxic T-lymphocytes (CTLs) in vitro using peptide pulsed dendritic cells from HLA-A2+ healthy donors as antigen-presenting cells. These CTLs efficiently lysed primary AML blasts that constitutively expressed both MUC1 and HLA-A2. The specificity of the CTLs was confirmed in a cold target inhibition assay. Our data demonstrate that MUC1-derived peptides are tumor antigens in AML which could potentially be used for immunotherapeutic approaches.

Acute Disease↗

PubMed2001

Hymenoptera-venom-induced upregulation of the basophil activation marker ecto-nucleotide pyrophosphatase/phosphodiesterase 3 in sensitized individuals.

BACKGROUND: Bee and wasp venoms are potent allergens capable of inducing severe clinical reactions. To detect immediate-type hypersensitivity to these allergens, a rapid in vitro test was developed that relies on the upregulation of ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) on activated basophils. METHODS: Blood basophils of 13 healthy donors and 22 patients allergic to bee or wasp venom were analyzed for E-NPP3 (CD203c) expression using monoclonal antibody 97A6. Basophils were analyzed by flow cytometry after activation with anti-IgE antibody or allergen. Venom-induced E-NPP3 upregulation on basophils was compared with diagnostic parameters, including skin tests and assessment of specific IgE. In selected samples, the increase in E-NPP3 expression on activated basophils was compared with histamine release and CD63 upregulation. RESULTS: In 20/22 patients sensitized to wasp or bee venom, E-NPP3 expression on basophils was upregulated in response to activation by allergen or anti-IgE. The maximum increase in E-NPP3 expression (above ten times of baseline) was achieved after 15 min of stimulation with 1 microg/ml of allergen or anti-IgE antibody. Sensitized individuals who failed to upregulate E-NPP3 in response to IgE receptor cross-linking also failed to induce histamine release and CD63 upregulation. CONCLUSIONS: Flow cytometric determination of hymenoptera-venom-induced upregulation of E-NPP3 is a novel in vitro test to identify sensitized individuals.

Adult↗

PubMed2000

CD164 monoclonal antibodies that block hemopoietic progenitor cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor.

The novel sialomucin, CD164, functions as both an adhesion receptor on human CD34+ cell subsets in bone marrow and as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation. These diverse effects are mediated by at least two functional epitopes defined by the mAbs, 103B2/9E10 and 105A5. We report here the precise epitope mapping of these mAbs together with that of two other CD164 mAbs, N6B6 and 67D2. Using newly defined CD164 splice variants and a set of soluble recombinant chimeric proteins encoded by exons 1-6 of the CD164 gene, we demonstrate that the 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164. The N6B6 and 67D2 mAbs, in contrast, recognize closely associated and complex epitopes that rely on the conformational integrity of the CD164 molecule and encompass the cysteine-rich regions encoded by exons 2 and 3. On the basis of their sensitivities to reducing agents and to sialidase, O-sialoglycoprotease, and N-glycanase treatments, we have characterized CD164 epitopes and grouped them into three classes by analogy with CD34 epitope classification. The class I 105A5 epitope is sialidase, O-glycosidase, and O-sialoglycoprotease sensitive; the class II 103B2/9E10 epitope is N-glycanase, O-glycosidase, and O-sialoglycoprotease sensitive; and the class III N6B6 and 67D2 epitopes are not removed by such enzyme treatments. Collectively, this study indicates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type specific post-translational modifications and suggests a role for epitope-associated carbohydrate structures in CD164 function.

Alternative Splicing↗

PubMed2000

Functionally defined CD164 epitopes are expressed on CD34(+) cells throughout ontogeny but display distinct distribution patterns in adult hematopoietic and nonhematopoietic tissues.

Three distinct classes of epitopes on human CD164 have been identified. Two of these, recognized by the monoclonal antibodies 105A5 and 103B2/9E10, are the CD164 class I and class II functionally defined epitopes, which cooperate to regulate adhesion and proliferation of CD34(+) cell subsets. In this article, we demonstrate that these 2 CD164 epitopes are expressed on CD34(+) cells throughout ontogeny, in particular on CD34(+ )cell clusters associated with the ventral floor of the dorsal aorta in the developing embryo and on CD34(+) hematopoietic precursor cells in fetal liver, cord blood, and adult bone marrow. While higher levels of expression of these CD164 epitopes occur on the more primitive AC133(hi)CD34(hi)CD38(lo/-) cell population, they also occur on most cord blood Lin(-)CD34(lo/-)CD38(lo/- )cells, which are potential precursors for the AC133(hi)CD34(hi)CD38(lo/-) subset. In direct contrast to these common patterns of expression on hematopoietic precursor cells, notable differences in expression of the CD164 epitopes were observed in postnatal lymphoid and nonhematopoietic tissues, with the class I and class II CD164 epitopes generally exhibiting differential and often reciprocal cellular distribution patterns. This is particularly striking in the colon, where infiltrating lymphoid cells are CD164 class I-positive but class II-negative, while epithelia are weakly CD164 class II-positive. Similarly, in certain lymphoid tissues, high endothelial venules and basal and subcapsular epithelia are CD164 class II-positive, while lymphoid cells are CD164 class I-positive. It therefore seems highly likely that these CD164 class I and II epitopes will mediate reciprocal homing functions in these tissue types.

Adult↗

PubMed2000

Cutting edge: signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells.

Signal-regulatory proteins (SIRPs) are cell-surface glycoproteins expressed on myeloid and neural cells that have been shown to recruit SH2 domain-containing protein phosphatase 1 (SHP-1) and SHP-2 and to regulate receptor tyrosine kinase-coupled signaling. One SIRP of unknown function, designated SIRP beta 1, contains a short cytoplasmic domain that lacks sequence motifs capable of recruiting SHP-1 and SHP-2. Using a SIRP-specific mAb, we show that SIRP beta 1 is expressed in monocytes and dendritic cells and associates with the signal transduction molecule DAP12. SIRP beta 1/DAP12 complex formation was required for efficient cell-surface expression of SIRP beta 1. Stimulation of this complex induced tyrosine phosphorylation, mitogen-activated protein kinase activation, and cellular activation. Thus, SIRP beta 1 is a new DAP12-associated receptor involved in the activation of myeloid cells.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Association of signal-regulatory proteins beta with KARAP/DAP-12.

The signal-regulatory proteins (SIRP) are Ig-like cell surface receptors detected in hematopoietic and non-hematopoietic cells. SIRP are classified as SIRPalpha molecules, containing a 110- to 113-amino acid long, or SIRPbeta molecules, with a 5-amino acid long intracytoplasmic domain. SIRPalpha molecules belong to inhibitory immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing molecules. The majority of ITIM-bearing receptors are paired with activating isoforms, which share highly related extracytoplasmic domains but harbor a shorter cytoplasmic domain devoid of ITIM and contain a charged amino acid residue in their transmembrane domain. Activating receptors are associated with immunoreceptor tyrosine-based activation motif (ITAM)-bearing proteins, such as KARAP/DAP-12 and FcRgamma. In this report, we show that human SIRPbeta1 is included in an oligomeric complex with KARAP/DAP-12 in hematopoietic and non-hematopoietic transfectant cells as well as in human monocytes. The physical association between SIRPbeta1 and KARAP/DAP-12 results in the functional coupling of SIRPbeta1 engagement to the recruitment of the protein tyrosine kinase Syk and to serotonin release in RBL cell transfectants. Therefore our results show that SIRPbeta1 acts as an activating isoform of SIRPalpha molecules, confirming the co-existence of inhibitory ITIM-bearing molecules, recruiting SHP-1 and SHP-2 protein tyrosine phosphatases, and activating counterparts, whose engagement couples to protein tyrosine kinases via ITAM-bearing molecules.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Ex vivo manipulation of hematopoietic stem and progenitor cells.

Ex vivo manipulations of hematopoietic stem and progenitor cells are Increasingly used in the context of autologous and allogeneic stem-cell transplantation. These manipulations include the positive selection of CD34+ cells for tumor-cell reduction and/or T-cell depletion, the ex vivo expansion of hematopoietic progenitor and stem cells under appropriate cytokine-stimulated culture conditions, and the ex vivo generation of myeloid or megakaryocytic postprogenitor cells and Immune effector cells. This article summarizes both the preclinical data on the ex vivo expansion of hematopoietic progenitor and stem cells from purified CD34+ cells and the Initial clinical studies with ex vivo-expanded stem and progenitor cells for hematopoietic support after high-dose chemotherapy.

Animals↗

PubMed2000

Effective ex vivo generation of granulopoietic postprogenitor cells from mobilized peripheral blood CD34(+) cells.

OBJECTIVE: Neutropenia following high-dose chemotherapy and peripheral blood progenitor cell (PBPC) transplantation might be abrogated by an additional transplantation of ex vivo generated granulopoietic postprogenitor cells (GPPC). Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for optimum GPPC production. MATERIALS AND METHODS: CD34(+) PBPC were cultured in serum-free medium comparing different (n = 32) combinations of stem cell factor (S), interleukin 1 (1), interleukin 3 (IL-3) (3), interleukin-6 (6), erythropoietin (E), granulocyte colony-stimulating factor (G), granulate-macrophage colony-stimulating factor (GM), daniplestim (D, a novel IL-3 receptor agonist), and Flt3 ligand (FL) under various culture conditions. Ex vivo generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. RESULTS: Addition of G +/- GM but not GM alone to cultures stimulated with S163E effectively induced the generation of GPPC. GPPC production was maximum after 12 to 14 days. Best expansion rates were observed when cells were cultured at 1.5x10(4)/mL in 21% O(2). Modifications of culture conditions were either less or equally effective (i.e., modification of starting cell concentrations, low oxygen, addition of serum albumin or autologous plasma, repetitive feeding). Comparison of different cytokine combinations revealed that the optimum GPPC expansion cocktail consisted of S6GD+FL (day 12: 130-fold cellular expansion, 32% myeloblasts/promyelocytes, 49.4% myelocytes/metamyelocytes, 12.4% bands/segmented), which furthermore expanded CD34(+) cells (3.4-fold) and clonogenic progenitors (13.4-fold). CONCLUSION: Using the S6DG+FL expansion cocktail, GPPC could be effectively produced ex vivo starting from positively selected CD34 PBPC, possibly enabling amelioration or even abrogation of posttransplant neutropenia.

Adjuvants, Immunologic↗