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Biomedical subjects

R Henschler

Publications and source records attributed to R Henschler.

At least 19 recordsLinked to original sources
PubMed2005

Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

Blood Cells↗
PubMed2004

Plasma quality after whole-blood filtration depends on storage temperature and filter type.

The study evaluated the quality of plasma obtained after whole-blood filtration with four different polyester filters and one polyurethane filter. The activities of coagulation factors and proteinase inhibitors were not or only negligibly affected by filtration, in all experiments. Filtration did not increase markers of clotting and fibrinolysis. Only a strong neutrophil and complement activation was observed, which depended on the type of filter and whole-blood storage conditions. However, as neutrophil elastase-specific degradation products did not increase and the complement-derived anaphylatoxin C3a was found in its inactivated form, C3a-desArg, these filtration-dependent changes apparently have little impact on the therapeutic quality of whole-blood-filtered fresh frozen plasma for transfusion.

Antithrombins↗
PubMed2004

A mouse model to study organ homing behaviour of haemopoietic progenitor cells reveals high selectivity but low efficiency of multipotent progenitors to home into haemopoietic organs.

To study the homing behaviour of an enriched multipotent primitive haemopoietic progenitor cell (HPC) population in mice, undifferentiated murine factor-dependent multipotent HPCs (FDCP-mix), stably transfected with the green fluorescence protein gene, were intravenously injected into congenic mice. After 2 or 24 h, cell suspensions were prepared from bone marrow, spleen, lung, liver, muscle, colon, kidney, brain or blood of the mice and analysed by flow cytometry. Using direct quantifiable determination of total HPC numbers homed per organ and a method to estimate the degree of organ contamination by HPC that were present in blood vessels within the organs before preparation, the highest absolute numbers of HPC were detected in the liver and lungs at 2 h but this was sharply decreased at 24 h, whereas HPC selectively accumulated in the bone marrow and spleen at 24 h after transplantation. Only a few HPC were detected in other organs. The seeding efficiency of homed FDCP-mix HPC to the bone marrow and spleen was approximately 1.5% and ranged between that of primary whole bone marrow cells and lineage-depleted freshly isolated bone marrow cells. Pretreatment of HPC with inhibitors of signal transduction indicated that short-term homing of multipotent HPC into haemopoietic organs is an active process requiring co-ordinated intracellular signalling through Rho family small GTPases and protein kinases. Thus, short-term homing of FDCP-mix HPC into haemopoietic organs is of low efficiency but high selectivity, and provides a system to analyse the mechanisms and manipulation of primitive HPC which saves large numbers of donor animals.

Animals↗
PubMed2002

Rho family small GTPases control migration of hematopoietic progenitor cells into multicellular spheroids of bone marrow stroma cells.

Seeding of hematopoietic progenitor cells (HPC) into the bone marrow requires a complex interaction between cell membrane and adhesion systems and cell signaling pathways. We established a multicellular, spheroid coculture model to study HPC migration in a three-dimensional stromal environment. Here, entry of primary CD34(+) cells into stroma cell spheroids was independent of the integrins very late antigen (VLA)-4, VLA-5, lymphocyte function-associated antigen-1, and the chemokine receptor CXCR4. Experiments using a panel of bacterial toxins selectively targeting key regulators of cellular locomotion, the Rho family small GTPases Rho, Rac, and Cdc42, revealed a considerable reduction or even abrogation of TF-1 cell migration without an increase of apoptosis or impairment of proliferation. Pertussis toxin, an inhibitor of Galpha(i) proteins, showed a similar effect. In some in vitro invasion assays, phosphatidylinositol-3 kinase (PI-3K) was shown to mediate Rac- and Cdc42-induced cell motility and invasion. However, inhibition of the PI-3K pathway by LY294002 did not impair TF-1 cell migration in our three-dimensional model system.

Bacterial Proteins↗
PubMed2001

Proliferation and differentiation of murine haemopoietic progenitor cells in stroma-free culture in the presence of metabolites of chlorinated pesticides.

We have studied the influence of metabolites of chlorinated pesticides (lindane, pentachlorophenol, hexachlorobenzene) on proliferation and differentiation in two stroma-free murine bone marrow culture models, a multipotent progenitor cell line (FDCP-mix) and primary lineage-depleted bone marrow cells. Tetrachlorohydroquinone (Cl(4)pHQ), tetrachloro-p-benzoquinone (Cl(4p)BQ), but not their positional isomers, tetrachlorocatechol (Cl(4)oHQ) and tetrachloro-o-benzoquinone (Cl(4)oBQ), nor 2,4,6-trichlorophenol (2,4,6-Cl(3)P), were much more toxic to FDCP-mix cells cultured under conditions which lead to self-renewal than under conditions which lead to granulocyte-macrophage differentiation. Under the latter conditions, Cl(4)pHQ and Cl(4p)BQ even stimulated growth at intermediate concentration levels. In the primary cell cultures, pronounced differences were observed in the sensitivity between individual developmental pathways and between the different compounds. The percent of cells differentiating into the granulocytic lineage was increased at high concentration levels of each test compound. However, stimulatory effects on the macrophage lineage were observed at intermediate concentration levels of Cl(4)pHQ, Cl(4p)BQ and 2,4,6-Cl(3)P, and differentiation into erythrocytes was stimulated at low concentrations of 2,4,6-Cl(3)P. It is concluded that chlorinated monocyclic pesticides, after biotransformation to quinoid metabolites, may interact directly with haemopoietic progenitor cells with differential effects on self-renewal and differentiation. These mechanisms could lead to myeloplastic disorders.

Animals↗
PubMed2001

Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy.

Ex vivo culture of hematopoietic progenitor cells for autologous transplantation has generated world-wide interest, since it offers the prospect of using a limited cell number, and may allow more efficient gene transfer and passive elimination of contaminating tumor cells. In this study, we expanded bone marrow (BM) cells from 10 breast cancer patients to determine whether small BM aliquots can durably restore hematopoiesis, and whether thrombopoietin (TPO) improves hematopoietic reconstitution after myeloablative chemotherapy. We used the AastromReplicell System (ARS), performing a computer-controlled, stromal-based cell expansion process with frequent medium, cytokine and gas exchange. For the inoculation of 9 x 10(8) MNC, a median BM volume of 97.8 ml (range, 72.4-272) was harvested. We found a median 4.5-fold nucleated cell expansion, an 18-fold CFU-GM expansion, and 69% of input LTC-IC numbers. Nucleated and Lin-/CD34+ cells were infused with a median of 43.5 x 10(6)/kg (range, 34.1-71.7) and 2.8 x 10(5)/kg (range, 0.95-5.9), respectively. Despite tumor cell detection by immunocytochemical staining in 3/10 patients before expansion, tumor cells were not detectable in 9/10, and in one patient 1 log reduced post ARS culture. Following high-dose STAMP V chemotherapy, all patients received 12-day expanded BM cells. The median time to engraftment was 17 days (range, 11-20) for WBC >1000/microl, and 28 days (range, 21-55) for platelets >20,000/microl. A correlation between post-expansion Lin-/CD34+ cells and engraftment for ANC >500/microl, WBC >1000/microl and platelets >20,000/microl was observed. Hematopoiesis has been maintained for a median of 15 (range, 6-24) months. Our results demonstrate that transplantation of ex vivo expanded small BM aliquots allows hematopoietic reconstitution after myeloablative chemotherapy. Ex vivo generated ARS cells can reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which only small stem cell doses are available, eg when using cord blood transplants or in non-mobilizing patients.

Adult↗
PubMed2000

Donor stromal cells from human blood engraft in NOD/SCID mice.

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)

Animals↗
PubMed2000

Interleukin-6 and interleukin-11 act synergistically with thrombopoietin and stem cell factor to modulate ex vivo expansion of human CD41+ and CD61+ megakaryocytic cells.

BACKGROUND AND OBJECTIVE: Thrombopoietin (TPO), the ligand for the c-mpl receptor, regulates in vivo platelet production and increases the number of colony-forming unit megakaryocytes (CFU-MK). Other cytokines including interleukin (IL) -3, IL-6, IL-11 and stem cell factor (SCF) can stimulate megakaryopoiesis. The aim of this study was to evaluate the effects of different combinations of cytokines involved in megakaryocytopoiesis on stroma-free liquid cultures of purified human CD34+ cells. DESIGN AND METHODS: Peripheral blood cells were collected after mobilization with granulocyte colony-stimulating factor (G-CSF). Purified CD34+ cells were then cultured with different combinations of TPO, SCF, IL-3, IL-6 and IL-11. RESULTS: The addition of TPO and SCF alone generated a population positive for the antigens CD41 (5.5+/-2.9%) and CD61 (6. 1+/-2.2%) but induced a low amplification of cell number (8.1+/-0.9 fold expansion). The presence of IL-6 or IL-11 was associated with MK progenitor cell expansion, and up to 7-10% of cultured cells were found to be CD41 and CD61 positive by flow cytometry. Conversely, the addition of IL-3 to this cytokine combination was associated with a prominent expansion of the myeloid lineage (70+/-10% of CD33+ cells) but only 0.9% and 2% of cultured cells were positive for CD61 and CD41 respectively. INTERPRETATION AND CONCLUSIONS: Our study supports the idea that IL-6 and IL-11 play crucial roles in the proliferation of MK progenitors and the use of SCF, TPO, IL-6 and IL-11 for ex vivo expansion of this cell population.

Antigens, CD↗
PubMed1999

Upstream elements bestow T-cell and haemopoietic progenitor-specific activity on the granzyme B promoter.

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

Animals↗
PubMed1999

Proliferation and survival of mammary carcinoma cells are influenced by culture conditions used for ex vivo expansion of CD34(+) blood progenitor cells.

Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34(+) blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1beta (IL-1beta), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34(+) BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-beta1 (TGF-beta1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1beta + IL-3 + IL-6 + EPO, but was suppressed by TGF-beta1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 x 10(5)/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34(+) BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34(+) BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.

Antigens, CD34↗
PubMed1999

Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.

Animals↗
PubMed1998

Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells.

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.

Animals↗
PubMed1998

Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.

Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.

Animals↗
PubMed1998

Purging of mammary carcinoma cells during ex vivo culture of CD34+ hematopoietic progenitor cells with recombinant immunotoxins.

Tumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncated Pseudomonas exotoxin A fragment devoid of its cell-binding domain. CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting. Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures. In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti-Erb-B2 IT. This included elimination of the subpopulations with regrowth potential. Similarly, addition of either anti-Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.

ADP Ribose Transferases↗
PubMed1998

Minimal residual disease in autologous hematopoietic harvests from breast cancer patients.

The increasing use of high-dose chemotherapy with autologous hematopoietic transplantation for the treatment of solid malignancies has raised concern about the role of tumor cells contaminating the grafts. Minimal residual disease (MRD) in autologous grafts has became a dynamic and intensively studied field in oncology. This review discusses the current status of MRD in breast cancer autografts and presents existing data on detection methodology, clinical relevance, biologic characteristics and purging techniques.

Antineoplastic Agents↗