Biomedical subjects

Zwi N Berneman

Publications and source records attributed to Zwi N Berneman.

At least 19 recordsLinked to original sources

PubMed2006

Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells.

BACKGROUND: Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity. RESULTS: In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711-20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-gamma ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-gamma-secreting CD8+ T cells in 5/5 healthy donors. CONCLUSION: We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Antigens, Viral, Tumor↗

PubMed2006

Gene expression signatures in CD34+-progenitor-derived dendritic cells exposed to the chemical contact allergen nickel sulfate.

The detection of the sensitizing potential of chemicals is of great importance to industry. A promising in vitro alternative to the currently applied animal assays for sensitization testing makes use of dendritic cells (DCs) that have the capability to process and present antigens to naive T cells and induce their proliferation. Here, we studied changes in gene expression profiles after exposing DCs to the contact allergen nickel sulfate. CD34+-progenitor-derived DCs, initiated from 3 different donors, were exposed to 60 microM nickel sulfate, during 0.5, 1, 3, 6, 12 and 24 h. cDNA microarrays were used to assess the transcriptional activity of about 11,000 genes. Significant changes in the expression of 283 genes were observed; 178 genes were up-regulated and 93 down-regulated. These genes were involved in metabolism, cell structure, immune response, transcription, signal transduction, transport, and apoptosis. No functional information was found for 74 genes. Real-time RT-PCR was used to confirm the microarray results of 12 genes. In addition, 3 DC maturation markers not present on the microarrays (DEC205, DC LAMP and CCR7) were analyzed using real-time RT-PCR and found to be up-regulated at several time points. Our data indicate that a broad range of biological processes is influenced by nickel. Some processes are clearly linked to the immune response and DC maturation, others may indicate a toxic effect of nickel.

Allergens↗

PubMed2005

Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins.

Infection with human immunodeficiency virus type 1 (HIV-1) is characterized by dysfunction of HIV-1-specific T cells. To control the virus, antigen-loaded dendritic cells (DCs) might be useful to boost and broaden HIV-specific T-cell responses. In the present study, monocyte-derived DCs from nontreated HIV-1-seropositive patients were electroporated with codon-optimized ("humanized") mRNA encoding consensus HxB-2 (hHXB-2) Gag protein. These DCs elicited a strong HIV-1 Gag-specific interferon-gamma (IFN-gamma) response by an HLA-A2-restricted CD8+ T-cell line. Moreover, hHXB-2 gag mRNA-electroporated DCs also triggered IFN-gamma secretion by autologous peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD8+ T cells from all patients tested. Next, a novel strategy was developed using autologous virus sequences. Significant specific IFN-gamma T-cell responses were induced in all patients tested by DCs electroporated with patients' autologous polymerase chain reaction (PCR)-amplified and in vitro-transcribed proviral and plasma viral mRNA encoding either Gag or Env. The stimulatory effect was seen on PBMCs, CD8+ T cells, and CD4+ T cells, demonstrating both major histocompatibility complex (MHC) class I and MHC class II antigen presentation. Moreover, a significant interleukin-2 (IL-2) T-cell response was induced by DCs electroporated with hHxB-2 or proviral gag mRNA. These findings open a major perspective for the development of patient-specific immunotherapy for HIV-1 disease.

Adoptive Transfer↗

PubMed2005

Synaptopodin and 4 novel genes identified in primary sensory neurons.

We performed differential gene expression profiling in the peripheral nervous system by comparing the transcriptome of sensory neurons with the transcriptome of lower motor neurons. Using suppression subtractive cDNA hybridization, we identified 5 anonymous transcripts with a predominant expression in sensory neurons. We determined the gene structures and predicted the functional protein domains. The 4930579P15Rik gene encodes for a novel inhibitor of protein phosphatase-1 and 9030217H17Rik was found to be the mouse gene synaptopodin. We performed in situ hybridization for all genes in mouse embryos, and found expression predominantly in the primary class of sensory neurons. Expression of 4930579P15Rik and synaptopodin was restricted to craniospinal sensory ganglia. Neither synaptopodin, nor any known family member of 4930579P15Rik, has ever been described in sensory neurons. The identification of protein domains and expression patterns allows further functional analysis of these novel genes in relation to the development and biology of sensory neurons.

Animals↗

PubMed2005

Apoptosis: mechanisms and relevance in cancer.

Apoptosis or programmed cell death is a process with typical morphological characteristics including plasma membrane blebbing, cell shrinkage, chromatin condensation and fragmentation. A family of cystein-dependent aspartate-directed proteases, called caspases, is responsible for the proteolytic cleavage of cellular proteins leading to the characteristic apoptotic features, e.g. cleavage of caspase-activated DNase resulting in internucleosomal DNA fragmentation. Currently, two pathways for activating caspases have been studied in detail. One starts with ligation of a death ligand to its transmembrane death receptor, followed by recruitment and activation of caspases in the death-inducing signalling complex. The second pathway involves the participation of mitochondria, which release caspase-activating proteins into the cytosol, thereby forming the apoptosome where caspases will bind and become activated. In addition, two other apoptotic pathways are emerging: endoplasmic reticulum stress-induced apoptosis and caspase-independent apoptosis. Naturally occurring cell death plays a critical role in many normal processes like foetal development and tissue homeostasis. Dysregulation of apoptosis contributes to many diseases, including cancer. On the other hand, apoptosis-regulating proteins also provide targets for drug discovery and new approaches to the treatment of cancer.

Animals↗

PubMed2005

Capacity of CD34+ progenitor-derived dendritic cells to distinguish between sensitizers and irritants.

In an attempt to develop an in vitro test to identify contact sensitizers, mostly dendritic cells (DCs) derived from monocytes (Mo-DC) have been used. Less is known about the potency of DC derived from CD34+ progenitors (CD34-DC) for in vitro allergen testing. CD34+ progenitor derived DC were exposed to nine well-known allergens (one weak, three moderate and five strong allergens) and two irritants. Surface marker expression (CD86, CD83 and HLA-DR) and cytokine production (IL-6, IL-12 and TNF-alpha) were analyzed after 24 h exposure to these chemicals. All allergens tested induced a significant increase in at least one of the DC surface markers. In contrast, none of the irritants tested were able to significantly upregulate membrane marker expression in exposed DC. The level of upregulation of CD86, CD83 and HLA-DR was dependent on the nature and concentration of the chemical, but not on the classification of the allergen. Changes in cytokine production (IL-6, IL-12 and TNF-alpha) were not consistently related to exposure to an allergen. Based on these results, we conclude that the in vitro test using CD34-DC has the capacity to distinguish between allergens and irritants based on altered phenotypic characteristics.

Allergens↗

PubMed2005

Cytokine transcript profiling in CD34+-progenitor derived dendritic cells exposed to contact allergens and irritants.

We here investigated wether genes encoding the interleukins IL-1beta, IL-6 and IL-8, and the chemokines CCL2, CCL3, CCL3L1 and CCL4 are useful markers for sensitization testing in CD34+-progenitor derived dendritic cells (CD34-DC). CD34-DC from at least three donors were exposed during 0.5 up to 24h to the chemical sensitizers nickel sulphate, oxazolone, 2,4-dinitrochlorobenzene (DNCB) and eugenol, and to the irritants sodium dodecyl sulphate (SDS) and benzalkonium sulphate (BC). mRNA expression was evaluated using real-time RT-PCR. We observed a large inter-individual variation in mRNA expression in CD34-DC exposed to the chemicals. No or limited effects on expression were observed for the irritant BC and the weak sensitizer eugenol. All other chemicals modulated the transcript levels of most cytokines that were investigated. Most of the time, no clear-cut distinctions could be made between the sensitizers and SDS. After 24 h, consistent upregulatory effects of all sensitizing compounds on transcript expression of CCL2, CCL3 and CCL4 were observed, whereas SDS (and BC) had no effect. Our findings suggest that the CCL2, CCL3 and CCL4 genes may be selective end-point markers in the CD34-DC model to discern chemical sensitizers from irritants.

Allergens↗

PubMed2004

Pathophysiology and treatment of platelet-mediated microvascular disturbances, major thrombosis and bleeding complications in essential thrombocythaemia and polycythaemia vera.

Essential thrombocythaemia (ET) is associated with a broad spectrum of microvascular circulation disturbances including erythromelalgia and its ischaemic complications, episodic neurological symptoms of atypical and typical transient ischaemic attacks (TIAs), transient ocular ischaemic attacks, acute coronary syndromes, and superficial 'thrombophlebitis'. The microvascular circulation disturbances are caused by spontaneous activation and aggregation of hypersensitive thrombocythaemic platelets at high shear stress in the endarterial microcirculation involving the peripheral, cerebral and coronary circulation. As this microvascular syndrome is a pathognomonic feature of essential thrombocythaemia and of thrombocythaemia associated with polycythaemia vera (PV) in complete remission with normal haematocrit, we have labelled these two variants of thrombocythaemia as thrombocythaemia vera. The arterial thrombophilia of microvascular circulation disturbances in thrombocythaemia vera already occur at platelet counts in excess of 400 x 10(9)/l. Complete relief of microvascular circulation disturbances in thrombocythaemia vera is obtained with the platelet cyclooxygenase inhibitor aspirin 50-100 mg/day, but not with dipyridamole, ticlopidine, coumarin or heparin. Haemorrhagic thrombocythaemia (HT) is a clinical syndrome of recurrent spontaneous mucocutaneous and secondary haemorrhages associated with extremely high platelet counts far in excess of 1000 x 10(9)/l. The paradoxical occurrence of microvascular circulation disturbances and mucocutaneous bleeding is usually seen at platelet counts between 1000 and 2000 x 10(9)/l. At increasing platelet counts from below 1000 to in excess of 2000 x 10(9)/l, the arterial thrombophilia of thrombocythaemia vera changes into a spontaneous bleeding tendency of HT as a consequence of platelet-mediated increased proteolysis of the large von Willebrand factor multimers leading to a type 2 acquired von Willebrand syndrome. As PV is usually associated with thrombocythaemia, the vascular complications in PV patients are microvascular circulation disturbances typical of thrombocythaemia. On top of this, major arterial and venous thrombotic events and haemorrhages are related to increased haematocrit, red cell mass and its concomitant increased blood viscosity. Correction of increased blood viscosity and haematocrit to normal values (0.40-0.44) by bloodletting alone will significantly reduce the risk of major thrombotic complications, but does not prevent the microvascular circulation disturbances because thrombocythaemia persists. The microvascular syndrome associated with thrombocythaemia in PV patients in remission after bloodletting is best controlled by low-dose aspirin (50-100 mg/day) or by reduction of platelet count to normal (< 350 x 10(9)/l).

Blood Platelets↗

PubMed2004

Highly efficient mRNA-based gene transfer in feeder-free cultured H9 human embryonic stem cells.

Several protocols have been described for virus-based gene transfer in human embryonic stem (hES) cells, while efficient non-viral methods are currently non-existing. In this study, we investigated the efficiency of mRNA-based gene transfer in feeder-free cultured H9 hES cells, based on electroporation of in vitro transcribed mRNA encoding the enhanced green fluorescent protein (EGFP). Optimisation of culture and electroporation conditions for feeder-free cultured H9 hES cells resulted a highly pure, transgene-expressing (90% positive cells) H9 hES cell population.

Cell Survival↗

PubMed2004

Bleeding prophylaxis for major surgery in patients with type 2 von Willebrand disease with an intermediate purity factor VIII-von Willebrand factor concentrate (Haemate-P).

The parameters to diagnose von Willebrand disease (vWD) include factor VIII coagulant activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), von Willebrand factor ristocetin cofactor activity (vWF:RCo), and von Willebrand factor collagen binding activity (vWF:CB). Type 2 vWD is associated with a moderate bleeding diathesis due to low levels of vWF:RCo and vWF:CB as compared with near normal or normal values for FVIII:C and vWF:Ag. As the factor VIII/von Willebrand factor (vWF) concentrate, Haemate-P, is featured by a vWF:RCo/FVIII:C ratio of about 2.2, the recommended loading dose of 50 U/kg FVIII:C followed by 25 U/kg FVIII:C every 12 h for several days for bleeding prophylaxis in type 2 vWD patients undergoing major surgery demonstrated a predicted significant over-treatment reaching vWF:RCo levels above 2 U/ml. Therefore, we restricted Haemate-P substitution for major surgery to one loading dose of 40-50 U/kg FVIII:C (88-110 U/kg vWF:RCo) followed by 15-20 U/kg FVIII:C (33-44 U/kg vWF:RCo) every 12 h for several days and evaluated this strategy in a prospective pharmacokinetic and efficacy study for bleeding prophylaxis in five type 2 vWD patients. Pre-treatment and peak levels (1 h after Haemate-P loading dose) rose from 0.43-0.66 to 1.5-2.5 U/ml for FVIII:C, from 0.23-0.45 to 1.5-2.5 U/ml for vWF:Ag, from 0.10-< 0.20 to 1.5-2.5 U/ml for vWF:RCo, and from < 0.05-0.10 to 1.0-2.0 U/ml for vWF:CB. Mean in vivo recoveries per transfused IU FVIII:C/kg body weight were 3.2% for FVIII:C, 3.9% for vWF:RCo, and 2.8% for vWF:CB. Mean in vivo recoveries per transfused IU vWF:RCo/kg were 1.45% for FVIII:C, 1.7% for vWF:RCo and 1.25% for vWF:CB. The biological half-life times after transfused Haemate-P were about 12 h for both vWF:RCo and vWF:CB. Based on these pharmacokinetic data, we propose to adapt the loading dose factor VIII/vWF concentrate (Haemate-P) to 60-80 U/kg vWF:RCo followed by 30-40 U/kg vWF:RCo every 12 h for no longer than several days (less than 1 week) for bleeding prophylaxis during major surgery or trauma, and to one loading dose of 40-60 U/kg vWF:RCo for minor surgery, trauma or mucotaneous bleedings in patients with type 2 vWD unresponsive to DDAVP.

Adult↗

PubMed2003

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA.

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.

Electroporation↗

PubMed2003

A novel alternative spliced chondrolectin isoform lacking the transmembrane domain is expressed during T cell maturation.

Chondrolectin (CHODL) is a novel type I transmembrane protein containing one carbohydrate recognition domain (CRD) of C-type lectins. Recently, data base searching revealed a variant of CHODL (AK022689) with a different 5' leader sequence derived from a new putative upstream alternative promoter (P2). The P2 promoter gives rise to at least three additional alternatively spliced isoforms, designated as CHODLf, CHODLf Delta E, and CHODL Delta E. Of all variants, the alternative exon E-splicing isoforms (CHODLf Delta E/CHODL Delta E) are expressed exclusively in the T lymphocyte lineage and are regulated during T lymphopoiesis. Peripheral T lymphocytes demonstrated a unique exon E-splicing pattern in comparison with end maturation stage thymocytes, suggesting its association with the post-thymic maturation of T cells. Since exon E encodes the transmembrane domain of CHODL, the exon E-skipping variant results in a non-transmembrane domain-containing isoform (CHODLf Delta E/CHODL Delta E) terminating in the QDEL sequence, thus suggesting different functional attributes of CHODL isoforms during the development of T cells. Double label immunofluoresence experiments demonstrated that the transmembrane-containing isoform (CHODLf) colocalizes with rBet1 to the endoplasmic reticulum-Golgi apparatus. In summary, this study describes the molecular characterization of novel members of the chondrolectin family associated with T cell maturation and a subcellular localization of CHODLf in the endoplasmic reticulum-Golgi apparatus.

Alternative Splicing↗

PubMed2003

Comparison of turbidimetric aggregation and in vitro bleeding time (PFA-100) for monitoring the platelet inhibitory profile of antiplatelet agents in patients undergoing stent implantation.

The present study compared classical ADP-induced platelet aggregation vs. PFA-100 closure times using collagen/ADP membrane cartridges to monitor the degree of platelet-inhibiting effect of three drug regimens: ticlopidin, abciximab/ticlopidin and loading dose clopidogrel, each on top of aspirin (acetylsalicylic acid, ASA) during and after elective stent placement (intervention) in a total of 31 patients with acute coronary syndrome. Ticlopidin was started directly after stent implantation, abciximab was started before coronary intervention and given intravenously for 12 h, and a clopidogrel loading dose was given before intervention. The 10 patients treated with ticlopidin (500 mg daily) showed no significant prolongation of PFA closure times and a slight increase of ADP-induced platelet aggregation shortly after intervention. In 11 patients treated with abciximab/ticlopidin, the PFA closure times were significantly prolonged, and ADP-induced platelet aggregation was reduced by more than 80% during the 12-h abciximab infusion after intervention. The 10 patients pretreated with loading dose clopidogrel (450 mg followed by 75 mg daily) showed an intermediate but significant prolongation of PFA closure times and reduction of ADP-induced platelet aggregation at levels between the ticlopidin/aspirin- and the abciximab/ticlopidin/aspirin-treated groups. At 20 h after intervention, a similar degree of PFA closure time prolongation and inhibition of ADP-induced aggregation was observed in the abciximab/ticlopidin/aspirin- and the clopidogrel/aspirin-treated patient groups. Both measurement of PFA-100 closure times and inhibition of ADP-induced platelet aggregation showed a similar degree of platelet inhibition, but had rather broad SD ranges, which limit their precision for the follow-up of individual patients. In conclusion, abciximab on top of ticlopidin/aspirin showed a stronger antiplatelet effect for only less than 20 h, as compared to loading dose clopidogrel/aspirin in acute coronary syndrome patients undergoing stent implantation. Whether such a short-term superiority of abciximab, as compared to loading dose clopidogrel, translates into an overall clinical benefit of thombotic and bleeding complications remains to be established in a randomized clinical trial.

Abciximab↗

PubMed2003

The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer.

The cell cycle is controlled by numerous mechanisms ensuring correct cell division. This review will focus on these mechanisms, i.e. regulation of cyclin-dependent kinases (CDK) by cyclins, CDK inhibitors and phosphorylating events. The quality checkpoints activated after DNA damage are also discussed. The complexity of the regulation of the cell cycle is also reflected in the different alterations leading to aberrant cell proliferation and development of cancer. Consequently, targeting the cell cycle in general and CDK in particular presents unique opportunities for drug discovery. This review provides an overview of deregulation of the cell cycle in cancer. Different families of known CDK inhibitors acting by ATP competition are also discussed. Currently, at least three compounds with CDK inhibitory activity (flavopiridol, UCN-01, roscovitine) have entered clinical trials.

Animals↗

PubMed2003

Cell cycle and apoptosis.

Apoptosis and proliferation are intimately coupled. Some cell cycle regulators can influence both cell division and programmed cell death. The linkage of cell cycle and apoptosis has been recognized for c-Myc, p53, pRb, Ras, PKA, PKC, Bcl-2, NF-kappa B, CDK, cyclins and CKI. This review summarizes the different functions of the proteins presently known to control both apoptosis and cell cycle progression. These proteins can influence apoptosis or proliferation but different variables, including cell type, cellular environment and genetic background, make it difficult to predict the outcome of cell proliferation, cell cycle arrest or cell death. These important decisions of cell proliferation or cell death are likely to be controlled by more than one signal and are necessary to ensure a proper cellular response.

Animals↗

PubMed2002

Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells.

Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.

Antigens↗

PubMed2002

Phenotypic alterations and IL-1beta production in CD34(+) progenitor- and monocyte-derived dendritic cells after exposure to allergens: a comparative analysis.

Dendritic cells (DC) have been shown to capture and process antigens and play an initiating role in contact sensitization. Cells with dendritic morphology can be generated in vitro either from CD34(+) cord blood cells or from CD14(+) peripheral monocytes. The aim of this study was to determine the state of maturation/activation of both populations after exposure to several concentrations of four well-established model allergens (nickel sulfate, eugenol, alpha-hexylcinnamaldehyde and 2,4,6-trinitrobenzene sulfonic acid) or the irritant sodium dodecyl sulfate. We analyzed the surface expression of CD86, CD83 and HLA-DR and the production of IL-1beta. DC from the two sources were generated separately in two laboratories, but challenged using identical test protocols. Using both DC populations it was possible to detect the allergens under investigation, though minor differences regarding effective concentrations were noted. The non-responsiveness of CD34-DC to CIN was probably due to non-optimal concentrations. Ni(2+), known as a moderate allergen in vivo, showed the most prominent effect in both cell systems. CD86 expression was the most reliable phenotypic marker for the in vitro identification of allergens. Due to substantial individual variations it was difficult to draw any definite conclusions as to the relevance of IL-1beta production as an activation endpoint. We conclude that both test systems are able to respond to allergens, but CD34-DC must be exposed to higher concentrations to demonstrate significant phenotypic changes. On the other hand, Mo-DC from only some of the donors reacted to allergens, in contrast to CD34-DC, which responded to allergens irrespective of the donor, thus necessitating the use of Mo-DC cultures from several blood donors.

Allergens↗

PubMed2002

Extracellular nucleoside diphosphate kinase NM23/NDPK modulates normal hematopoietic differentiation.

OBJECTIVE: We previously demonstrated the presence of nucleoside diphosphate kinase NDPK/NM23 in normal human plasma. It also was reported that extracellular NM23 could inhibit differentiation of certain hematopoietic cell lines. We further investigated the extracellular effect of NM23 on hematopoiesis by adding recombinant NM23-H1, NM23-H2, and NM23-H3 proteins to in vitro differentiation assays of normal human hematopoietic progenitors. MATERIALS AND METHODS: To study the effect on the earlier stages of hematopoietic maturation, NM23 was added to serum-free pre-colony-forming unit (pre-CFU) assays starting from immature CD34++CD38- bone marrow cells. Serum-free CFU assays starting from CD34+ CD38+ bone marrow cells were used as a model for terminal hematopoietic differentiation. RESULTS: In pre-CFU assays, none of the NM23 isoforms used significantly changed the expansion of CD34++CD38- cells, nor did NM23 alter the CD34++ CD38- cell lineage commitment. In contrast, terminal differentiation of CD34+CD38+ progenitor cells in CFU assays was significantly altered by addition of NM23 protein. More erythroid burst-forming units and fewer macrophage colonies were observed in cultures containing any of the NM23 isoforms examined. Similar effects were observed using the enzymatically inactive H118N mutant of NM23-H1, strongly suggesting that the observed effect is independent of the nucleoside diphosphate kinase activity of NM23. CONCLUSION: We demonstrated a modulating effect of extracellular NM23 proteins on the terminal stages of normal hematopoietic differentiation. Therefore, the fairly high concentrations of NM23 constitutively present in plasma could have a physiologic role in supporting erythropoiesis and inhibiting excessive macrophage formation.

Antigens, CD34↗