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At least 1,009 records · Page 56Linked to original sources
PubMed2000

Osteoprotegerin ligand regulates osteoclast adherence to the bone surface in mouse calvaria.

The stimulators of bone resorption, prostaglandin E(2) (PGE(2)) and 1,25-dihydroxyvitamin D(3) (1,25D(3)), act through osteoblast-like cells to activate osteoclasts. One candidate for the intermediary produced by osteoblasts that subsequently stimulates the osteoclast is osteoprotegerin ligand (OPGL). OPGL has been shown to stimulate osteoclast differentiation and activation. The aim of the work reported here was to determine if soluble recombinant extracellular domain of human OPGL would bring about the change in osteoclast adhesion from the periosteum of mouse calvaria to the adjacent bone surface that occurs with the above-mentioned stimulators of resorption. This change in adherence or translocation of osteoclasts onto the bone surface required the expression and functioning of the integrin subunit, beta 3. We show that this soluble OPGL, like PGE(2) and 1,25D(3), stimulated the release of osteoclasts from the periosteum and their adherence to the bone surface accompanied by an increase in staining for immunolocalized integrin subunit beta 3. Recombinant human osteoprotegerin (OPG), which binds strongly to OPGL, inhibited this translocation of osteoclasts that occurred with PGE(2) and 1,25D(3), leaving integrin beta-3-negative osteoclasts on the periosteum. PGE(2) and 1,25D(3) increased the expression of messenger RNA for OPGL compared with indomethacin-treated controls after 6 h exposure. Evidence is presented that the change in the adhesion of osteoclasts from the periosteum to the bone surface, resulting in osteoclast activation, is mediated by OPGL.

Acid Phosphatase↗
PubMed1995

The P1A alloantigen system is a sensitive indicator of the structural integrity of the amino-terminal domain of the human integrin beta 3 subunit.

Within the native integrin alpha pi b beta 3, the conformation of the amino-terminal domain of the beta 3 subunit has a significant influence on the availability of the Leu33/Pro33 polymorphism that defines the A1 and A2 alleles, respectively, of the P1A alloantigen system. The majority of anti-P1A1 IgG antibodies, affinity-purified by adsorption to either (A1/A1)-platelets or purified alpha pi b(A1) beta 3, fail to bind to the Leu33 polymorphic loop within Cys26-Cys38 in native beta 3 unless this sequence is maintained in a proper orientation by two noncontiguous human beta 3 sequences. By comparing IgG binding to recombinant beta 3 molecules composed of segments of human, Xenopus and avian sequences expressed by Spodoptera frugiperda cell lines, we have found that neither region alone is sufficient for full expression of the epitope. One sequence, that most proximal to Leu33/Pro33, lies within beta 3[54-133], and a second distal sequence is located within beta 3[435-490]. Proximity of the distal sequence to the Cys26-Cys38 loop in native beta 3 is confirmed by the fact that both anti-P1A1 IgG and the murine monoclonal antibody SZ21, specific for beta 3[28-35], completely inhibit the binding of rabbit polyclonal IgG specific for the sequence beta 3[479-485]. Anti-P1A1 IgG antibodies from all of the 7 donors selectively bind to the epitope within the native conformation of beta 3. However, in one of the seven donors, roughly 60% of the anti-P1A1IgG binds exclusively to denatured (A1) beta 3, and not to either denatured (A2) beta 3 or nondenatured (native) (A1) beta 3. This IgG subpopulation cannot be purified by adsorption to intact (A1/A1)-platelets or to native alpha pi b(A1) beta 3 integrin. The physiologic relevance of this exceptional type of anti-P1A1 antibody is debatable, but the presence of IgG with this specificity only in an immunized subject suggests that the epitope may well be presented on a "denatured" form of beta 3 in vivo. Regardless, our results indicate that the amino-terminal domain of the native beta 3 molecule associates with noncontiguous beta 3 sequences, and the epitope recognized by a majority of anti-P1A1 antibodies is a sensitive indicator of correct tertiary structure within this region of beta 3.

Amino Acid Sequence↗
PubMed1997

The neurite-promoting effect of laminin is mediated by different mechanisms in embryonic and adult regenerating mouse optic axons in vitro.

Retinal explants from embryonic or adult mice were placed on laminin or merosin substrates and the outgrowth of optic fibers was assayed under serum-free conditions. Both substrates strongly promoted outgrowth. A blocking antibody to the beta1/beta3 integrin subunits completely blocked laminin-dependent growth of embryonic optic fibers but had no detectable effect on adult fibers. Similarly, a blocking antibody against the main neurite-promoting region within the globular domain of the E8 fragment of laminin inhibited growth of embryonic fibers but had no effect on adult optic fibers. The beta1 integrin subunit was identified immunohistochemically on both embryonic and adult fibers. These findings indicate that adult fibers have lost the beta1 function which dominates laminin-dependent growth in embryonic fibers but express a receptor for laminin-dependent growth that is not detectable in embryonic fibers. These findings suggest that there are intrinsic differences between embryonic and adult optic fibers that may have implications for regenerative failure in the central nervous system of adult mammals .

Age Factors↗
PubMed1999

Effects of mutations in the cytoplasmic domain of integrin beta(1) to talin binding and cell spreading.

Integrins are transmembrane proteins linking the extracellular matrix or certain cell-cell contacts to the cytoskeleton. To study integrin-cytoskeleton interactions we wanted to relate talin-integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin beta(1) (GST-cytobeta(1)) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of beta(3) linked to the cytoplasmic domain of beta(1) were expressed in CHO cells as a dimer with the alpha(IIb) subunit. Point mutations in the amino acid sequence N(785)PIY(788) of beta(1) disrupted both the integrin-talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of beta(1) at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of beta(1) (W(780)DTGENPIYKSAV(792)) bound to purified talin and inhibited talin binding to GST-cytobeta(1). The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin beta(1) tail resides at or close to the central NPXY motif and suggest that the integrin-talin interaction is necessary but not sufficient for integrin-mediated cell spreading.

3T3 Cells↗
PubMed1999

Extensive clear zone and defective ruffled border formation in osteoclasts of osteopetrotic (ia/ia) rats: implications for secretory function.

The cellular distribution of osteoclast integrin subunits alpha(v) and beta(3), the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits alpha(v) and beta(3) were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane. In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.

Acid Phosphatase↗
PubMed1999

Expression and function of beta(1) and beta(3) integrins of human mesothelial cells in vitro.

Mesothelial cells (MC) and extracellular matrix (ECM) components are thought to play a pivotal regulatory role during the inflammatory-reparative response of serosal membranes. Integrins are known to serve as cellular ECM receptors, but mesothelial integrin expression and its function, particularly its role for attachment to different ECM components, remain to be elucidated. The aim of the present study was to characterize the integrin expression of human omentum majus derived MC (HOMC) in vitro by immunohistochemistry and to investigate their functional significance with regard to HOMC adhesion to fibronectin (fn), vitronectin (vn), collagen IV (coll IV), and laminin (ln). Mesothelial cells in vitro strongly expressed beta(1), beta(3), alpha(2), alpha(3), alpha(5), and alpha(v) chains. A weak reactivity was found for alpha(1) and alpha(6), but no alpha(4) reactivity was detectable. Compared to the control, fn, vn, coll IV, and ln caused a significant 2.6-, 2.2-, 2-, and 1.6-fold increase of HOMC adhesion, respectively. Inhibition studies revealed that HOMC attachment to fn is mediated by alpha(5)beta(1), alpha(v)beta(1), and alpha(v)beta(3), with a synergistic effect of alpha(5)beta(1) and alpha(v)beta(3). Adhesion to vn is mediated by alpha(v)beta(1) and alpha(v)beta(3). Integrins alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) mediate adhesion to coll IV and ln. We suggest that the integrin expression and function of mesothelial cells described here play an important role in the interaction of MC with the ECM, particularly during the acute and chronic inflammatory-reparative response of serosal membranes.

Antigens, CD↗
PubMed1996

The human PECAM1 gene maps to 17q23.

We have determined the chromosomal and regional location of the gene encoding PECAM-1 (termed PECAM1 by GBD nomenclature) using a polymerase chain reaction (PCR)-based analysis of somatic cell hybrids. Analysis of a somatic cell hybrid chromosome panel established that the PECAM1 gene is on chromosome 17. Interestingly, several adhesion molecules expressed on platelets and endothelium also localize to chromosome 17: the GP1BA locus (glycoprotein (GP) Ibalpha) has been provisionally mapped to the region 17p12-pter, the ITGA2B (GPIIb) and the ITGB3 (GPIIIa) loci have been confirmed to the region 17q21.32; and the ICAM2 locus has been provisionally mapped to the region 17q23-q25. To determine if the PECAM1 locus colocalizes with any of the loci for these adhesion molecules, PCR-based analysis of a regional mapping panel for human chromosome 17 was conducted. We found that the PECAM1 locus is on the long arm of chromosome 17, in the region q23-qter. To confirm this observation and obtain a more precise localization of the PECAM1 locus, fluorescence in situ hybridization was conducted. Together our data allowed assignment of the PECAM1 locus to the region 17q23.

Animals↗
PubMed2001

Localization of matrix metalloproteinases on endometrial cancer cell invasion in vitro.

OBJECTIVES: In this study, we have established an in vitro three-dimensional (3D) coculture, where normal endometrial stromal cells and endometrial cancer cells were cocultured under defined hormonal conditions, to investigate the potential paracrine effects on synthesis and secretion of matrix metalloproteinases (MMPs) and, thus, cancer invasion. METHODS: Endometrial stromal cells were obtained by biopsy, cultured in the presence of 100 nM estrogen for 3 days, and then mixed with extracellular matrix (ECM) composed of collagen I and matrigel in a 4:1 ratio. After 3 more days in culture, a 3D coculture was established with HEC-1A cells, an endometrial adenocarcinoma cell line, grown on top of the mixture under various ovarian steroids (no steroid, 100 nM beta-estradiol (E2), or 1.0 microM progesterone (P4)) for 10 days. The expression and localization of MMP-2, MMP-9, and integrin beta 3 subunit were visualized by immunocytochemistry and analyzed by reverse transcription polymerase chain reaction (RT-PCR). The extent of cancer invasion was quantified by Boyden's chamber assay. RESULTS: Integrin beta 3 subunit was localized along the cell surface of HEC-1A cell under all three hormonal conditions. MMP-2 was located in the cytoplasm of stromal cells and on the surface of HEC-1A cells. Synthesis and secretion of stromal MMP-2 were increased in the presence of ovarian steroids. In contrast, expression of stromal MMP-9 was suppressed in the presence of ovarian steroids. No MMPs were synthesized in HEC-1A cells. Invasion assay revealed that HEC-1A cells achieved high tumoral invasiveness in the presence of beta-estradiol. CONCLUSIONS: These findings suggest that stromal cell-derived MMP-2 is translocated to the surface of HEC-1A cells. Integrin beta 3 subunit might contribute, in part, to providing a binding site for MMP-2. Thus, HEC-1A cells invade by recruiting MMP-2 secreted by stromal cells, which is greatly enhanced in the presence of beta-estradiol. The 3D coculture provides an excellent experimental system in which single parameters can be isolated from a complex in vivo system in the process of endometrial cancer invasion.

Adenocarcinoma↗
PubMed2001

A new genetic isolate of gray platelet syndrome (GPS): clinical, cellular, and hematologic characteristics.

Gray platelet syndrome (GPS) is a disorder characterized by thrombocytopenia and large platelets that lack alpha granules and their contents. We describe two siblings with GPS who are members of a Moslem Bedouin genetic isolate. The children, an 8-year-old girl and a 5-year-old boy, had characteristic pale blue platelets lacking alpha granules on electron microscopy. Platelet aggregation studies were normal. The girl underwent a bone marrow aspiration and biopsy which showed mild myelofibrosis and extensive emperipolesis, i.e., the passage of other hematopoietic cells through megakaryocytes. Both children lacked high-molecular-weight multimers of von Willebrand factor (vWF) and had reduced activity and antigens of vWf. Platelet activation was approximately normal when ADP was employed as agonist, but significantly impaired using the thrombin receptor-activating peptide (TRAP). These findings are explained in light of the mechanism of action of each agonist. In addition, we propose that the emperipolesis was caused by increased P-selectin in megakaryocytes, and resulted in release of fibroblastic growth factors, explaining the myelofibrosis. The detailed description of these cases provides a basis for future differentiation of the various types of GPS, and for our current attempts to isolate the gene causing GPS in this genetic isolate.

Adenosine Diphosphate↗
PubMed2001

Cellular receptors and hantavirus pathogenesis.

Hantaviruses cause two potentially lethal diseases, HPS and HFRS, and both diseases result in defects in vascular permeability and platelet function. Human beta 3 integrins confer cellular susceptibility to HPS- and HFRS-causing hantaviruses, a fact directly linking platelets, endothelial cells, and hantavirus diseases to the use of [figure: see text] cellular receptors that maintain capillary integrity and regulate platelet function. The role of vitronectin, PAI-1, uPAR, and complement cascades in hantavirus pathogenesis are unstudied but may contribute to specific disease syndromes effected by hantaviruses. The divergence of hantavirus surface glycoproteins and common beta 3-integrin usage provides further insight into the interaction of hantaviruses with cells. G1 and G2 glycoprotein variation is likely to contribute to additional interactions that determine pathogenic responses to individual viruses. beta 3-integrin usage also suggests that common elements exist on G1 or the more highly conserved G2 surface glycoprotein, which mediate viral attachment to integrins. Although there is currently no data defining the virion attachment protein, the development of antibodies that recognize the hantavirus attachment protein and block integrin interactions is of interest since it is likely to provide an additional point for therapeutic intervention and vaccine development. There are a plethora of effects that could be elicited by hantavirus regulation of cellular beta 3 integrins and their ligands that are consistent with hantavirus diseases. Since beta 3 integrins are critical adhesive receptors on platelets and endothelial cells and regulate both vascular permeability and platelet activation and adhesion, the use of these receptors by hantaviruses is likely to be fundamental to hantavirus pathogenesis. The lack of an animal model for hantavirus pathogenesis has prevented a systematic analysis of immune and cellular responses to hantavirus infections, and it impedes our ability to study protective or therapeutic approaches to hantavirus diseases. However, recent findings suggest that human beta 3 integrins within transgenic mice may provide animal models of hantavirus pathogenesis and have the potential to radically alter the ability to investigate hantavirus disease.

Animals↗
PubMedDate not supplied

Down-regulation of human NDR gene in megakaryocytic differentiation of erythroleukemia K562 cells.

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues. NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34+ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.

Adult↗
PubMed1995

Localization of the human homolog of the yeast cell division control 27 gene (CDC27) proximal to ITGB3 on human chromosome 17q21.3.

The human homolog of the Saccharomyces cerevisiae cell division control 27 gene (CDC27) was mapped to human chromosome 17q12-q21 using a panel of human/rodent somatic cell hybrids and localized distal to the breast cancer susceptibility gene, BRCA1, using a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of human CDC27 on human chromosomes 17 is between the marker D17S409 and the beta 3 subunit of integrin (ITGB3). Further confirmation of this localization comes from the sequence tagged site (STS) mapping of human CDC27 to the same yeast artificial chromosomes (YACs) positive for ITGB3. The estimated distance between ITGB3 and human CDC27 is less than 600 kb.

Animals↗
PubMed1999

The effect of in vitro activation and platelet interaction on the CD9 distribution and adhesion properties of human eosinophils.

OBJECTIVE AND DESIGN: The redistribution of CD9 in peripheral blood eosinophils was investigated with respect to the interaction with platelets during in vitro activation, and whether this interaction exerts influence on eosinophil adhesion properties. MATERIALS AND METHODS: Flow cytometry was used to investigate the CD9 expression in purified eosinophils or eosinophils from different whole blood preparations, with or without platelets present. To confirm an eosinophil/platelet interaction fluorescence microscopy was used, and to demonstrate release/shedding of CD9 molecules a biosensor technique was performed. RESULTS: Our results show that both intracellular and surface expression of CD9 decrease upon in vitro activation in the absence of platelets, a phenomenon probably caused by release/shedding of soluble forms of CD9 and not due to intracellular degradation. Increased expression of CD9 on eosinophils, stimulated in the presence of platelets, is partly a result of interacting platelets, judged by the increase in platelet specific marker CD61. In our adhesion assay a significant increase in eosinophil adhesion properties to fibronectin was obtained when eosinophils were PMA stimulated and interacting with platelets, as compared to activated eosinophils without platelets. CONCLUSIONS: Our findings, that CD9 expression on eosinophils is dynamically regulated, support our previous suggestion that CD9 may be a useful activity marker and that platelet interaction acts on eosinophil adhesion.

Adolescent↗
PubMed2000

Expression of platelet P-selectin and detection of soluble P-selectin, NPY and RANTES in patients with inflammatory bowel disease.

OBJECTIVE AND DESIGN: P-selectin, a membrane glycoprotein which is expressed on activated platelets and endothelial cells, plays a crucial role in the inflammatory response. The main action is adhesion of leukocytes, facilitation of diapedesis and induction of cytokine production from monocytes (MCP-1 and IL-8), mediated via RANTES released from activated platelets. An abnormal platelet activity has been reported in patients with ulcerative colitis (UC) and Crohn's disease (CD), jointly referred to as inflammatory bowel disease (IBD), which could have an aggravating influence on the inflammatory response. In addition, an up-regulation of platelet IL-8 receptors among patients with IBD has been reported. To reveal a presumptuous platelet dysfunction we analysed the expression of platelet surface P-selectin at resting state and after stimulation with thrombin, collagen, epinephrine and interleukin 8 (IL-8), and plasma levels of soluble P-selectin, neuropeptide Y (NPY) and RANTES in patients with IBD. SUBJECTS: Blood from twelve healthy subjects (control group) and twenty-one patients with IBD who had not taken any anti-platelet drugs or steroids were analysed. METHODS: Patients were sub-grouped according to disease entity, disease activity and 5ASA medication. Surface P-selectin expression on isolated human platelets and plasma P-selectin, NPY and RANTES were analysed with ELISA. All values are presented as mean +/- standard error of the mean (SEM). Mann-Whitney U test and Wilcoxon matched rank test were used for statistical analyses. RESULTS: Patients with IBD in remission (n = 9) had higher basal P-selectin expression, 0.38+/-0.04, compared to the control group (n = 12), 0.22+/-0.03,p < 0.01. UC patients (n = 16) showed down-regulation of P-selectin expression after stimulation with IL-8, 0.26+/-0.03 to 0.22+/-0.02, p < 0.05. No significant differences could be observed concerning soluble P-selectin and NPY in plasma. Patients with 5ASA (n = 12) had lower levels of plasma RANTES, 2.39+/-0.06 microg/l, compared to the control group (n = 12), 3.29+/-0.19 microg/l, p < 0.01, and patients without 5ASA (n = 9), 2.90+/-0.17 microg/l, p < 0.05. CONCLUSIONS: Patients with IBD in remission have higher basal platelet surface P-selectin expression. An exaggerated platelet activity with increased expression of platelet P-selectin and release of inflammatory mediators such as RANTES, which is chemotactic and induce chemokine production, could have a reinforcing and aggravating influence on the inflammatory response and increase the susceptibility to IBD. In addition IL-8 has a down-regulating effect on platelet surface P-selectin expression and 5ASA medication seems to lower plasma RANTES. If 5ASA is responsible for lowering the concentration of RANTES this could be one of the beneficial outcomes of 5ASA medication.

Adult↗
PubMed2004

The PlA1/A2 polymorphism of platelet glycoprotein IIIa is not associated with the risk of type 2 diabetes. The Ludwigshafen Risk and Cardiovascular Health study.

AIMS/HYPOTHESIS: The PlA1/A2 polymorphism of platelet glycoprotein IIIa (GPIIIa) has been implicated in the pathogenesis of type 2 diabetes. We studied this polymorphism in a homogenous, extensively phenotyped cohort using the candidate gene approach. METHODS: The PlA1/A2 polymorphism was determined in 1051 patients with type 2 diabetes and in 2247 individuals without type 2 diabetes. RESULTS: In patients with type 2 diabetes, genotype frequencies were as follows: PlA1/A1 71.4%, PlA1/A2 26.0%, and PlA2/A2 2.7%. In individuals without type 2 diabetes, genotype frequencies were 71.6%, 25.7% and 2.8%, respectively. The PlA2 allele was not associated with fasting and postprandial glucose, glycated haemoglobin, insulin, proinsulin, C-peptide and calculated indices of insulin resistance or pancreatic beta cell function. The PlA2 allele was also not significantly associated with angiographic CHD (adjusted odds ratio [OR] 1.13; 95% CI, 0.93-1.39) or with a history of previous myocardial infarction (adjusted OR 1.09; 95% CI, 0.87-1.37). CONCLUSIONS/INTERPRETATION: The GPIIIa PlA1/A2 polymorphism is not associated with type 2 diabetes, glucose metabolism, angiographic CHD or myocardial infarction.

Aged↗
PubMed2002

In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation.

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.

Antigens, CD↗
PubMed1999

Intracellular expression of CD61 precedes surface expression.

We report a case of acute myeloid leukemia which we have classified as acute megakaryoblastic leukemia because of intracytoplasmic expression of CD61. Light microscopy demonstrated agranular blasts which, by cytochemical staining, were negative for myeloperoxidase. Using flow cytometry, the blast cells were seen to be positive for HLA-DR, CD7, CD13, CD33, and CD34, thus revealing their myeloid origin. Immunophenotyping on fixed blood smears additionally showed positive reaction with the CD61 antibody, demonstrating the megakaryoblastic differentiation of the blasts. After permeabilization of the cell membrane, the intracytoplasmic CD61 expression was confirmed by flow cytometry. Cytogenetic analysis disclosed a del(7)(q21-22). Our findings suggest that cytoplasmic expression of CD61 may precede the cell-surface expression of this antigen and should therefore be investigated in all cases of acute leukemias with undifferentiated morphology and negative cytochemistry to set apart early FAB-M7 from FAB-M0.

Aged↗