BACKGROUND: Cell adhesion molecules (CAMs) expressed on hematopoietic progenitor cells (HPCs), endothelial cells, and stromal cells play a pivotal role in the mobilization of CD34+ cells. Herein, we conducted a non-randomized peripheral blood stem cell (PBSC) mobilization study aimed to compare the potential differences in the expressions of several CAMs and chemokines on CD34+ cells obtained from bone marrow aspirate before and after HPC mobilization from patients with hematologic malignancies and healthy donors. METHODS: Three-color cytofluorometric analysis was used to compare the expressions of CAMs and chemokines in the bone marrow before and after mobilization. RESULTS: For all studied groups, CAM expression among those with good and poor yields of CD34+ cells was significantly correlated with VCAM-1 (P=0.007), CD44 (P=0.027), and VLA-4 (P=0.014) expressions. VCAM-1 (P=0.001), FLT-3 (P=0.001), CD44 (P=0.011), VLA-4 (P=0.001), and LFA-1 (P=0.001) expressions were higher before HPC mobilization than after HPC mobilization. By contrast, the expression of CXCR4 significantly varied before and after mobilization only among those with successful PBSC mobilization (P=0.002). CONCLUSION: We attempted to identify particular aspects of CAMs involved in CD34+ cell mobilization, which is a highly complex mechanism that involves adhesion molecules and matrix metalloproteases. The mechanism by which CD34+ cell mobilization is activated through proteolytic enzymes is not fully understood. We believe that CXCR4, VLA-4, CD44, and VCAM-1 are the most important molecules implicated in HPC mobilization, particularly because they show a correlation with the yield of CD34+ cells collected via large volume leukapheresis.
Bone Marrow* ; Cell Adhesion Molecules ; Chemokines ; Endothelial Cells ; Hematologic Neoplasms ; Hematopoietic Stem Cells ; Humans ; Integrin alpha4beta1 ; Leukapheresis ; Lymphocyte Function-Associated Antigen-1 ; Lymphoma, Non-Hodgkin ; Metalloproteases ; Multiple Myeloma ; Peptide Hydrolases ; Stem Cells ; Stromal Cells ; Tissue Donors ; Vascular Cell Adhesion Molecule-1

Macrophages have two major roles in regulating the dynamic equilibrium in erythropoiesis, promoting the differentiation and maturation of nucleated red blood cells into reticulocytes and removing old red blood cells. A recent mouse study has demonstrated that the phenotype of macrophages in erythroblastic islands is CD169+ VCAM-1+ ER-HR3+ CD11b+ F4/80+ Ly-6G+. Molecular connections between erythroid progenitor cells and central macrophages help to maintain the function and integrity of erythroblastic islands. New research advances in Kruppel-like factor 1 (KLF1) provide new evidence for the important role of macrophages in erythroblastic islands. Macrophages play an important role in erythropoiesis both in sickness and in health, and provide a potential targeted therapy for diseases such as polycythemia vera and beta-thalassemia in the future.
Animals ; Erythropoiesis ; Humans ; Integrin alpha4beta1 ; physiology ; Kruppel-Like Transcription Factors ; physiology ; Macrophages ; physiology ; Mice ; Phenotype ; Vascular Cell Adhesion Molecule-1 ; physiology

To demonstrate the effect of AB serum on terminal erythroid differentiation ex vivo.
 Methods: After separation of CD34+ cells from cord blood, the cells were cultured and divided into a control group and an experimental group. The effects of AB serum were examined by the expressions of different markers (GPA, Band3 and α4-integrin) for erythroblast differentiation and enucleation by flow cytometry. 
 Results: The CD34+ cells were successfully differentiated to enucleated red blood cells. There were evident differences among the expressions of GPA, Band3 and α4-integrin between the 2 groups. The percentage of GPA positive cells in the experimental group was bigger than that in the control group in every time point. The expression of Band3 in the experimental group was higher than that in the control group. The expression of α4-integrin in the experimental group was lower than that in the control group. In addition, the enucleation rate in the experimental group was higher than that in the control group.
 Conclusion: AB serum can promote the cell differentiation and enucleation during terminal erythroid differentiation in vitro.
ABO Blood-Group System ; blood ; physiology ; Anion Exchange Protein 1, Erythrocyte ; metabolism ; Antigens, CD34 ; blood ; Cell Differentiation ; genetics ; physiology ; Cell Nucleus ; Cells, Cultured ; Erythrocytes ; physiology ; ultrastructure ; Erythropoiesis ; genetics ; physiology ; Fetal Blood ; cytology ; physiology ; Flow Cytometry ; Glycophorins ; metabolism ; Humans ; Integrin alpha4beta1 ; metabolism

There is growing evidence that crosstalk between mantle cell lymphoma (MCL) cells and stromal microenvironments, such as bone marrow and secondary lymphoid tissues, promotes tumor progression by enhancing survival and growth as well as drug resistance of MCL cells. Recent advances in the understanding of lymphoma microenvironment have led to the identification of crucial factors involved in the crosstalk and subsequent generation of their targeted agents. In the present study, we evaluated the combinatory effect of blocking antibodies (Ab) targeting CXCR4 and VLA-4, both of which were known to play significant roles in the induction of environment-mediated drug resistance (EMDR) in MCL cell line, Jeko-1. Simultaneous treatment with anti-CXCR4 and anti-VLA-4 Ab not only reduced the migration of Jeko-1 cells into the protective stromal cells, but also enhanced sensitivity of Jeko-1 to a chemotherapeutic agent to a greater degree than with either Ab alone. These combinatorial effects were associated with decreased phosphorylation of ERK1/2, AKT and NF-kappaB. Importantly, drug resistance could not be overcome once the adhesion of Jeko-1 to the stromal occurred despite the combined use of Abs, suggesting that the efforts to mitigate migration of MCLs should be attempted as much as possible. Our results provide a basis for a future development of therapeutic strategies targeting both CXCR4 and VLA-4, such as Ab combinations or bispecific antibodies, to improve treatment outcomes of MCL with grave prognosis.
Antibodies, Bispecific ; Antibodies, Blocking ; Bone Marrow ; Cell Line ; Drug Resistance ; Drug Therapy* ; Integrin alpha4beta1* ; Lymphoid Tissue ; Lymphoma ; Lymphoma, Mantle-Cell* ; NF-kappa B ; Phosphorylation ; Prognosis ; Stromal Cells

Multiple sclerosis (MS) is the most common demyelinating disease affecting the central nervous system of young adults living in the western world. MS should be strongly suspected when a young adult develops one or more neurological episodes consistent with damage to white matter within the central nervous system (CNS), especially when these affect the optic nerves, brainstem, or spinal cord. The patient with relapses, each of which can be attributed to demyelination in the CNS, requires no investigation prior to establishing the diagnosis of clinically definite MS. For a diagnosis of MS, separate anatomical sites within the CNS must have been affected on different occasions, typically three. MS in Asian populations is characterized by the selective and dominant involvement of the optic nerve and spinal cord with some incidence of brainstem lesions. 35-40% of MS cases in Korea are of this optico-spinal type with or without brainstem lesions. Reported cases of neuromyelitis optica spectrum disease (NMOSD), causing severe optic neuritis (ON) and/or longitudinally extensive transverse myelitis, either monophase or with a relapse-remitting pattern, some of which were diagnosed previously as the optico-spinal form of MS in Asia, have increased annually in Korea with the development of the NMO-IgG or aquaporin4-antibody detecting technique. NMO-IgG detection is very important in the diagnosis of early stage of NMOSD and the differentiation of MS and other demyelinating disease. Many new convenient oral drugs or very potent intravenous monoclonal antibodies for targeting VLA-4, CD20, and CD52 may decrease the annual relapse rate and burden of brain-spinal cord lesionsin MS.
Antibodies, Monoclonal ; Asia ; Asian Continental Ancestry Group ; Brain Stem ; Central Nervous System ; Demyelinating Diseases ; Humans ; Incidence ; Integrin alpha4beta1 ; Korea ; Multiple Sclerosis ; Myelitis, Transverse ; Neuromyelitis Optica ; Optic Nerve ; Optic Neuritis ; Recurrence ; Spinal Cord ; Western World ; Young Adult

OBJECTIVETo study the protective effect of bone marrow stromal cells (BMSCs) upon childhood leukemia cells and the influence of VLA-4 antibody in vitro on leukemia cell apoptosis.

METHODSBMSCs from children with acute leukemia-were isolated by human lymphocyte separation medium. BMSCs (adherent) and leukemia cells (suspended) were cultured in vitro. This study included four groups: leukemia cells alone (control), leukemia cells+BMSCs, leukemia cells+BMSCs supernatant and leukemia cells+BMSCs+VLA-4 antibody. The apoptosis rate of leukemia cells in the four groups was determined by Annexin Ⅴ-FITC double-labeled flow cytometry. The expression of survivin and bcl-2 genes in leukemia cells was ascertained by RT-PCR.

RESULTSThe apoptosis rate of leukemia cells in the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups was lower than that in the control group (P<0.05). Compared with the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups, the apoptosis rate of leukemia cells in the VLA-4 antibody group increased significantly (P<0.05). In the VLA-4 antibody group, the apoptosis rate of leukemia cells increased with prolonged culture time. There were significant differences in the apoptosis rate between 12 hrs and 24 hrs after VLA-4 antibody treatment (P<0.01). The expression of survivin and bcl-2 genes in leukemia cells from the VLA-4 antibody groups was reduced compared with that from the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups (P<0.05).

CONCLUSIONSBMSCs play protective roles on leukemia cells. VLA-4 antibody can block the adhesion between BMSCs and leukemia cells and promote leukemia cell apoptosis.

Adolescent ; Antibodies ; therapeutic use ; Apoptosis ; Bone Marrow Cells ; physiology ; Child ; Child, Preschool ; Female ; Genes, bcl-2 ; Humans ; Infant ; Inhibitor of Apoptosis Proteins ; Integrin alpha4beta1 ; immunology ; Leukemia ; pathology ; therapy ; Male ; Microtubule-Associated Proteins ; genetics ; Stromal Cells ; physiology

To investigate whether lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) are involved in vasoendothelial adhesion and transendothelial migration of high proliferative potential endothelial progenitor cells (HPP-EPCs), flow cytometry was used to analyze the expression of integrin beta1 and beta2, the expression of intercellular adhesion molecule (ICAM-1, 2) and vascular cell adhesion molecule (VCAM-1) in mouse bone marrow endothelial cells (mBMECs). The adhesion and transmigration through endothelial cells of the HPP-EPCs blocked by functional grade neutralizing antibodies of VLA-4 and LFA-1 were studied in vitro. The results revealed that HPP-EPCs were positive for CD11a and CD49d in HPP-EPCs. The expression of ICAM-1and VCAM-1 of mBMECs increased after activated by IL-1beta and TNF-alpha. The results of adhesion in vitro revealed that the numbers of the adhered and migrated cells in the CD11a antibody group, in the CD49d antibody group and in the combinational antibody group were less than those in the isotype control antibody group. Furthermore, the number of adhered and migrated cells in the combinational antibody group was less than that in the CD11a or the CD49d antibody group (p < 0.05). It is concluded that both LFA-1 and VLA-4 are involved in vasoendothelial adhesion and transendothelial migration of HPP-EPCs.
Animals ; Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Cell Adhesion ; Cell Adhesion Molecules ; metabolism ; Cell Movement ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Integrin alpha4beta1 ; physiology ; Intercellular Adhesion Molecule-1 ; metabolism ; Lymphocyte Function-Associated Antigen-1 ; physiology ; Mice ; Stem Cells ; cytology ; Vascular Cell Adhesion Molecule-1 ; metabolism

The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired alpha and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.
Protein Binding ; Paxillin/*metabolism/physiology ; Models, Biological ; Leukocytes/cytology/*metabolism ; Integrin alpha4beta1/metabolism/physiology ; Integrin alpha4/*metabolism/physiology ; Humans ; Cell Movement/*physiology ; Cell Adhesion/physiology

To explore the effect of ligustrazine on the expression of adherent molecule VCAM-1/VLA-4 of bone marrow cells in syngenic bone marrow transplantation (BMT) mice, the mice were divided into 3 groups: normal group (which received no treatment), BMT control group and ligustrazine-treated groups. BMT mouse models were established. The BMT control group and the ligustrazine-treated group were orally administered 0.2 ml saline per mouse and 2 mg ligustrazine per mouse, respectively, twice a day. On the day 7, 14, 21, 28 after BMT, mice were respectively killed. Bone marrow nucleated cells were detected, and then the expression of VCAM-1/VLA-4 was assayed by immunohistochemistry, RT-PCR and flow cytometry analysis, respectively. The results showed that in ligustrazine-treated group, the accounts of bone marrow nucleated cells on the day 7, 14, 21, 28 after BMT were all higher than that in BMT control group. The expression level in the ligustrazine-treated group was significantly higher than that in the BMT control group (P < 0.05 or P < 0.01). It is concluded that ligustrazine can enhance VCAM-1/VLA-4 expression in bone marrow after syngenic bone marrow transplantation in mice, which may be related to the mechanisms underlying the ligustrazine accelerating hematopoietic reconstitution in allogenic bone marrow transplantation.
Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Bone Marrow Transplantation ; methods ; Flow Cytometry ; Immunohistochemistry ; Integrin alpha4beta1 ; biosynthesis ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Pyrazines ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Isogeneic ; Vascular Cell Adhesion Molecule-1 ; biosynthesis ; genetics

We assessed the cytokine combinations that are best for ex vivo expansion of cord blood (CB) and the increment for cell numbers of nucleated cells, as well as stem cells expressing homing receptors, by an ex vivo expansion of cryopreserved and unselected CB. Frozen leukocyte concentrates (LC) from CB were thawed and cultured at a concentration of 1x10(5)/mL in media supplemented with a combination of SCF (20 ng/mL)+TPO (50 ng/mL)+FL (50 ng/mL)+/-IL-6 (20 ng/mL)+/-G-CSF (20 ng/mL). After culturing for 14 days, the expansion folds of cell numbers were as follows: TNC 22.3+/-7.8~26.3+/-4.9, CFU-GM 4.7+/-5.1~11.7+/-2.6, CD34+CD38- cell 214.0+/-251.9~464.1+/-566.1, CD34+CXCR4+ cell 4384.5+/-1664.7~7087.2+/-4669.3, CD34+VLA4+ cell 1444.3+/-1264.0~2074.9+/-1537.0, CD34+VLA5+ cell 86.2+/-50.9~ 113.2+/-57.1. These results revealed that the number of stem cells expressing homing receptors could be increased by an ex vivo expansion of cryopreserved and unselected CB using 3 cytokines (SCF, TPO, FL) only. Further in vivo studies regarding the engraftment after expansion of the nucleated cells, as well as the stem cells expressing homing receptors will be required.
ADP-ribosyl Cyclase/metabolism ; Antigens, CD/metabolism ; Antigens, CD34/metabolism ; Cryopreservation ; Fetal Blood/*cytology ; Humans ; Integrin alpha4beta1/metabolism ; Integrin alpha5beta1/metabolism ; Membrane Proteins ; Receptors, CXCR4/metabolism ; Receptors, Lymphocyte Homing/*metabolism ; Research Support, Non-U.S. Gov't ; Stem Cell Factor ; Stem Cells/*cytology/*metabolism ; Thrombopoietin