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

Migration of donor T cells to the host second lymphoid organs and homing of activated donor T cells into the target tissues play crucial role in the pathophysiology of acute graft-versus-host disease (aGVHD). More deep understanding of the mechanisms for donor T cell migration and homing reveals an important significance in preventing the initiation of aGVHD. In this review, the migration of donor T cells to host second lymphoid organs, homing of donor activated T cells into the target organs, homing of regulating T cells into target organs, the mechanisms of T cell migration and homing in process of aGVHD and its study prospects were summarised.
Cell Movement ; Graft vs Host Disease ; Humans ; Receptors, Lymphocyte Homing ; T-Lymphocytes ; cytology ; immunology

PURPOSE: The CD44 has been known a lymph node homing receptor on circulating lymphocytes. CD44 spliced variants have been found to be overexpressed in human cancers and metastatic cancers. The variant CD44v 6-7 in particular has been suggested to have a potential role in tumor metastasis. It has been reported that histopathological examination could occaisionally miss lymph node micrometastasis. PURPOSE: The aim of this study was to investigate the role of CD44v in metastasis of rectal carcinoma to the pelvic lateral lymph nodes around the obturator nerve, obturator vessel and superior vesical artery. METHODS: Thirty pelvic lateral lymph nodes reported normal histopathologically from 22 patients with rectal carcinomas, 22 rectal carcinomas and their corresponding colonic mucosas. We have used RT-PCR for the detection of CD44 gene products (CD44v and CD44 v6-7) in samples. RESULTS: The expression rates of CD44v were 2/22 (9%) for normal colonic mucosa, 20/22 (90%) for cancer tissues, and 4/30 (13.3%) for pelvic lateral lymph nodes. The rates of CD44v6-7 were also 2/22 (9%) for normal colonic mucosa 20/22 (90%) for cancer tissues, but 7/30 (23.3%) for pelvic lateral lymph nodes. CONCLUSIONS: The analysis of CD44v might be useful for determination of pelvic lateral lymph nodes metastasis, but it should not be used as a metastatic marker in general for rectal cancer patients.
Arteries ; Colon ; Humans ; Lymph Nodes ; Lymphocytes ; Mucous Membrane ; Neoplasm Metastasis ; Neoplasm Micrometastasis ; Obturator Nerve ; Receptors, Lymphocyte Homing ; Rectal Neoplasms*

Asthma is a complex immune mediated chronic inflammatory lung disease characterized by chronic inflammation of the airways, airway hyper-responsiveness and airway obstruction, and the prevalence of this disease has increased in recent years. It is well known that many features of allergic asthma are consequences of Th2 cell dominated immune responses against allergens, thus allergen specific Th2 cells play a critical role in the pathogenesis. In this review, we will discuss the properties of common indoor and outdoor allergens including house dust mite, fungus, pollen and cockroach, the activation and differentiation of naive CD4 T cells by protease allergens, how specific allergens modify host's immune system to mediate immune evasion, and regulation of homing receptor expression and trafficking of allergen specific Th2 cells. Lastly, we will also overview the general course of pathogenesis of allergic asthma and discuss prospects of development of novel immuno-therapies to asthma.
Airway Obstruction ; Allergens ; Antigens, Differentiation, T-Lymphocyte ; Asthma ; Cockroaches ; Fungi ; Immune Evasion ; Immune System ; Inflammation ; Lung Diseases ; Pollen ; Prevalence ; Pyroglyphidae ; Receptors, Lymphocyte Homing ; T-Lymphocytes ; Th2 Cells

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.
Antigens, CD34/analysis* ; Cells, Cultured ; DNA/analysis ; Fetal Blood/cytology* ; G2 Phase ; Hematopoietic Stem Cells/physiology* ; Human ; Immunophenotyping ; Infant, Newborn ; Integrins/analysis* ; Receptors, Lymphocyte Homing/analysis* ; S Phase

Immunotherapy with regulatory T lymphocytes is considered to be an attractive new therapeutic modality to prevent allograft rejection. The success of this new therapy is critically dependent on the preparation of highly effective and enough number of regulatory T cells. Here, we tried to establish a proper strategy for the ex vivo expansion of regulatory T cells and evaluated their characteristics. CD4+CD25h+CD62L+ T cells were isolated from the recipient mice and weekly stimulated with various stimuli in the presence of IL-2. The most efficient protocol for the expansion of regulatory T cells maintaining Foxp3 expression and regulatory activity was the three cycles stimulation with donor bone marrow-derived dendritic cells (BM-DCs) which yielded around 400 fold expansion of regulatory T cells. The in vitro-expanded regulatory T cells expressing lymph node homing receptors on their cell surface, were composed of polyclonal population, and did not acquire the ability to produce effector cytokines. Importantly, these expanded regulatory T cells induced a modest prolongation of skin allograft survival when combined with transient T cell depletion in recipient mice. These data indicate that our protocol could be used to obtain an effective population of natural regulatory T cells available for the regulatory T cell therapy to prevent allograft rejection.
Animals ; Cytokines ; Dendritic Cells ; Humans ; Immunotherapy ; Interleukin-2 ; Mice ; Receptors, Lymphocyte Homing ; Rejection (Psychology) ; Skin ; T-Lymphocytes ; T-Lymphocytes, Regulatory ; Tissue Donors ; Tissue Therapy ; Transplantation, Homologous

OBJECTIVETo study the effect of ex vivo expansion on the adhesion activities of umbilical cord blood hematopoietic stem and progenitor cells (HSPC).

METHODSFresh UCB CD(34)(+) cells were cultured in a serum and stroma-free culture system. At day 7, day 10 and day 14, CD(34)(+) cells were re-selected from the expanded products. The expression of adhesion molecules (CAMs) such as VLA-4, VLA-5, LFA-1, ICAM-1, HCAM, L-selectin and PECAM-1, and the adhesion activity of the expanded CD(34)(+) cells were evaluated and compared with those of precultured fresh CD(34)(+) cells.

RESULTS(1) The CD(34)(+) cells expressing homing-related CAMs were increased (from 15-fold increase for CD(34)(+) CD(54)(+) subset to 72-fold increase for CD(34)(+) CD(49e)(+) subset at day 14). (2) The expressions of CD(49d), CD(44), CD(11a) and CD(49e) on the expanded CD(34)(+) cells were increased or sustained the same levels as those on fresh UCB CD(34)(+) cells, while the expression of CD(62L), CD(54) and CD(31) on expanded CD(34)(+) cells declined with the cultivating. (3) Spontaneous adhesion and SDF-1-induced adhesion tended to be increased in the course of the first 10 day's culture.

CONCLUSIONSThe culture system used in this study could substantially support the expansion of HSPCs expressing the above CAMs, and the expanded HSPCs would sustain their intrinsic adhesion potentials.

Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Cell Adhesion ; Cell Adhesion Molecules ; biosynthesis ; Cell Division ; Fetal Blood ; cytology ; immunology ; metabolism ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; metabolism ; Humans ; Receptors, Lymphocyte Homing ; biosynthesis

OBJECTIVETo compare the expression of cell adhesion molecules (CAMs) among VLA-4 (CD49 d), VLA-5 (CD49e), LFA-1 (CD11a), L-selectin (CD62L), and PECAM-1 (CD31) which are more related to the homing of hematopoietic stem and progenitor cells (HSPC) on the ex vivo expanded CD34+ subset with that of fresh isolated AC133+ cells.

METHODSAC133+ cells selected from fresh cord blood (CB) samples were cultured in QBSF-60 serum-free media in the presence of Flt-3 ligand + SCF + TPO (FST), with initial addition of IL-3 for up to 2 week. Expansion potential and the expression of above CAMs were evaluated at day 0, day 7, day 10 and day 14.

RESULTS(1) Simultaneously numerical expansion of various HSPC was constantly observed during the culture, and the fold expansion of AC133+ cells and CD34+ cells on day 14 were 33.50 and 64.56 respectively; (2) The number of CD34+ subsets expressing the above adhesions were all increased at different degrees (from 20 fold to 160 fold). (3) The expressions of CD11a, CD49d, and CD49e on ex vivo expanded CD34+ cells were increased as compared to their baseline levels, but the percentage of CD62L+ and CD31+ subpopulations in CD34+ cells were decreased.

CONCLUSIONSOur short-term culture system can not merely support the simultaneous expansion of CB derived AC133+ cells, but the expanded hematopoietic progenitors may well sustained the expression of homing-related adhesion molecules.

AC133 Antigen ; Antigens, CD ; Antigens, CD34 ; metabolism ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cell Division ; drug effects ; Cells, Cultured ; Culture Media, Serum-Free ; Cytokines ; physiology ; Female ; Fetal Blood ; cytology ; metabolism ; Glycoproteins ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Interleukin-3 ; pharmacology ; Lymphocyte Subsets ; Peptides ; metabolism ; Pregnancy ; Receptors, Lymphocyte Homing ; metabolism

BACKGROUND: There has been contradictory reports regarding the homing potential of hematopoietic cells briefly exposed to hematopoietic growth factors in vitro. To get a resolution to this controversy, we investigated the effects of short-term growth factor treatment of hematopoietic cells on the expression of CXCR4 and adhesion molecules, and the chemotaxis in response to stromal cell-derived factor-1 (SDF-1), which is widely accepted to play a critical role in bone marrow (BM) homing of hematopoietic stem cells. METHODS: BM and cord blood(CB) CD34+ cells were incubated with various hematopoietic growth factors including IL-1beta, IL-3, IL-6, G-CSF, GM-CSF, stem cell factor (SCF), flk-2 ligand, and thrombopoietin, alone or in combination for up to 48 hours. Before and after the incubation, the expression of CXCR4 and adhesion molecules of CD34+ cells was analyzed using flow cytometry. SDF-1-mediated transmembrane or transendothelial migration of CD34+ cells, cobblestone area-forming cells (CAFCs), and/or long-term culture-initiating cells (LTC-ICs) was measured using Transwell(TM) system. RESULTS: VLA-4 was moderately up-regulated by the incubation of the cells with IL-3 and SCF, and ICAM-1 was slightly up-regulated by IL-1 and IL-3. The expression of L-selectin, PECAM-1 or LFA-1 was not altered by any growth factors. With the incubation of the cells in the absence of growth factors or SDF-1, CXCR4 expression of CD34+ cells was rapidly increased, reaching a plateau at 24 hours. The spontaneous up-regulation was abrogated with the addition of SDF-1. In agreement with the up-regulation of CXCR4, CD34+ cells incubated for 40 hours showed much enhanced chemotaxis in response to SDF-1 compared to non-incubated cells (24.7 3.5% vs. 7.0 1.6%, P=0.01). Any growth factors examined in this study did not alter the CXCR4 expression of CD34+ cells. Neither did growth factors affect the transendothelial migration of LTC-ICs toward bone marrow stromal cells as well as the SDF-1-induced transmembrane chemotaxis of CD34+ cells and CAFCs. CONCLUSION: Short-term treatment of hemo-topoietic cells with hematopoietic growth factors does not alter the expression of CXCR4 or SDF-1-mediated transendothelial chemotaxis.
Antigens, CD31 ; Bone Marrow ; Chemotaxis ; Flow Cytometry ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells ; Integrin alpha4beta1 ; Intercellular Adhesion Molecule-1 ; Intercellular Signaling Peptides and Proteins* ; Interleukin-1 ; Interleukin-3 ; Interleukin-6 ; L-Selectin ; Lymphocyte Function-Associated Antigen-1 ; Mesenchymal Stromal Cells ; Stem Cell Factor ; Thrombopoietin ; Transendothelial and Transepithelial Migration* ; Up-Regulation

BACKGROUND: It is well known that harmonious interactions among adhesion molecules and stromal cell-derived factor-1 (SDF-1)-mediated chemoattraction signalling via CXCR4 are needed for bone marrow homing of hematopoietic stem cells and progenitor cells. The aim of this study was to define the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta 1 (TFG-beta1), known as hematopoiesis-inhibitory cytokines, in the regulation of the molecules in relation to the homing. METHODS: We investigated the effects of these cytokines on the expression of CXCR4 and adhesion molecules and the production of SDF-1 in bone marrow cells including CD34+ cells, bone marrow endothelial cells (BMEC-1 cells), and bone marrow stromal cells (BMSCs). We also examined whether the cytokines influence in vitro transmigration of hematopoietic progenitors. RESULTS: None of the cytokines influenced CXCR4 expression on CD34+ cells or SDF-1- mediated chemotaxis of the cells. IFN-gamma and TNF-alpha, but not TGF-beta up-regulated the expression of L-selectin, ICAM-1, and VLA-4 on CD34+ cells. However, the up-regulation was not translated into the enhanced transendothelial migration. IFN-gamma and TNF-alpha up-regulated the expression of VCAM-1 and ICAM-1 on BMEC-1 cells, and rendered the endothelium more suitable for transendothelial migration of hematopoietic progenitors. IFN-gamma and TNF-alpha also up-regulated the expression of VCAM-1 and ICAM-1 on primary human BMSCs. All three cytokines significantly attenuated SDF-1 production from primary BMSCs, and TNF-alpha diminished SDF-1 production from BMEC-1 cells. CONCLUSION: These data indicate that IFN-gamma, TNF-alpha, and TGF-beta1 play a role in the regulation of bone marrow homing of hematopoietic cells via up-regulation of adhesion molecule expression and down-modulation of SDF-1 production in bone marrow cells.
Bone Marrow Cells* ; Bone Marrow* ; Chemotaxis ; Cytokines ; Endothelial Cells ; Endothelium ; Hematopoietic Stem Cells ; Humans* ; Integrin alpha4beta1 ; Intercellular Adhesion Molecule-1 ; Interferon-gamma* ; L-Selectin ; Mesenchymal Stromal Cells ; Stem Cells ; Transendothelial and Transepithelial Migration ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha* ; Up-Regulation ; Vascular Cell Adhesion Molecule-1