Very Late Activation Antigen-4 Research Articles

Effects of Natalizumab Treatment on Foxp3+ T Regulatory Cells

BackgroundNatalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.MethodologyA combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.Principal FindingsBinding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25highCD127lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.ConclusionsWe provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

Histone deacetylase inhibitor treatment downregulates VLA-4 adhesion in hematopoietic stem cells and acute myeloid leukemia blast cells

The alpha4beta1 integrin very late activation antigen-4 (VLA-4) is an alpha4 (CD49d)/beta1 (CD29) heterodimer. It plays a key role in the adhesion of both hematopoietic progenitor cells and leukemic blast cells to bone marrow stromal cells which express the vascular cell adhesion molecule-1 (VCAM-1) or produce fibronectin. VLA-4 expression has been associated with bone-marrow minimal residual disease, which causes relapse after chemotherapy in patients with acute myelogenous leukemia. Conversely, the absence of VLA-4 reduces bone marrow retention of both hematopoietic progenitor and leukemic blast cells. We report on the downregulation of VLA-4/CD49d for various acute myelogenous leukemia cells lines, on primary cells from patients with acute myelogenous leukemia, and on hematopoietic stem cells and peripheral blood mononuclear cells from healthy donors on treatment with the histone deacetylase inhibitors suberoylanilide hydroxamic acid and valproic acid, which is associated with decreased adhesion to mesenchymal stromal cells. These findings suggest that HDAC-inhibitor treatment may on the one hand impair stem cell homing, while on the other it may improve peripheral blood stem cell mobilization and significantly help to reduce minimal residual disease from acute myelogenous leukemia.

Treatment with Natalizumab in Patients with Multiple Sclerosis Is Associated with Increased Numbers of Circulating CD34+ Cells in the Peripheral Blood.

Introduction: Under physiological condition there is a small proportion of CD34+ hematopoietic stem and progenitors cells present in the peripheral blood. The underlying mechanisms of blood stem cell mobilisation and trafficking are only partially understood. It is apparently a multistep process involving cytokines and chemokines as well as endothelial cells and compounds of the bone marrow's extracellular matrix. Here, we analysed the role of the adhesion molecule very late activation antigen 4 (VLA-4), an a4/b1-integrin (CD49d), on CD34+ cells for blood stem and progenitor cell trafficking. For this purpose, patients with Multiple Sclerosis (MS) were examined who received the humanized anti-VLA-4 monoclonal antibody Natalizumab, which is directed against the a4 chain of the integrin.Patients and Methods: A total of 15 MS patients (11 females / 4 males) with a median age of 31 years (range: 21–42) were included in the study. At the time of examination patients had received a median number of 4 infusions (range: 1–9) of Natalizumab at a standard dose of 300 mg i.v. monthly. In all 15 patients the number of CD34+ cells and the immunophenotype were analysed in the peripheral blood using flow-cytometry. The samples were taken four weeks following the last Natalizumab infusion. For comparison, peripheral blood samples from 5 healthy volunteers (4 females / 1 male) were also analysed. In addition sequential analysis, including colony-forming unit assays, were performed in 3 patients before and at 1, 24, 48 and 72 hours after initial administration of Natalizumab.Results: With a median proportion of 0.07% CD34+ cells (range: 0.03 – 0.3) and a corresponding median concentration of 6.8 CD34+ cells/μl blood (range: 2.2 – 32.4) the Natalizumab treated MS patients showed significantly higher CD34+ cell-counts compared to the 5 healthy volunteers who had a median proportion of 0.03% CD34+ cells (range: 0.01–0.03) and a median concentration of 1.4 CD34+ cells/μl blood (range: 0.6 – 2.5) (p=0.019). In all 15 patients a second CD34+ cell measurement was performed 1h after Natalizumab infusion, without showing a significant change in the number of circulating CD34+ cells which argues against an immediate release. Dual colour phenotyping showed that the vast majority (>85%) of circulating CD34+ cells belonged to the subset of more committed progenitors coexpressing CD38. Sequential analysis in 3 patients showed, that a single-dose administration of Natalizumab at 300 mg iv caused a 4-fold increase in circulating CD34+ cells, representing pluripotent hematopoietic progenitors in in vitro colony-forming unit assays, with a peak value at 48 to 72 hours without declining to baseline levels within 31 days.Conclusion: Natalizumab treatment is associated with an increased number of circulating CD34+ cells in the peripheral blood in patients with MS. This effect of Natalizumab might be useful for patients with hematological malignancies responding poorly to G-CSF based mobilisation protocols.

Inflammatory Markers of Atherosclerosis Are Decreased after Moderate Consumption of Cava (Sparkling Wine) in Men with Low Cardiovascular Risk ,

Atherosclerosis is considered a low-grade inflammatory disease. Polyphenol-rich alcoholic beverages (red wine) have shown a more pronounced antiinflammatory effect than polyphenol-free alcoholic beverages (gin). However, no studies to our knowledge have evaluated the antiinflammatory effects of alcoholic beverages with medium-level polyphenol content such as cava (sparkling wine). We enrolled 20 healthy men (aged 34 ± 9 y) in a randomized crossover study to receive 30 g ethanol/d as cava or gin for 28 d. Before both interventions, subjects abstained from alcohol for 2 wk. Inflammatory biomarkers of atherosclerosis and expression of adhesion molecules on peripheral leukocytes were measured before and after each intervention. Likewise, dietary intake and exercise were also evaluated. Expression of lymphocyte function-associated antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), Sialyl-Lewisx (SLex), and CD40 on monocytes decreased after cava intake (all P < 0.05), whereas only SLex was reduced after gin intake (P = 0.036). Circulating markers of atherosclerosis including vascular cell adhesion molecule-1, E-selectin, and P-selectin decreased after both interventions (all P < 0.05). High-sensitivity C-reactive protein, intercellular adhesion molecule-1 (ICAM-1), IL-6, monocyte chemoattractant protein-1 (MCP-1), and CD40L were diminished only after cava intake (all P < 0.05). The effects of cava on circulating CD40L, ICAM-1, and MCP-1, and monocyte surface expression of CD40, LFA-1, and VLA-4 were greater than those of gin (all P < 0.05). In conclusion, both cava and gin showed antiinflammatory properties; however, cava had a greater protective effect, probably due its polyphenol content.

Modulation of dendritic cell development by immunoglobulin G in control subjects and multiple sclerosis patients

Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 alpha4-integrin], the adhesion molecule required for mDCs to cross the blood-brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.

Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models

BackgroundToll-like receptor (TLR)3 ligands serve as natural inducers of pro-inflammatory cytokines capable of promoting Type-1 adaptive immunity, and TLR3 is abundantly expressed by cells within the central nervous system (CNS). To improve the efficacy of vaccine strategies directed against CNS tumors, we evaluated whether administration of a TLR3 ligand, polyinosinic-polycytidylic (poly-IC) stabilized with poly-lysine and carboxymethylcellulose (poly-ICLC) would enhance the anti-CNS tumor effectiveness of tumor peptide-based vaccinations.MethodsC57BL/6 mice bearing syngeneic CNS GL261 glioma or M05 melanoma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes- mEphA2 (671–679), hgp100 (25–33) and mTRP-2 (180–188) for GL261, or ovalbumin (OVA: 257–264) for M05. The mice also received intramuscular (i.m.) injections with poly-ICLC.ResultsThe combination of subcutaneous (s.c.) peptide-based vaccination and i.m. poly-ICLC administration promoted systemic induction of antigen (Ag)-specific Type-1 CTLs expressing very late activation antigen (VLA)-4, which confers efficient CNS-tumor homing of vaccine-induced CTLs based on experiments with monoclonal antibody (mAb)-mediated blockade of VLA-4. In addition, the combination treatment allowed expression of IFN-γ by CNS tumor-infiltrating CTLs, and improved the survival of tumor bearing mice in the absence of detectable autoimmunity.ConclusionThese data suggest that poly-ICLC, which has been previously evaluated in clinical trials, can be effectively combined with tumor Ag-specific vaccine strategies, thereby providing a greater index of therapeutic efficacy.

Chemical modulation of VLA integrin affinity in human breast cancer cells

The fact that disruption of integrin–extracellular matrix contacts leads to cell death, has converted cell adhesion into a potential target for the control of invasive cancer. In this work, we studied the functional consequences of the interference with the activity of the very late activation antigen (VLA) family of integrins in human breast cancer cell lines of distinct malignancy. The α2β1-mediated adhesion reduced the entry of highly malignant, hormone-independent breast cancer cells into apoptosis. Adhesion of breast cancer cells through the VLA integrins α2β1 and α5β1 was significantly reduced by an apoptosis-inducing natural triterpenoid, dehydrothyrsiferol (DT), when studied on low amounts of extracellular matrix. This effect was dose-dependent, not related to cell toxicity and not shared with apoptosis-inducing standard chemotherapeutics, such as doxorubicin and taxol. The compound did not affect either the cell surface expression level of VLA integrins or cell distribution of vinculin and actin during cell spreading. In addition, neither phosphorylation of the focal adhesion kinase pp125FAK on Tyr397 nor the protein kinase B (Akt/PKB) on Ser473 was significantly altered by DT. The integrin activation level, assessed by binding of soluble collagen to the α2β1 integrin, was reduced upon cell treatment with DT. Importantly, the TS2/16, an anti-β1 activating monoclonal antibody was able to rescue DT-treated cells from apoptosis. Since the activation state of integrins is increasingly recognized as an essential factor in metastasis formation, findings presented herein reveal that the chemical regulation of integrin affinity may be a potential therapeutic strategy in cancer therapy.

Expression of VLA1, VLA2 and VLA3 Integrin Molecules in Uterine Endometrium of Infertile Women with Unexplained Aetiology in Ahwaz-Iran.

Recent attention has focused on the expression of integrin molecules within the endometrium, and their relation to infertility. The present prospective study was undertaken to determine whether the endometrium of women with unexplained infertility differs in the expression of very late activation antigens (VLA) from the endometrium of normal fertile women. Thirty samples of endometrial biopsies from hysterectomies with nonendometrial pathology and 28 endometrial samples by uterine curetting from infertile women in secretary phase at implantation time were collected, stained with three monoclonal antibodies against beta1 integrin subunits including VLA-1 to VLA-3 by immunohistochemical technique and then assessed semiquantitively by microscope. Chi-Square test was used to compare the expression of VLA antigens on epithelial cells, stromal cells, lymphocytes and vessels within endometrial tissues between two groups. The results showed that most VLA integrins were present in fertile and infertile endometrium tissues. There were similarities and differences in the expression of VLA molecules in different compartments. VLA-2, VLA-3 expression on endometrial compartments showed an unaltered pattern of staining during the putative window of implantation in either fertile or infertile women with no significant differences (P-value> 0.5). VLA-1 expression on endometrial compartments changed in fertile and unexplained infertile women, the differences were related to the presence or lack of the molecules on epithelial and stromal cells respectively. Differences may explain causes of unexplained infertility, and suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation and early placental development which requires more investigations.

Structure–Function Relation of Efomycines, a Family of Small-Molecule Inhibitors of Selectin Functions

Selectin-mediated leukocyte adhesion to endothelia, the crucial first step initiating the pathogenic cascade of inflammation, is an attractive target for specific therapies. The small-molecule macrolide, efomycine M, inhibits selectin-mediated leukocyte adhesion in vitro and in vivo, and effectively alleviates inflammatory disorders in vivo. To define the molecular basis of the therapeutically relevant antiadhesive properties of efomycines, several new species of this family were purified and/or synthesized. Efomycines E and G were isolated from Steptomyces BS1261. Efomycine O was synthesized by Lewis acid-catalyzed acetalization and efomycine M was generated by base-catalyzed deglycosylation. Efomycine S resulted from ester cleavage of the macrolide ring system, and efomycine T represents the peracetylated form of efomycine M. When the functional activity of efomycines on adhesion of leukocytes to vascular endothelium was studied, some remarkable differences between the compounds became apparent, inasmuch as efomycines E, G, M, and O significantly inhibited adhesion of both human and porcine leukocytes to the vascular endothelium, whereas efomycines S and T did not show any biological activity. A novel docking engine (ProPose), generating an improved, fully configurable protein-ligand interaction model, demonstrated that biological activities of efomycines can be predicted in silico, thus highlighting the utility of such combinatorial approaches.

Proinflammatory Cytokines, Adhesion Molecules, and Lymphocyte Integrin Expression in the Internal Jugular Blood of Migraine Patients Without Aura Assessed Ictally

The aim of the present research was to verify the levels of the soluble adhesion molecules sL- and sE-selectins, intercellular adhesion molecule (sICAM)-1, and vascular cell adhesion molecule-1 in serial samples of internal jugular venous blood taken from migraine patients without aura (MWoA) during attacks. The expression of leukocyte function antigen (LFA)-1 and very late activation antigen (VLA)-4 was also assessed on lymphocytes obtained from jugular venous blood. Levels of certain proinflammatory cytokines (tumor necrosis factor-alpha[TNF-alpha], interleukin-1beta[IL-1beta], IL-4, and IL-6) were also determined and correlated with those of adhesion molecules. Seven MWoA patients were admitted in the hospital during attacks and blood samples were taken immediately after catheter insertion, at 1, 2, and 4 hours after attack onset, and within 2 hours after its termination. The levels of adhesion molecules and cytokines were measured with ELISA method. The expression of LFA-1 and VLA-4 was assessed by flow cytometry. A parallel transient increase of sICAM-1, TNF-alpha, and IL-6 was observed in the first 2 hours after attack onset compared with the time of catheter insertion (P < .0001, <.001, and <.003, respectively). The proportion of CD4+ and CD8+ T-cells expressing high levels of LFA-1 showed instead a progressive down-regulation with significantly lower percentages at 2 and 4 hours after attack onset (P < .01 and <.022, respectively). No variation in the percentage of VLA-4 expressing cells was observed at any time of the study. The transient increase in sICAM-1 and TNF-alpha found in the internal jugular blood of MWoA patients assessed ictally can be induced by sensory neuropeptides released from activated trigeminal endings. The progressive decrease in sICAM-1 levels during attacks and the down-regulation of LFA-1 expression by lymphocytes could antagonize their transvascular migration, supporting the hypothesis of sterile inflammation in the dura mater during migraine attacks.

Identification of IL-8 as a key factor in breast cancer invasion and angiogenesis

Human umbilical cord blood derived stromal cells (hUCBDSCs), a novel resource isolated by our laboratory, have been shown to exert an immunologic regulation. Very late activation antigen-4 (VLA-4) has been associated with graft-versus-host disease (GVHD). This study aimed to investigate the possible mechanism by in vitro co-cultured splenocytes of donor mice with hUCBDSCs and in haploidentical stem cell transplantation in mice with acute GVHD. Both hUCBDSCs and human bone marrow stromal cells (hBMSCs) elicited decreased lymphocyte expression of VLA-4, but this decrease was stronger with hUCBDSCs than with hBMSCs (p < 0.05). Cotransplantation of bone marrow with hUCBDSCs significantly decreased the expression of VLA-4 compared with control mice (p < 0.05). A significant reduction of VLA-4 labeling in the target organs of GVHD was evident in haploidentical mice cotransplanted with hUCBDSCs. Our study shows that hUCBDSCs may protect mouse recipients of haploidentical stem cell transplantation from aGVHD via downregulating the expression of VLA-4.

Anti-α4 integrin therapy for multiple sclerosis

The symptoms, severity, and course of multiple sclerosis (MS) vary among patients, leading to complex treatment issues. In recent years, research has focused on specific adhesion molecules that participate in the activation and function of lymphocytes, especially the migration of these cells to sites of inflammation. In particular, the integrin, very late activation antigen (VLA)-4, has been implicated in mediating adhesion and migration of immune cells through interaction with its ligand, vascular cell adhesion molecule (VCAM)-1. VLA-4 is comprised of alpha4/beta1 and is critical in mediating Th-1 cell migration in the animal model of MS, experimental autoimmune encephalomyelitis, and has been the target of several recent clinical trials in MS. The humanized monoclonal antibody to alpha4 integrin, natalizumab (Tysabri, Biogen Idec/Elan), was recently approved in the United States for the treatment of relapsing MS. The authors discuss the mechanisms by which alpha4 integrins alter lymphocyte function as a rationale for anti-alpha4 integrin use in MS.

Different effects of red wine and gin consumption on inflammatory biomarkers of atherosclerosis: a prospective randomized crossover trial: Effects of wine on inflammatory markers

Background: No intervention studies have explored the anti-inflammatory effects of different alcoholic beverages on markers of atherosclerosis. We embarked on a randomized, crossover, single-blinded trial to evaluate the effects of wine and gin on inflammatory biomarkers of atherosclerosis. Methods and results: Forty healthy men (mean age, 37.6 years) consumed 30 g ethanol per day as either wine or gin for 28 days. Before and after each intervention, we measured the expression of lymphocyte function-associated antigen 1 (LFA-1), Mac-1, very late activation antigen 4 (VLA-4), and monocyte chemoattractant protein (MCP-1) in monocytes, as well as the soluble vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-1α (IL-1α), C-reactive protein (hs-CRP) and fibrinogen. After either gin or wine consumption, plasma fibrinogen decreased by 5 and 9%, respectively, and cytokine IL-1α by 23 and 21%. The expression of LFA-1 (−27%), Mac-1 (−27%), VLA-4 (−32%) and MCP-1 (−46%) decreased significantly after wine, but not after gin. Wine reduced the serum concentrations of hs-CRP (−21%), VCAM-1 (−17%) and ICAM-1 (−9%). Conclusions: Both wine and gin showed anti-inflammatory effects by reducing plasma fibrinogen and IL-1α levels. However, wine had the additional effect of decreasing hs-CRP, as well as monocyte and endothelial adhesion molecules.

Mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.

Adhesion molecules and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling play key roles in homing and mobilization of hematopoietic stem cells (HSC). Active signaling through SDF-1/CXCR4 and upregulation of adhesion molecules are required for homing, whereas downregulation of adhesion molecules and disruption of SDF-1/CXCR4 signaling are required for mobilization of HSC. We studied the surface expression of CXCR4 very late activation antigen (VLA)-4 and VLA-5 on myeloma cells mobilized with cyclophosphamide and GM-CSF in 12 multiple myeloma patients undergoing HSC mobilization for autologous transplantation. We also studied the plasma levels of SDF-1 in apheresis collection of these patients. We observed a statistically significant decrease in the levels of SDF-1 and surface expression of CXCR4 on myeloma cells in four consecutive apheresis collections compared with premobilization bone marrow specimens. We also observed a statistically significant decrease in surface expression of VLA-4 in myeloma cells in the apheresis collections compared with premobilization bone marrow samples. Furthermore, myeloma cells derived from apheresis collections had decreased adhesion and trans-stromal migration in response to SDF-1, which could be reversed by short incubation with interleukin-6. Hence, mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.

A novel genetic leukocyte adhesion deficiency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow

AbstractLeukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective β2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte β1 and β2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein–coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of β2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fail to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow.

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

Ex vivo expansion of stem cells from umbilical cord blood: expression of cell adhesion molecules.

Expansion of stem cells from cord blood has been demonstrated to increase the numbers of CD34+ cells, CD34+ subsets, long-term culture-initiating cells, and severe combined immunodeficient mouse, repopulating cells. However, reports suggest that the ex vivo expanded population behaves differently than freshly isolated cells and shows a delayed or diminished engraftment. In this study, we investigated the effects of the cytokines flt3 ligand, stem cell factor, and thrombopoietin on expansion of CD34+ and CD34+/CD38- cells. In addition, we studied the expression of adhesion molecules, very late activation antigen-4 (VLA-4) and leukocyte function antigen-1 (LFA-1), on CD34+ cells from cord blood by flow cytometry. We also looked at the expression of an adhesion receptor, namely, vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stromal cells by Western blot analysis after exposure to low dose gamma irradiation. After culturing for 7 days, increases in the absolute numbers of CD34+, CD34+/CD38-, CD34+/VLA-4+, and CD34+/LFA-1+ cells were 5.67 +/- 2.91 (mean +/- standard deviation) fold, 7.21 +/- 4.38 fold, 99.56 +/- 101.5 fold, and 101.39 +/- 83.25 fold, respectively. There was a transient upregulation in the expression levels of VCAM-1 on stromal cells, which peaked at 4 hours. Though there was an increase in the absolute numbers of CD34+ cells expressing the adhesion molecules, the expression levels (antigen density) of the adhesion molecules on the CD34+ cells remained unaffected.

Role of LFA-1/ICAM-1, CLA/E-selectin and VLA-4/VCAM-1 Pathways in Recruiting Leukocytes to the Various Regions of the Chronic Leg Ulcer

The center, edge and distant regions of the venous leg ulcer differ in inflammatory cell composition, suggesting that these represent different developmental stages. Our goal was to determine which recruitment pathways contribute to the differences in leukocyte composition between the various ulcer regions. The multiple region biopsy approach, which enables to study the different development phases of the ulcer at one time-point, was employed to immunohistochemically identify the vascular adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and their counter-ligands on extravasated leukocyte cutaneous lymphocyte-associated antigen (CLA), lymphocyte function-associated antigen (LFA-1) and very late activation antigen-4 (VLA-4), respectively. E-selectin expression was highest at the ulcer edge, while ICAM-1 was highest at the ulcer center. VCAM-1 expression was minor at all ulcer regions. CLA stained up to 80% of the epidermal Langerhans' cells, 62% of the T cells, and only 9% of the macrophages. LFA-1 did not stain Langerhans' cells, stained up to 89% of the T cells and up to 11% of the macrophages. VLA-4 stained up to 30% of the T cells and 71% of the macrophages. In conclusion, the results indicate that Langerhans' cells, T cells and macrophage are each recruited by more than one adhesion-molecule pathway to any of the chronic venous leg ulcer regions.

Eosinophil trafficking in asthma

Asthma is characterised by a 50–fold increase in the number of eosinophils relative to neutrophils in the bronchial mucosa. This is the result of the cumulative and sequential effects of several, approximately fourfold, increases in selective eosinophil versus neutrophil migration occurring at a number of stages in the life cycle of the eosinophil. These events, which are integrated and directed by allergen-specific T helper 2 lymphocytes through the generation of interleukin (IL)-5, IL-4 and IL-13, include:•effects on the bone marrow, mediated principally by IL-5, which result in a fourfold increase in circulating eosinophils•selective tethering of eosinophils to venular endothelium through the combined effects of P-selectin/P-selectin glycoprotein ligand (PSGL)-1 and very late activation antigen (VLA)-4/vascular cell adhesion molecule-1, which has the potential for an up to tenfold increase in eosinophil versus neutrophil adhesion•selective chemotaxis under the influence of CC chemokines•prolonged survival, again mediated by IL-5.The implications of this multistep process are that antagonists of IL-5, VLA-4, PSGL-1 and CC chemokine receptor 3, as well as IL-4 and IL-13, each have the potential markedly to inhibit eosinophil recruitment in asthma.

Recovery of purification-associated reduction in antigen-induced histamine release from rat peritoneal mast cells.

Immunoglobulin E (IgE)-dependent histamine release from purified rat peritoneal mast cells (PMC) is very low in comparison to that from a non-purified preparation (PEC). The reduced histamine release from PMC is recovered or potentiated by reconstitution with separated non-mast cells (NMC). In the present study, further characterization was undertaken to elucidate the mechanisms involved. Sensitized mast cells were recovered from peritoneal cavities of rats, and purified by density gradient centrifugation with Percoll. Effects of NMC reconstitution, membrane fraction of NMC, NMC incubation supernatant, adhesion molecules and extracellular matrix proteins on IgE-dependent histamine release from PMC were examined. IgE-dependent histamine release was significantly potentiated by NMC reconstitution to PMC. The potentiation was dependent on the concentration of NMC reconstituted and reached a plateau after 30 min incubation. Increasing concentration of PMC did not affect the histamine release. Membrane fraction prepared from NMC also potentiated PMC histamine release in a dose-dependent manner. The potentiation reached a plateau in 5 min. Furthermore, incubation supernatant of NMC potentiated PMC histamine release. Antibodies against intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-1, very late activation antigen (VLA)-1, VLA-4 and VLA-6, and fibronectin did not affect the potentiation of PMC histamine release by NMC reconstitution. Fibronectin, laminin and collagen failed to potentiate PMC histamine release. These results indicate that the membrane component(s) of NMC in the rat peritoneal cavity seems to modulate IgE-dependent histamine release from peritoneal mast cells of rats, and that the active molecule(s) may be released from NMC. Adhesion molecules such as ICAM-1, LFA-1 and VLA are not involved.