Expression Of Adhesion Receptors Research Articles

Human CMV Infection of Porcine Endothelial Cells Increases Adhesion Receptor Expression and Human Leukocyte Recruitment

Potential xenozoonosis is a concern for the clinical application of xenotransplantation. Human cytomegalovirus (HCMV) is one of the most important pathogens in allotransplantation, but the consequences of HCMV cross-species infection of porcine xenografts are unknown. Therefore, we investigated the effects of HCMV infection of porcine endothelial cells (pEC) on cell surface molecule expression and human leukocyte recruitment. Infection of pEC inoculated with untreated, UV-inactivated, or heparin-treated HCMV at a multiplicity of infection (MOI) of 1 was analyzed by immediate early (IE) antigen expression. Cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and differentially on IE-positive and -negative pEC. Adhesion of human leukocytes was tested on pEC monolayers. pEC supernatants were analyzed for cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures. At day 2 postinfection, IE staining was evident in 10% to 20% of HCMV-infected cells. Cell-surface expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) was upregulated in both IE-negative and -positive fractions of HCMV-infected pEC. In contrast, porcine major histocompatibility complex class I expression was upregulated in IE-negative cells, but reduced in IE-positive cells. The receptor alterations in the IE-negative fraction were mediated by pEC-derived soluble factors. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin, but not UV-inactivation, prevented adhesion-receptor modulation and reversed the increased adhesion and chemotaxis. After pig-to-human solid organ transplantation HCMV may infect and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection.

The Release of Soluble Factors Contributing to Endothelial Activation and Damage after Hematopoietic Stem Cell Transplantation Is Not Limited to the Allogeneic Setting and Involves Several Pathogenic Mechanisms

This study evaluated the relative impact of the intensity of the conditioning regimen and the alloreactivity in the endothelial dysfunction occurring after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It involved a comparative analysis of the effect of incubating human umbilical vein endothelial cells (ECs) with serum samples from patients receiving autologous HSCT (auto-HSCT) or unrelated donor allo-HSCT. In both groups, blood samples were collected through a central line before conditioning (Pre), before transplantation (day 0), and at days 7, 14, and 21 after transplantation. Changes in the expression of EC receptors and adhesion proteins, adhesion of leukocytes and platelets under flow, and signaling pathways were analyzed. Endothelial activation and damage were observed in both groups, but with differing patterns. All markers of endothelial dysfunction demonstrated a progressive increase from day Pre to day 14 in the auto-HSCT group and exhibited 2 peaks of maximal expression (at days 0 and 21) in the allo-HSCT group. Both treatments induced a proinflammatory state (ie, expression of adhesion receptors, leukocyte adhesion, and p38 MAPK activation) and cell proliferation (ie, morphology and activation of ErK42/44). Prothrombotic changes (ie, von Willebrand factor expression and platelet adhesion) predominated after allo-HSCT, and a proapoptotic tendency (ie, activation of SAPK/JNK) was seen only in this group. These findings indicate that endothelial activation and damage after HSCT also occur in the autologous setting and affect macrovascular ECs. After the initial damage induced by the conditioning regimen, other factors, such as granulocyte colony-stimulating factor (G-CSF) toxicity, engraftment, and alloreactivity, may contribute to the endothelial damage seen during HSCT. Further studies are needed to explore the association between this endothelial damage and the vascular complications associated with HSCT.

Angiostasis as a way to improve immunotherapy

Tumours express tumour-associated antigens that are recognised as self-antigens precluding the induction of effective anti-tumour immune responses. Inflammatory conditions which facilitate appropriate antigen presentation and reduce the immuno-suppressive micro-milieu may break tolerance. However, tumours have evolved mechanisms to escape cytotoxic T-cell attack by expressing inhibitory molecules on their surface, secreting suppressive factors, attracting regulatory T cells to the tumour environment or downregulating MHC molecules. Induction of angiogenesis by tumours may represent another mechanism by which tumours escape from immune attack. It provides an anti-inflammatory milieu that will prevent appropriate activation and maturation of antigen presenting cells, allow tumours to secrete suppressive factors and inhibit expression of tumour endothelial adhesion receptors, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin, needed for appropriate interactions with immune cells. Inhibition of angiogenesis may, apart from its direct detrimental effects on the tumour, reverse these processes and contribute to anti-tumour immune reactivity. Without trying to give a complete overview of the field, this paper reviews insights on angiogenesis inhibition in relation to tumour immune responsiveness, mainly based on the Maastricht-Amsterdam experience. This review adds to the hypothesis of improvement of immuno-directed therapies for cancer by angiostasis.

FTY720 Abrogates the Therapeutic Potential of Adoptively Transferred Treg Via Inhibition of IL-2 Induced in Vivo Expansion

CD4+CD25+ T cells (Treg) entry into secondary lymphoid organs (SLO) and local expansion after activation is at least in part responsible for their immunosuppressive action. Thus we hypothesized that trapping of adoptively transferred Treg in SLO would be an effective means to tip the balance towards a more immunosuppressive milieu within the LN microenvironment. Systemic application of the sphingosine-phosphate receptor agonist FTY720 has been proven to trap harmful effector T cells in SLO, thereby inhibiting their migration and destruction of target tissue. Here we provide first evidence that selective entrapment of adoptively transferred Treg in inflammatory LN can be achieved by blockade of SP-receptors upon ex vivo exposure of Treg to FTY720 before adoptive transfer. FTY720 exposure did not interfere with proper Treg localization within the T-cell areas of SLO as determined by immunofluorescent microscopy after co-transfer of either FTY720- or solvent exposed and subsequently differentially labelled Treg. However, despite the fact that the in vitro phenotype (including expression of adhesion and chemokine receptors), function (including anergy and suppressive activity) and survival (determined by Annexin/PI staining) of Treg remained unaltered by FTY720, it abrogated their protective effect after adoptive transfer in a murine model of acute experimental glomerulonephritis (determined by quantification of proteinuria and histological analysis) as well as in an acute GvHD model (determined by survival analysis and quantification of the in vivo expansion of luciferase-transgenic effector T cells by bioluminiscence technology). Notably, adoptive transfer of CFSE-labelled Treg revealed a markedly impaired proliferation of Treg in inflammatory SLO when pre-exposed to FTY720 ex vivo. Accordingly, FTY720 blocked Treg-proliferation induced by TCR-stimulation in combination with IL-2 in vitro. In line with this observation, FTY720 completely abolishes IL-2 induced phosphorylation of STAT-5. Thus, SP-1P receptors induce Treg trapping in inflammatory SLO but abrogate their in vivo immunosuppressive potential by inhibition of local Treg expansion.

Erythrocyte Adhesion and Phosphatidylserine Exposure in HbSC Disease: Baseline Data from the CHAMPS Study

Red blood cells (RBCs) in sickle cell anemia are characterized by increased adhesion to laminin and endothelial cells, due both to increased adhesion receptor expression as well as receptor activation. However, these RBC properties have not been comprehensively evaluated in HbSC disease. In the on-going multi-center CHAMPS trial, all subjects have a diagnosis of HbSC disease and have had at least one significant vaso-occlusive event in the previous 12 months (but none in the previous 4 weeks). All subjects are at least 5 years of age (median 13.4 yr, range = 5.2–53.3 yr) at enrollment. Subjects receive hydroxyurea (HU) and/or magnesium pidolate (Mg) (or their placebos); endpoints include the effect of study drugs on erythrocyte density and RBC-endothelial interactions. To characterize the adhesive properties of RBCs at baseline in these patients, we performed the following analyses on blood samples shipped to a central lab at Duke University from 11 participating sites: adhesion to laminin at 1 dyne/cm2 shear stress; adhesion to endothelial cells after epinephrine (epi) stimulation of RBCs at shear stress of 1 dyne/cm2; and phosphatidylserine exposure, as measured by mean fluorescence intensity after binding of fluoresceinated annexin V. Most subjects had two pre-treatment specimens analyzed. Analysis of the first 83 samples (47 patients) showed that a mean of 1.64% (± 0.15% SEM) of circulating HbSC RBCs expressed phosphatidylserine, or a little less than half as many as in HbSS RBC samples (4.12% positive ± .91% SEM, n = 29) tested as controls (HbSC vs HbSS p=.0009). However HbSC RBCs were also different from HbAA RBCs (1.076% positive, n = 5), and all three sets of values were significantly different by ANOVA (p = .0017) (Figure). The two baseline visits for HbSC subjects resulted in nearly identical means of 1.64% and 1.65%, respectively. Adhesion of HbSC red cells to laminin at baseline was quite variable among patients, with a mean of 36.1 adherent cells/mm2 (range 2–256). This was slightly but not significantly lower than the values obtained using HbSS control RBCs (mean 58.8 cells/mm2, range 0.2–258). After epi stimulation of RBCs, 9.6% of cells adhered to endothelial monolayers at 1 dyne/cm2, whereas 14.8% of HbSS RBCs were adherent (p = .186). HbAA RBCs showed minimal adhesion in both assays (< 5 cells/mm2 and <3%, respectively). Finally, no correlation was found by linear regression analysis between phosphatidylserine exposure and either adhesion to laminin or adhesion to endothelial cells. In summary, HbSC RBCs show appreciable adhesion to both laminin and endothelial cells, as well as phosphatidylserine exposure. While in general these abnormalities are less pronounced than for HbSS RBCs, they are still well outside the normal range and provide potential targets for therapeutic intervention. [Display omitted]

Dynamic Pattern of Adhesion Receptor Expression during the Maturation of Ex-Vivo Generated Human Adult and Neonatal Erythroid Cells.

The maturation of erythroid cells occurs in specialized areas of the marrow in close proximity to macrophages. The mature cell, the reticulocyte, loses its association with the macrophages and egresses into the blood stream. This dynamic pattern of cellular interactions is mediated by specific adhesion receptors, such as CXCR4 (CD184), VLA- 4 (α4 integrin, CD49d) and P-selectin glycoprotein ligand-1 (PSGL-1 or CD162). Culture conditions capable to generate ex vivo human erythroblasts in numbers sufficient for transfusion have been recently established by several investigators. The aim of this study was to evaluate whether these ex vivo-generated erythroblasts would express the adhesion receptor profile required for establishing, once injected in vivo, the cellular interactions necessary to complete their maturation. For this purpose, the pattern of CD184, CD49d and CD162 expression during the maturation of human erythroblasts generated ex vivo from adult and cord blood was investigated. Erythroblasts were divided into 4 classes of maturation by cytofluorimetrical criteria based on the levels of CD36 and CD235a (glycophorin A) expression: class 1, CD36highCD235aneg (CFU-E); class 2, CD36highCD235alow (pro-erythroblasts); class 3, CD36highCD235ahigh (basophilic-polychromatic erythroblasts) and class 4, CD36lowCD235ahigh (orthochromatic erythroblasts). The transition of the different cell populations through the maturation process was tracked by cell cycle analyses and CFSE staining. Large numbers (>5 x 107) of erythroblasts were generated from as little as 10 mL of either cord- or adult blood after 10–11 days of culture in the presence of hematopoietic growth factors, dexamethasone and estradiol (Migliaccio et al, BCMD 28: 169, 2002). Cord blood-derived cells remained significantly more immature than the adult blood-derived ones (e.g. 60% vs 10% in class 1). Class 1–2 cells were mostly in G1 (G1=74%, S=21% and G2=3–5%) while a large proportion of the class 3 cells were in S (G1=34–56, S=43–56% and G2=10–11%). Changes in the levels of CSFE staining indicated that class 3 cells completed one division within 24 hrs and did not divide further. On the other hand, class 1–2 cells completed one division in 24 hr and their progeny was composed both by class 1–2 and class 3 cells (in a 50% ratio). The class 1–2 progeny divided at least one more time in the following 24 hrs while the class 3 progeny did not divide progressing directly toward the mature class 4. The majority of class 1–2 cells expressed low level of CD184 (80–85% CD184dim and 15–20% CD184high) and high levels of CD49d and CD162. When these cells were induced to mature by exposure to EPO alone, they rapidly (within 24 hrs) expressed high levels of CD184 and CD49d while the expression of CD162 was reduced. By the end of 4 days of the maturation culture in the presence of EPO, most of the cells had progressed to the mature class 3–4 phenotype. These mature class 4 cells were CD184dim, CD49dlow and CD162low. Therefore, in vitro maturation of ex vivo-generated cord and adult blood erythroblasts was associated with a dynamic pattern of adhesion receptor expression. Although, the changes observed with cord and adult blood-derived erythroblasts were similar, they occurred more rapidly and with a higher magnitude in cord blood-derived cells. In conclusion, the pattern of CD184, CD49d and CD162 expressed by ex vivo-derived human erythroblasts suggests that these cells might be capable to establish proper cellular interactions and to progress in their maturation following in vivo infusion.

A role for the integrin 6 1 in the differential distribution of CD4 and CD8 T-cell subsets within the rheumatoid synovium

CD4 and CD8 T-cell subsets accumulate in distinct microdomains within the inflamed rheumatoid synovium. The molecular basis for their differential distribution remains unclear. Since chemokines and adhesion molecules play an important role in the positioning of leucocytes at sites of inflammation, we tested the hypothesis that the differential expression and function of chemokine and/or adhesion molecules explains why CD4(+) T cells accumulate within perivascular cuffs, whereas CD8(+) T cells distribute diffusely within the tissue. Expression of an extensive panel of chemokine receptors and adhesion molecules on matched CD4(+) and CD8(+) T cells from peripheral blood (PB) and synovial fluid (SF) was analysed by multicolour flow cytometry. Migration assays and flow-based adhesion assays were used to assess the functional consequences of any differences in the expression of chemokine and adhesion receptors. CD4(+) and CD8(+) T cells from PB and SF expressed unique yet consistent patterns of chemokine and adhesion receptors. SF CD8(+) T cells were much less promiscuous in their expression of chemokine receptors than SF CD4(+) T cells. The alpha(6)beta(1) integrin was highly expressed on PB CD4(+) T cells, but not on PB CD8(+) T cells. Laminin, the ligand for alpha(6)beta(1), retained CD4(+) T cells, but less so CD8(+) T cells, within inflamed synovial tissue. Infiltrating PB CD4(+) T cells, but not CD8(+) T cells, express functional levels of the alpha(6)beta(1) integrin. We propose that this leads to their retention within the rheumatoid synovium in perivascular cuffs, which are defined and delineated by the expression of laminin.

Loss of E-Cadherin Promotes Ovarian Cancer Metastasis via α5-Integrin, which Is a Therapeutic Target

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of alpha(5)-integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an alpha(5)beta(1)-integrin-blocking antibody. When E-cadherin is silenced, alpha(5)-integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogen-activated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/beta-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of alpha(5)-integrin, i.p. treatment with an alpha(5)beta(1)-integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). alpha(5)-Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had alpha(5)-integrin overexpression, and 39% had some level of alpha(5)-integrin expression. The median survival for patients with high alpha(5)-integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified alpha(5)-integrin up-regulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

Modulation of erythroid adhesion receptor expression by hydroxyurea in children with sickle cell disease

We investigated adhesion receptor levels on red blood cells, reticulocytes and erythroid progenitors from children with sickle cell disease treated or not with hydroxyurea. Four groups of patients were investigated: (i) children receiving hydroxyurea for severe vaso-occlusive events (n=26); (ii) untreated children with a history of vaso-occlusive events (n=20); (iii) children with no history of vaso-occlusive events (n=28); and (iv) healthy African controls (n=27). Expression of adhesion receptors was analyzed by flow cytometry with specific mono-clonal antibodies. Reticulocytes and/or red blood cells from the children with sickle cell disease showed significantly higher expression of CD36, alpha 4beta 1, Lu/BCAM than those from controls, whatever the severity of the disease, as well as less marked increases in expression of ICAM-4, CD47 and CD147. Under hydroxyurea treatment, the expression of CD36, alpha 4beta 1 and ICAM-4 (to a lesser extent) was decreased, but surprisingly the expression of Lu/BCAM (and also CD47 and CD147 to a lesser extent) was significantly increased. Alterations of levels of adhesion receptors could be recapitulated in two-phase liquid cultures of erythroid progenitors from controls and untreated children with a history of vaso-occlusive disease, grown in the absence or presence of hydroxyurea. Our results suggest that hydroxyurea acts during erythroid development and modulates adhesion receptor expression and function differently, possibly by acting on gene expression and the signaling cascade leading to receptor activation.

Platelet hyperactivity in type 2 diabetes: role of antiplatelet agents

Type 2 diabetes mellitus increases atherothrombotic risk. Platelets in individuals with diabetes show increased activity at baseline and in response to agonists, ultimately leading to increased aggregation. Increased expression of platelet surface adhesion molecules and receptors, enhanced production of thromboxane and thrombin and disturbances in platelet calcium homeostasis are well documented. As intra-arterial thrombi are initiated by platelets, strategies to limit acute thrombotic events have largely focused on antiplatelet agents. Aspirin remains the cornerstone of antiplatelet therapy but appears to have limited benefit in diabetes. Use of thienopyridines and platelet glycoprotein IIb/IIIa receptor inhibitors has been shown to benefit high-risk patient populations. This review summarises the different platelet abnormalities characterised in diabetes and the role of currently used antiplatelet agents.

Cytokine-pretreatment of CD34 + cord blood stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice

Stem cell homing, engraftment and organ regeneration are controlled by cytokines, chemokines and cell–cell interactions. In this paper, cytokine effects on homing- and engraftment-related characteristics of CD34 + cord blood cells were examined. Untreated CD34 + cells were mainly in the G 0/G 1 cell cycle phase, expressed adhesion receptors on a low level, were positive for vimentin, and negative for the epithelial marker cytokeratin 8/18. Treatment with stem cell factor (SCF) stimulated cell proliferation, increased the number of cells in S and G 2/M cell cycle phase as well as the expression of adhesion receptors. The expression of cytokeratin 8/18 was increased and that of vimentin remained unchanged. Hepatocyte growth factor (HGF) did not stimulate cell proliferation and expression of adhesion receptors, but increased expression of cytokeratin 8/18. In NOD/SCID mice, kinetics of stem cell distribution revealed a fast elimination of human cells from blood. An increase in the number of engrafted cells was observed in different mouse organs in a time-dependent manner, preferentially in bone marrow, spleen and liver. Pretreatment with SCF resulted in reduction of long-term engraftment in bone marrow. HGF pretreatment of cord blood cells showed no significant effects on long-term engraftment capacity in mouse organs compared to untreated cells. Our data provide in vivo evidence that pretreatment of CD34 + cells with SCF reduces long-term cell engraftment in NOD/SCID mice.

Compartmentalization in membrane rafts defines a pool of N‐cadherin associated with catenins and not engaged in cell–cell junctions in melanoma cells

Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.

Expression of cell adhesion receptors in human osteoblasts cultured on biofunctionalized poly-( ε-caprolactone) surfaces

This study was aimed to investigate whether the activation of poly-( ε-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine–glycine–aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5×10 15He +/cm 2 (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm 2). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24 h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed α1, α2, α3, α5, β1, and αVβ3; after 24 h, cells cultured on tissue-plastic expressed high levels of β1 and αVβ3, while α1 showed a low intensity and α2, α3, and α5 were negative. β1 and αV β3 were selected to evaluate the interaction between cells and PCL samples. The β1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in αVβ3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.

T-Cell Distribution and Adhesion Receptor Expression in Metastatic Melanoma

Metastatic malignant melanoma is a devastating disease with a poor prognosis. Recent therapeutic trials have focused on immunotherapy to induce development of endogenous antitumor immune responses. To date, such protocols have shown success in activation of tumor-specific CTL but no overall improvement in survival. To kill tumor, antigen-specific CTL must efficiently target and enter tumor tissue. The purpose of this study was to examine the pathway of leukocyte migration to metastatic melanoma. Peripheral blood and metastatic melanoma tissues (n = 65) were evaluated for expression of adhesion molecules using immunohistochemistry of tumor sections and flow cytometry of tumor-associated and peripheral blood CTL and compared with healthy controls. CTL expressing T-cell receptors for the melanoma antigen MART-1 were identified in a subset of samples by reactivity with HLA-A2 tetramers loaded with MART-1 peptide. Results show that the majority of metastatic melanoma samples examined do not express the vascular adhesion receptors E-selectin (CD62E), P-selectin (CD62P), and intercellular adhesion molecule-1 (CD54) on vessels within the tumor boundaries. Strong adhesion receptor expression was noted on vessels within adjacent tissue. Tumor-associated T lymphocytes accumulate preferentially in these adjacent areas and are not enriched for skin- or lymph node-homing receptor phenotype. Expression of leukocyte homing receptors is dysregulated on the vasculature of metastatic melanoma. This results in a block to recruitment of activated tumor-specific CTL to melanoma metastases and is a likely factor limiting the effectiveness of current immunotherapy protocols.

Gene expression shift towards normal B cells, decreased proliferative capacity and distinct surface receptors characterize leukemic blasts persisting during induction therapy in childhood acute lymphoblastic leukemia

In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.

Effect of cytokine treatment on the in vitro expression of the P. falciparum adhesion receptor chondroitin-4-sulphate on the surface of human choriocarcinoma (BeWo) cells

BeWo human choriocarcinoma cells have recently been identified as an in vitro model of adhesion of Plasmodium falciparum-infected erythrocytes to the major placental receptor chondroitn-4-sulphate (CSA). In this study, we show that treatment of BeWo cells with tumour necrosis factor-alpha and/or interferon-gamma, cytokines linked with pregnancy-associated malaria and poor pregnancy outcome, does not alter the expression of cell surface CSA. BeWo cells do not express the common P. falciparum adhesion receptor cluster of differentiation 36 (CD36) on the cell surface, and this was unchanged after treatment with cytokines. These data demonstrate that in vitro cultured BeWo cells mimic the P. falciparum adhesion receptor expression profile of ex vivo placental cytotrophoblast cells.

Platelets as Immune Cells

Beyond an eminent role in hemostasis and thrombosis, platelets are characterized by expert functions in assisting and modulating inflammatory reactions and immune responses. This is achieved by the regulated expression of adhesive and immune receptors on the platelet surface and by the release of a multitude of secretory products including inflammatory mediators and cytokines, which can mediate the interaction with leukocytes and enhance their recruitment. In addition, platelets are characterized by an enormous surface area and open canalicular system, which in concert with specialized recognition receptors may contribute to the engulfment of serum components, antigens, and pathogens. Platelet-dependent increases in leukocyte adhesion may not only account for an exacerbation of atherosclerosis, for arterial repair processes, but also for lymphocyte trafficking during adaptive immunity and host defense. This review compiles a selection of platelet-derived tools for bridging inflammation and vascular disease and highlights the molecular key components governing platelet-mediated mechanisms operative in immune surveillance, vascular remodeling, and atherosclerosis.

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

The role of skin-homing T cells in extrinsic atopic dermatitis

T cells that express Cutaneous Lymphocyte-Associated antigen (CLA) have the potential of migrating to the skin, and are hypothesized to play a role in cutaneous atopic disease. To investigate the immune phenotype and cytokine responses to Der p 1 stimulation of CLA+ T cells in extrinsic atopic dermatitis (EAD). In vitro testing, with controls. Peripheral blood mononuclear cells (PBMC) were obtained from EAD patients (n=27) and non-atopic healthy individuals (n=22). Phenotypic analysis of naive, CLA+ and non-CLA+ memory/effector CD4+ and CD8+ T cells used markers of cell activation, differentiation, adhesion, apoptosis and chemokine receptor expression. Cytokine responses in these cells were studied following Der p 1 stimulation. CLA+ T cells from EAD patients expressed significantly higher levels of CD25, HLA-DR, CD38, CD71, CXCR1, CXCR2 and lower levels of bcl2, CCR5, CCR7, CXCR3, and CD62L (p<0.05). In EAD patients, CLA+ T cells express increased levels of markers associated with activation, adhesion and apoptosis, show differences in the level of expression of differentiation markers and display a distinct chemokine receptor preference, compared with cells from healthy controls. These data suggest a significant role for CLA+ T cells in the pathogenesis of cutaneous atopic disease.

Selectin, Platelet Plays a Critical Role in Granulocyte Access to the Pregnant Mouse Uterus Under Physiological and Pathological Conditions1

Leukocyte recruitment to the pregnant mouse uterus is associated with highly regulated patterns of expression of vascular adhesion receptors. One striking observation is the localized expression of mucosal vascular addressin cell adhesion molecule (MADCAM1) and selectin, platelet (SELP, formerly P-selectin) by maternal vessels in the vascular zone (VZ) during the first half of pregnancy. From midgestation onwards, endothelial cells lining the maternal vessels of the VZ in addition express vascular cell adhesion molecule-1 (VCAM1). The predominant cell population within these vessels is monocyte-like cells. Granulocytes and low numbers of lymphocytes are also present. Murine fetal trophoblast cells are almost devoid of adhesion molecules, including SELP. In contrast, spontaneous abortions of allogeneic pregnancies are characterized by dramatic upregulation of SELP on maternal VZ vessels and on fetal trophoblast cells. Upregulation of SELP is associated with a dramatic influx of highly activated granulocytes, which infiltrate the vessels and tissue of the VZ and the trophoblast. The majority of the activated granulocytes within the trophoblast undergo nuclear fragmentation, which can be detected by TUNEL staining. To demonstrate that SELP is involved in the recruitment of granulocytes to the pregnant uterus, we undertook long-term in vivo inhibition studies using a monoclonal antibody to inhibit the contribution of SELP to leukocyte trafficking to the decidua. In addition, the pregnant uteri of syngeneic Selp(-/-) x Selp(-/-) mice were investigated and compared to the controls. Our results clearly demonstrate the importance of SELP for granulocyte access to the pregnant mouse uterus under physiological and pathological conditions.