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R Alon

Publications and source records attributed to R Alon.

At least 55 records · Page 3Linked to original sources
PubMed1996

CD44 and hyaluronan-dependent rolling interactions of lymphocytes on tonsillar stroma.

Little is known about how lymphocytes migrate within secondary lymphoid organs. Stromal cells and their associated reticular fibers form a network of fibers that radiate from high endothelial venules to all areas of the lymph node and may provide a scaffold for lymphocyte migration. We studied interactions of lymphocytes with cultured human tonsillar stromal cells and their extracellular matrix using shear stress to distinguish transient interactions from firm adhesion. Tonsillar lymphocytes and SKW3 T lymphoma cells tethered and rolled on monolayers of cultured tonsillar stromal cells and their matrix. A significant proportion of these rolling interactions were independent of divalent cations and were mediated by CD44 binding to hyaluronan, as shown by inhibition with mAb to CD44, soluble hyaluronan, as hyaluronidase treatment of the substrate, and O-glycoprotease treatment of the rolling cells. O-glycoprotease treatment of the substrate also blocked binding completely to stromal matrix and partially to stromal monolayers. SKW3 cells tethered and rolled on plastic-immobilized hyaluronan, confirming the specificity of this interaction. By contrast, monolayers of resting or stimulated human umbilical vein endothelial cells failed to support CD44- and hyaluronan-dependent rolling. SKW3 cells added under flow conditions to frozen sections of human tonsil bound and rolled along reticular fibers in the presence of EDTA. Rolling was blocked by either CD44 mAb or hyaluronan. We propose that lymphocytes migrating through secondary lymphoid organs may use CD44 to bind to hyaluronan immobilized on stromal cells and reticular fibers.

Antigens, Surface↗
PubMed1996

Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions.

Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L-selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L-selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo-glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation.

Animals↗
PubMed1996

Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow.

We demonstrate an additional step and a positive feedback loop in leukocyte accumulation on inflamed endothelium. Leukocytes in shear flow bind to adherent leukocytes through L-selectin/ligand interactions and subsequently bind downstream and roll on inflamed endothelium, purified E-selectin, P-selectin, L-selectin, VCAM-1, or peripheral node addressin. Thus adherent leukocytes nucleate formation of strings of rolling cells and synergistically enhance leukocyte accumulation. Neutrophils, monocytes, and activated T cell lines, but not peripheral blood T lymphocytes, tether to each other through L-selectin. L-selectin is not involved in direct binding to either E- or P-selectin and is not a major counterreceptor of endothelial selectins. Leukocyte-leukocyte tethers are more tolerant to high shear than direct tethers to endothelial selectins and, like other L-selectin-mediated interactions, require a shear threshold. Synergism between leukocyte-leukocyte and leukocyte-endothelial interactions introduces novel regulatory mechanisms in recruitment of leukocytes in inflammation.

Cell Adhesion↗
PubMed1996

[Limitation of blood transfusions during hepatectomies. Study of 150 consecutive hepatic resections on healthy and pathological livers].

OBJECTIVE: Over the last 5 years, a policy to limit blood transfusions has been adopted in patients undergoing liver resection. The aim of this retrospective study was to report the results of 150 liver resections performed during this period. METHODS: There were 63 major (42%) and 87 minor hepatectomies (58%). Resection was performed for malignant lesions in 64% of the patients. Vascular exclusion of the liver was used in large (> or = 10 cm) tumors and those located at the cavohepatic junction. Clamping of the portal triad or selective clamping of the pedicle of the portal lobe was used in peripheral lesions < 10 cm in diameter. Anesthesia was adapted to the type of vascular clamping and blood transfusions were deliberately limited. Red blood cells were transfused to maintain the hematocrit level above 25% in healthy patients and above 30% in patients with risk of coronary artery disease. RESULTS: Ninety three patients (62%) did not receive blood transfusions. Three patients received more than 10 units of packed red blood cells (2%). 48% of patients with major hepatectomies and 72% with minor hepatectomies were not transfused. The rate of non transfused patients was 93% for benign lesions and 44% for malignant lesions. The presence of pathologic changes in non-tumor liver parenchyma did not influence the need for transfusions. Hospital mortality was 3% (5/150). There was no mortality in patients with normal non-tumorous livers, 14% in the presence of cirrhosis, and 12% in the presence of obstructive jaundice or steatosis > 50%. The specific morbidity rate was 7% in patients with normal livers and 54% in patients with abnormal livers. CONCLUSION: This series shows that more than 60% of liver resections can be performed without blood transfusions. These results require an appropriate surgical technique and collaboration between anesthesiologist and surgeon. Thus hepatectomies in normal non-tumorous livers can be performed without mortality. In contrast, the presence of abnormalities of the non-tumorous liver parenchyma remains a major risk factor.

Adult↗
PubMed1995

Glycolipid ligands for selectins support leukocyte tethering and rolling under physiologic flow conditions.

Selectin interactions with glycolipids have been examined previously under static conditions, whereas physiologic interactions mediated by selectins take place under flow. We find that under physiologic flow conditions, sialyl Lewis(x) (sLe(x)) glycolipid and sialyl Lewisa (sLe(a)) neoglycolipid support tethering and rolling adhesions of Chinese hamster ovary (CHO) cells expressing E-selectin and lymphoid and myeloid cells expressing L-selectin. These selectin-mediated adhesions persist at the highest shear stresses that occur in postcapillary venules in vivo and occur at lower site densities than found for sLe(x) on neutrophils. The interactions are Ca(2+)-dependent and can be specifically and completely blocked with anti-selectin mAbs. Asialo nonfucosylated glycolipids are inactive, and sulfatide supports weak tethering, but not rolling, of L-selectin-expressing cells. Rolling velocities and resistance to detachment are related to the glycolipid site density and fall within the range measured for neutrophil and myeloid cell rolling on substrates containing purified selectins. These observations are the first indication that glycolipids can interact with selectins in physiologic flow conditions, and can contribute to rolling adhesions.

Animals↗
PubMed1995

Lifetime of the P-selectin-carbohydrate bond and its response to tensile force in hydrodynamic flow.

Selectins tether to the blood vessel wall leukocytes that are flowing in the bloodstream and support subsequent labile rolling interactions as the leukocytes are subjected to hydrodynamic drag forces. To support this rolling, selectins have been proposed to have rapid bond association and dissociation rate constants, and special mechanical properties linking tensile forces and bond dissociation. We have visualized transient tethering and release of neutrophils in hydrodynamic flow on lipid bilayers containing densities of P-selectin below those required to support rolling. We report here that transient tethers had first-order kinetics and other characteristics suggesting a unimolecular interaction between P-selectin and its glycoprotein ligand (PSGL-1). The unstressed dissociation constant (off rate) was 1 s-1. Hydrodynamic shear stresses of up to 1.1 dyn cm-2, corresponding to a force on the bond of up to 110 pN, increased the off rate only modestly, to 3.5 s-1. The data was adequately matched by a proposed equation relating off rate to the exponential of tensile force on the bond and the bond interaction distance, and gave a bond interaction distance of 0.5 A. This distance is compatible with hydrogen and metal coordination bonds between P-selectin and PSGL-1. Fast on and off rates, together with the high tensile strength of the selectin bond, appear necessary to support rolling at physiological shear stresses.

Carbohydrates↗
PubMed1995

Intrahepatic cholangiocarcinoma. Results of aggressive surgical management.

OBJECTIVE: To report the results of a deliberately aggressive surgical management in patients with intrahepatic cholangiocarcinoma. DESIGN: A case series of patients with intrahepatic cholangiocarcinoma. SETTING: A tertiary care university hospital in a metropolitan area. PATIENTS: From 1989 to 1993, 19 patients with intrahepatic cholangiocarcinoma underwent laparotomy, with a 74% resectability rate (14 liver resections). In addition, two selected patients with a slow-growing tumor underwent orthotopic liver transplantation after limited recurrence following resection in one case and after exploratory laparotomy in the other. INTERVENTIONS: The 14 liver resections included six right or left hepatectomies and eight extended right or left hepatectomies. Total vascular exclusion of the liver was used in nine cases (64%) and resection of the biliary confluence with reconstruction was used in six cases (43%). RESULTS: There was one postoperative death (7%). There were four postoperative biliary fistulas (28%). Overall actuarial 1- and 2-year survival rates were 58% and 32%, respectively. The 1- and 2-year survival rates were 100% after curative resection (no lymph node invasion, clearance margin of < or = 1 cm, and solitary tumor [five cases]) and 48% and 10% after palliative resection. Median survival was 14 months for the whole series and 27 and 9 months following curative and palliative resections, respectively. The two liver transplant recipients are alive and free of disease at 25 and 31 months. CONCLUSION: These results support aggressive surgical management in patients with intrahepatic cholangiocarcinoma, including complex liver resection procedures and selective use of orthotopic liver transplantation.

Actuarial Analysis↗
PubMed1995

The integrin VLA-4 supports tethering and rolling in flow on VCAM-1.

Selectins have previously been shown to tether a flowing leukocyte to a vessel wall and mediate rolling. Here, we report that an intergrin, VLA-4, can also support tethering and rolling. Blood T lymphocytes and alpha 4 integrin-transfected cells can tether in shear flow, and then roll, through binding of the intergrin VLA-4 to purified VCAM-1 on the wall of a flow chamber. VLA-4 transfectants showed similar tethering and rolling on TNF-stimulated endothelium. Tethering efficiency, rolling velocity, and resistance to detachment are related to VCAM-1 density. Tethering and rolling did not occur on ICAM-1, fibronectin, or fibronectin fragments, and tethering did not require integrin activation or the presence of an alpha 4 cytoplasmic domain. Arrest of rolling cells on VCAM-1 occurred spontaneously, and/or was triggered by integrin activating agents Mn2+, phorbol ester, and mAb TS2/16. These agents, and the alpha 4 cytoplasmic domain, promoted increased resistance to detachment. Together the results show that VLA-4 is a versatile integrin that can mediate tethering, rolling, and firm arrest on VCAM-1.

Cell Adhesion↗
PubMed1995

Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1).

Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction.

Animals↗
PubMed1995

Specialized functional properties of the integrin alpha 4 cytoplasmic domain.

For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.

Animals↗
PubMed1994

TNF-alpha associated with fibronectin enhances phorbol myristate acetate- or antigen-mediated integrin-dependent adhesion of CD4+ T cells via protein tyrosine phosphorylation.

The effects of cytokines on immune cells may be influenced by their milieu, such as the extracellular matrix (ECM), in the vicinities of which cytokines and inflammatory cells interact and function. Previously, we demonstrated that TNF-alpha bound to fibronectin (FN) and augments the level of adhesion of activated CD4+ cells. Herein, we examined the mechanisms of this pro-adhesive activity of TNF-alpha and its putative physiologic consequences using human or rat CD4+ cells. A brief exposure of CD4+ cells to low dosages of soluble TNF-alpha or of FN- or laminin-bound TNF-alpha synergized with PMA to enhance the integrin-mediated binding of CD4+ cells to these immobilized ECM moieties. TNF-alpha-enhanced adhesion of CD4+ cells did not delay or inhibit the subsequent detachment of the cells from the substrate, and adhesion was increased provided the cells were treated with TNF-alpha immediately after their exposure to PMA. This indicates that the enhancing effect of TNF-alpha requires a previous activation of the cells. When TNF-alpha was immobilized on FN, less TNF-alpha was required to induce CD4+ cell binding to FN. Soluble, and to a greater extent FN-bound, TNF-alpha synergizes with PMA to intensify protein tyrosine phosphorylation in FN-bound CD4+ cells, and this effect of TNF-alpha was inhibited by inhibitors of tyrosine kinase. That FN-bound or soluble TNF-alpha also amplified the binding of an Ag-specific autoimmune rat T cell line to immobilized FN, emphasizes the physiologic relevance of our findings. Thus, the signal transduction and cell adhesive properties of ECM glycoproteins may be modulated upon their association with TNF-alpha, and matrix-linked TNF-alpha may recruit and direct immune cells to inflammatory sites.

Animals↗
PubMed1994

TNF-alpha binds to the N-terminal domain of fibronectin and augments the beta 1-integrin-mediated adhesion of CD4+ T lymphocytes to the glycoprotein.

Certain inflammatory cytokines and growth factors have been previously shown to interact with glycosaminoglycan moieties of the extracellular matrix (ECM). We have examined the association of the pleiotropic cytokine TNF-alpha with glycoprotein constituents of ECM. TNF-alpha interacted with fibronectin (FN) and laminin, and to a lesser degree with collagen. The major binding site for TNF-alpha on FN was localized to its 30-kDa N-terminal fragment (FN-N') with a Ki in the sub-nM range. The binding of 125I-labeled TNF-alpha to immobilized FN or FN-N' persisted for at least 24 h, and was specifically inhibited by antibodies to FN, mAb directed against the FN-N' domain, unlabeled TNF-alpha, and by the truncated forms of TNF-alpha receptors. Once bound to immobilized FN or FN-N', the cytokine could not be released by the soluble TNF-alpha-receptors, although it could be released by anti-TNF-alpha Ab. TNF-alpha was also found to interact with soluble FN, although with a lower affinity. Similar to the soluble cytokine, the FN-bound TNF-alpha appears to be functional; it augmented the beta 1-integrin-mediated adhesiveness of activated CD4+ human T cells to the glycoprotein. Hence, binding of TNF-alpha to immobilized FN, which modifies its functional accessibility to soluble TNF-alpha receptors, does not abolish but rather may locally restrict its activity. This study suggests that a major ECM glycoprotein can present, in a restricted manner, a functional adhesion-modulating cytokine to immune cells, and that ECM glycoproteins may regulate their intrinsic cell-adhesive properties by associating with cytokines.

Amino Acid Sequence↗
PubMed1994

Distinct cell surface ligands mediate T lymphocyte attachment and rolling on P and E selectin under physiological flow.

Memory T lymphocytes extravasate at sites of inflammation, but the mechanisms employed by these cells to initiate contact and tethering with endothelium are incompletely understood. An important part of leukocyte extravasation is the initiation of rolling adhesions on endothelial selectins; such events have been studied in monocytes and neutrophils but not lymphocytes. In this study, the potential of T lymphocytes to adhere and roll on endothelial selectins in vitro was investigated. We demonstrate that T cells can form tethers and rolling adhesions on P selectin and E selectin under physiologic flow conditions. Tethering and rolling on P selectin was independent of cell-surface cutaneous lymphocyte antigen (CLA) expression, which correlated strictly with the capacity of T cells to form rolling adhesions under flow on E selectin. T cell tethering to P selectin was abolished by selective removal of cell surface sialomucins by a P. haemolytica O-glycoprotease, while cutaneous lymphocyte antigen expression was unaffected. A sialomucin molecule identical or closely related to P selectin glycoprotein ligand-1 (PSGL-1), the major P selectin ligand on neutrophils and HL-60 cells, appears to be a major T cell ligand for P selectin. P selectin glycoprotein ligand-1 does not appear to support T cell rolling on E selectin. In turn, E selectin ligands do not appear to be associated with sialomucins. These data demonstrate the presence of structurally distinct ligands for P or E selectins on T cells, provide evidence that both ligands can be coexpressed on a single T cell, and mediate tethering and rolling on the respective selectins in a mutually exclusive fashion.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1994

Inhibition of CD4+ T lymphocyte binding to fibronectin and immune-cell accumulation in inflammatory sites by non-peptidic mimetics of Arg-Gly-Asp.

The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta 1 subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha 11b beta 3, and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4+ T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition.

Amino Acid Sequence↗
PubMed1994

Novel psi-S-CH2 peptide-bond replacement and its utilization in the synthesis of nonpeptidic surrogates of the Leu-Asp-Val sequence that exhibit specific inhibitory activities on CD4+ T cell binding to fibronectin.

The Leu-Asp-Val-(LDV)-containing amino acid sequence, derived from the alternatively spliced first connecting segment region of fibronectin (FN), was shown to be recognized primarily by the alpha 4 beta 1-integrin receptor expressed on the surface of various cell types. This adhesion epitope may therefore inhibit integrin-mediated cell interactions with the extracellular matrix glycoprotein, including adhesion, migration, activation and differentiation. To probe the structural requirements for LDV recognition by integrins and examine the feasibility of inhibition of LDV-dependent cell-FN interactions, we have designed and constructed a novel psi-S-CH2 peptide bond surrogate that was employed in the formation of LDV surrogates. The synthesis of the psi-S-CH2 surrogates reported herein is based on Michael addition of 4-methylpentane thiol to an itaconic acid diester to form an S-CH2 bond. We have found that the LDV surrogates comprises of 4-methylpentanoate-Asp-i-butyl amide and 8-methyl-3-(2-methylpropylaminocarbonyl)-5-thianonanoic acid interfered with CD4+ human T-cell adhesion to FN in vitro, with an ED50 of 280 micrograms/mL. A control structural mimetic of the Leu-Glu-Val (LEV) peptide did not interfere with the T-cell-FN interaction. The specificity of the reaction was substantiated by the finding that the LDV mimetics did not interfere with T-cell adhesion to laminin, another major cell-adhesive glycoprotein of the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1993

Inhibition of integrin-mediated platelet aggregation, fibrinogen-binding, and interactions with extracellular matrix by nonpeptidic mimetics of Arg-Gly-Asp.

The interaction of the activated platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) with fibrinogen and von-Willebrand factor (vWF) is essential for platelet aggregation. The minimal structure required for this integrin's binding to fibrinogen is the Arg-Gly-Asp (RGD) sequence. Inasmuch as normal level of GPIIb-IIIa-RGD interactions are required for maintaining hemostasis, elevated platelet aggregation can cause adverse pathological effects. We have previously reported that nonpeptidic mimetics of RGD, consisting of carboxylate and guanidinium groups of Asp and Arg divided by a linear 11-atom spacer, acquired a significant affinity for the GPIIb-IIIa integrin and inhibited platelet aggregation. The structural requirements for the interactions of the RGD sequence with GPIIb-IIIa and the inhibitory potential of a newly designed series of mimetics on platelet aggregation and interactions with extracellular matrix (ECM) were assayed herein. Adenosine-diphosphate (ADP)-induced platelet aggregation was inhibited in a dose-dependent manner by various RGD mimetics, with a maximal inhibition of 80-100% with an IC50 of 3 microM for the most potent inhibitor, NS-11, in which a six-membered ring was introduced into the spacer chain, which exceeded the IC50 attained with the original RGDS peptide. The inhibitory effect of the RGD mimetics was attributed to their specific interaction with the GPIIb-IIIa integrin, since these mimetics inhibited the binding of the PAC-1 mAb to GPIIb-IIIA. Furthermore, the binding of 125I-labeled fibrinogen to platelets was inhibited by the RGD surrogates in a dose-dependent and saturable manner. The RGD-mimetics also inhibited up to 70% the adhesion, aggregation, and deposition of platelets onto ECM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗