CD47 knockout mice exhibit improved recovery from spinal cord injury.

Recent data have implicated thrombospondin-1 (TSP-1) signaling in the acute neuropathological events that occur in microvascular endothelial cells (ECs) following spinal cord injury (SCI) (Benton et al., 2008b). We hypothesized that deletion of TSP-1 or its receptor CD47 would reduce these pathological events following SCI. CD47 is expressed in a variety of tissues, including vascular ECs and neutrophils. CD47 binds to TSP-1 and inhibits angiogenesis. CD47 also binds to the signal regulatory protein (SIRP)α and facilitates neutrophil diapedesis across ECs to sites of injury. After contusive SCI, TSP-1(-/-) mice did not show functional improvement compared to wildtype (WT) mice. CD47(-/-) mice, however, exhibited functional locomotor improvements and greater white matter sparing. Whereas targeted deletion of either CD47 or TSP-1 improved acute epicenter vascularity in contused mice, only CD47 deletion reduced neutrophil diapedesis and increased microvascular perfusion. An ex vivo model of the CNS microvasculature revealed that CD47(-/-)-derived microvessels (MVs) prominently exhibit adherent WT or CD47(-/-) neutrophils on the endothelial lumen, whereas WT-derived MVs do not. This implicates a defect in diapedesis mediated by the loss of CD47 expression on ECs. In vitro transmigration assays confirmed the role of SIRPα in neutrophil diapedesis through EC monolayers. We conclude that CD47 deletion modestly, but significantly, improves functional recovery from SCI via an increase in vascular patency and a reduction of SIRPα-mediated neutrophil diapedesis, rather than the abrogation of TSP-1-mediated anti-angiogenic signaling.

Functions of the extracellular matrix and matrix degrading proteases during tumor progression

Cell interactions with extracellular matrices are important to pathological changes that occur during cell transformation and tumorigenesis. Several extracellular matrix proteins including fibronectin, thrombospondin-1, laminin, SPARC, and osteopontin have been suggested to modulate tumor phenotype by affecting cell migration, survival, or angiogenesis. Likewise, proteases including the matrix metalloproteinases (MMPs) are understood to not only facilitate migration of cells by degradation of matrices, but also to affect tumor formation and growth. We have recently demonstrated an in vivo role for the RGD-containing protein, osteopontin, during tumor progression, and found evidence for distinct functions in the host versus the tumor cells. Because of the compartmentalization and temporal regulation of MMP expression, it is likely that MMPs may also function dually in host stroma and the tumor cell. In addition, an important function of proteases appears to be not only degradation, but also cleavage of matrix proteins to generate functionally distinct fragments based on receptor binding, biological activity, or regulation of growth factors.

Rôle de la molécule CD47 sur le lymphocyte T dans la régulation de la réponse immunitaire

L’importance respective des lymphocytes T régulateurs naturels générés dans le thymus ou induits en périphérie dans la régulation immunitaire et la résolution de l’inflammation est désormais bien établie. Nous avons contribué à mettre en évidence une nouvelle voie d’induction de lymphocytes T régulateurs périphériques à partir de cellules T humaines CD4+CD25- naïves et mémoires. Nous avons montré que l’engagement de la molécule ubiquitaire transmembranaire CD47 sur la cellule T par un anticorps monoclonal ou par le peptide 4N1K (peptide dérivé du domaine carboxy-terminal de la thrombospondine-1 et spécifique du site de liaison à CD47) induisait des lymphocytes T CD4+ régulateurs exerçant une fonction suppressive sur les lymphocytes T effecteurs. Les propriétés suppressives induites par la thrombospondine-1 confortent les fonctions anti-inflammatoires de cette protéine de la matrice extracellulaire. L’inhibition exercée par les lymphocytes T régulateurs induits dépend du contact intercellulaire entre les cellules T régulatrices et leurs cibles, et est indépendante du TGF-. Nos résultats démontrent également le rôle de CD47 sur le lymphocyte T CD4+ dans la réponse immunitaire spécifique de l’antigène in vivo. En effet, les souris BALB/c déficientes pour CD47 présentent un biais de la sécrétion d’anticorps et de cytokines de type Th1, alors que les souris BALB/c sont décrites comme exprimant un profil de production de cytokines de type Th2. Nos travaux mettent en évidence le rôle de CD47 dans l’inhibition du développement d’une réponse cellulaire et humorale de type Th1 in vivo, confirmant de précédentes études in vitro réalisées avec des cellules T CD4+ humaines. Nous présentons également le rôle inhibiteur de l’engagement de CD28 in vitro sur la différenciation en cellules Th17 des lymphocytes T CD4+ naïfs isolés de souris BALB/c. Le mécanisme proposé est dépendant de la production de l’IL-2 et de l’IFN- et indépendant de la présence de lymphocytes T régulateurs. Notre étude du rôle de deux molécules transmembranaires CD47 et CD28 exprimées sur la cellule T CD4+, contribue à une meilleure connaissance des mécanismes impliqués dans la tolérance immunologique, la résolution de l’inflammation et la différenciation des cellules T "helper" CD4+.

Application du SPR dans le criblage des ligands synthétiques du CD36 et sa validation

Le CD36 est un récepteur de type éboueur de classe B exprimé à la surface de nombreux types cellulaires dont les macrophages, les cellules endothéliales de la microvasculature et les plaquettes. Ce récepteur multiligand est impliqué dans plusieurs processus pathologiques notamment l’athérosclérose, l’angiogénèse et la malaria via la liaison spécifique de ligands comme les lipoprotéines oxydées de basse densité, la thrombospondine-1 et la protéine PfEMP-1, respectivement. Les peptides de la relâche de l’hormone de croissance (GHRP) ont été identifiés comme les premiers ligands synthétiques du CD36. Afin de développer de nouveaux ligands synthétiques du CD36, l’établissement d’une méthode de criblage est essentiel pour découvrir des composés avec une liaison de haute affinité pour ce récepteur. Pour y parvenir, nous avons surexprimé le domaine extracellulaire du CD36 humain dans les cellules d’insectes Sf9. La protéine soluble purifiée par chromatographie d’affinité fut immobilisée à la surface d’une plaque de résonance de plasmons de surface (SPR) pour les études de liaison. La méthodologie développée a permis de caractériser les ligands du CD36 en déterminant leurs constantes de dissociation (KD), et d’établir une relation structure-activité des ligands de la famille des azapeptides, des composés dérivés du GHRP-6. Afin de valider la méthode par spectroscopie SPR, une corrélation a été établie entre les valeurs de KD obtenues en SPR et les valeurs d’CI50 de courbes d’inhibition de la phosphorylation des MAP kinases JNK1/2 induite par un phospholipide oxydé, le POVPC, en présence de concentrations croissantes de ligands du CD36 dans les macrophages RAW 264.7.

Secuenciación de siguiente generación en mujeres afectadas por falla ovárica prematura no sindrómica para la identificación de nuevos genes etiológicos de la enfermedad y estudio funcional de la mutación p.thr943ile en adamts19

La infertilidad afecta en la actualidad a aproximadamente 1 de cada 7 parejas a nivel mundial. La falla ovárica prematura (FOP) es una condición común en la población femenina, afectando al 1% de mujeres menores de 40 años. La etiología de la FOP es idiopática entre el 50% y el 80% de los casos, lo que sugiere causas genéticas, epigenéticas y ambientales aún desconocidas. A pesar de los avances en las técnicas de cartografía genética y de sistematización de la técnica de Sanger, pocos genes etiológicos de FOP fueron identificados en los últimos 20 años. Este fracaso relativo se asoció principalmente a que cientos de genes, que abarcan grandes regiones del genoma, son candidatos pero la técnica de secuenciación directa sólo permite el análisis de unas 700bp en cada reacción. En el presente trabajo se empleó la secuenciación de siguiente generación (NGS) para la búsqueda de mutaciones en 70 genes candidatos que potencialmente contribuyen con el desarrollo de la patología. Se identificaron mutaciones en 3 de 12 pacientes. La paciente POF-7 presentaba una mutación no sinónima en el gen ADAMTS19 (c.2828C>T, p.Thr943Ile). La proteína ADAMTS19 se clasifica dentro de la familia ADAMTS como huérfana ya que no se ha identificado su sustrato. Mediante el sistema de doble hibrido en levaduras se buscó identificar las potenciales proteínas que interactúan con ADAMTS19. Permitió identificar, a partir de las versiones murinas, la interacción de Adamts19 y Col6a2. Para comprobar la interacción entre las proteínas ADAMTS19 y COL6A2 humanas se empleó el sistema de doble hibrido en células eucariotas. Los hallazgos no permitieron replicar los resultados obtenidos previamente. En síntesis de identificó una mutación potencialmente causal de FOP en un gen nuevo y una muy probable interacción entre ADAMTS19 y COL6A2.

The role of plasma membrane STIM1 and Ca(2+)entry in platelet aggregation. STIM1 binds to novel proteins in human platelets.

Ca(2+) elevation is essential to platelet activation. STIM1 senses Ca(2+) in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca(2+) entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca(2+) entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca(2+) entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca(2+)-sensing role of STIM1 is served by the protein in the ER.

Methylation pattern of THBS1, GATA-4, and HIC1 in pediatric and adult patients infected with helicobacter pylori

Background: Helicobacter pylori infection is usually acquired in childhood and persists into adulthood if untreated. The bacterium induces a chronic inflammatory response, which is associated with epigenetic alterations in oncogenes, tumor-suppressor genes, cell-cycle regulators, and cell-adhesion molecules. Aim: The aim of this study was to analyze the effect of H. pylori infection on the methylation status of Thrombospondin-1 (THBS1), Hypermethylated in cancer 1 (HIC1) and Gata binding protein-4 (GATA-4) in gastric biopsy samples from children and adults infected or uninfected with the bacterium and in samples obtained from gastric cancer patients. Methods: The methylation pattern was analyzed with methylation-specific PCR. Results: Our results showed that H. pylori infection was associated with methylation of the promoter regions of the THBS1 and GATA-4 genes in pediatric and adult samples (p < 0.01). HIC1 showed the lowest level of methylation, which was not an early event during gastric carcinogenesis. Conclusions: The results from this study indicate that methylation of THBS1 and GATA-4 occurs in the early stages of chronic gastritis and gastric cancer in association with H. pylori infection; however, in gastric cancer samples, other mechanisms cooperate with the down-regulation of these genes. Methylation of HIC1 may not be the principal mechanism implicated in its down-regulation in gastric cancer samples. © 2013 Springer Science+Business Media New York.

Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

Capillary growth, ultrastructure remodelling and exercise training in skeletal muscle of essential hypertensive patients

AIM The aim was to elucidate whether essential hypertension is associated with altered capillary morphology and density and to what extent exercise training can normalize these parameters. METHODS To investigate angiogenesis and capillary morphology in essential hypertension, muscle biopsies were obtained from m. vastus lateralis in subjects with essential hypertension (n = 10) and normotensive controls (n = 11) before and after 8 weeks of aerobic exercise training. Morphometry was performed after transmission electron microscopy, and protein levels of several angioregulatory factors were determined. RESULTS At baseline, capillary density and capillary-to-fibre ratio were not different between the two groups. However, the hypertensive subjects had 9% lower capillary area (12.7 ± 0.4 vs. 13.9 ± 0.2 μm(2)) and tended to have thicker capillary basement membranes (399 ± 16 vs. 358 ± 13 nm; P = 0.094) than controls. Protein expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 and thrombospondin-1 were similar in normotensive and hypertensive subjects, but tissue inhibitor of matrix metalloproteinase was 69% lower in the hypertensive group. After training, angiogenesis was evident by 15% increased capillary-to-fibre ratio in the hypertensive subjects only. Capillary area and capillary lumen area were increased by 7 and 15% in the hypertensive patients, whereas capillary basement membrane thickness was decreased by 17% (P < 0.05). VEGF expression after training was increased in both groups, whereas VEGF receptor-2 was decreased by 25% in the hypertensive patients(P < 0.05). CONCLUSION Essential hypertension is associated with decreased lumen area and a tendency for increased basement membrane thickening in capillaries of skeletal muscle. Exercise training may improve the diffusion conditions in essential hypertension by altering capillary structure and capillary number.

Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an “antiadhesive” matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.

Characterization of Cell–Matrix Adhesion Requirements for the Formation of Fascin Microspikes

Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell–substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix requirements for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent on fibronectin, vitronectin, collagen IV, or platelet factor 4. However, both fascin microspikes and focal contacts were assembled by cells adherent on laminin-1. Using mixed substrata containing different proportions of TSP-1, and fibronectin, fascin microspike formation by H9c2 and C2C12 cells was found to be reduced on substrata containing 25% fibronectin and abolished on substrata containing 75% fibronectin. Adhesion to intermediate mixtures of TSP-1 and fibronectin resulted in coassembly of fascin microspikes and focal contacts, colocalization of fascin with actin stress fiber bundles and altered distributions of β1 integrins, cortical α-actinin, and tropomyosin. In cells adherent on 50% TSP-1:50% fibronectin, GRGDSP peptide treatment decreased focal contact assembly and altered cytoskeletal organization but did not inhibit microspike assembly. Treatment with chondroitin sulfate A or p-nitrophenol β-d-xylopyranoside decreased microspike formation and modified cytoskeletal organization but did not inhibit focal contact formation. In polarized migratory and postmitotic C2C12 cells, fascin microspikes and ruffles were localized at leading edges and TSP matrix deposition was also concentrated in this region. Depletion of matrix TSP by heparin treatment correlated with decreased microspike formation and cell motility. Thus, the balance of adhesive receptors ligated at the cell surface during initial cell–matrix attachment serves to regulate the type of substratum adhesion contact assembled and subsequent cytoskeletal organization. A role for fascin microspikes in cell motile behavior is indicated.

Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members α, γ, and λ, and PKCα localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCα activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin α5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

PTEN controls tumor-induced angiogenesis

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions. (C) 2004 Elsevier Ltd. All rights reserved.