E-selectin is a transmembrane endothelium adhesion protein involved in rolling, arrest and migration of leukocytes as well as in the metastasis of many cancer types. Previous reports suggested that the interactions between E-selectin and its ligands transduce signals into migrating leukocytes and in E-selectin expressing endothelial cells. This study investigates the signaling pathways involved in E-selectin binding to ligands on leukocytes. Using recombinant soluble E-selectin constructs, we simulated the binding of E-selectin to its ligand(s) to reveal important signaling pathways triggered upon these interactions in acute myeloid leukemia (AML) cells. Since phosphorylation is the major post-translational modification, we examined the changes in the phosphorylation profile in tyrosine residues. We found a time-dependent reduction in the phosphotyrosine levels upon E-selectin binding to the AML cell line, KG-1a. The results of this study revealed two tyrosine phosphatases with altered activity after E-selectin treatment. The first is a cytoplasmic, dual-specific, phosphatase known as PTEN which is involved in controlling cell survival and proliferation. The second is CD45, which is a major component of the leukocytes cell membrane responsible for antigen receptor signaling. A more global phosphoproteomics analysis in AML cells revealed large scale changes in the phosphorylation levels after E-selectin treatment. In particular, 2259 phosphorylated proteins were identified, 530 of which portray significant changes in their phosphorylation status. The majority of those proteins are related to nuclear functions and are involved in pathways crucial for the cell cycle. Knowing that E-selectin binding stimulates chemoresistance in cancer cells, the findings of this project can contribute to the identification of multiple pathways responsible for this phenomenon and help towards the development of drugs that may inhibit such pathways in controlling disease.

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No description available.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / IntroduÃÃo e objetivos: Os neutrÃfilos desempenham importante papel na pancreatite aguda grave. InfiltraÃÃo de neutrÃfilos no pÃncreas à um processo complexo, coordenado por molÃculas de adesÃo especÃficas, tais como a P-selectina. Fucoidina à um polissacarÃdeo sulfatado que bloqueia a funÃÃo da L-e P-selectinas. No presente estudo avaliamos se o tratamento com fucoidina poderia impedir a infiltraÃÃo de neutrÃfilos e, assim, reverter a inflamaÃÃo sistÃmica e dismotilidades gastrintestinais associadas à pancreatite aguda grave. MÃtodos: A pancreatite aguda foi induzida em camundongos Swiss pela infusÃo retrÃgrada de Ãcido taurolitocÃlico (3,0%) (TLC-S) no ducto pancreÃtico ou por injeÃÃes intraperitoneais de ceruleÃna (50 Âg/kg/h). Os grupos experimentais receberam fucoidina (25 mg/kg, iv) antes da induÃÃo da pancreatite, e os grupos de controle receberam apenas soluÃÃo salina. ApÃs 24 horas, os nÃveis sÃricos de amilase, lipase, IL-1β, TNF-α, nitrito e de malondialdeÃdo (MDA) pancreÃtico foram medidos. AlÃm disso, a atividade de mieloperoxidase (MPO) (pulmÃo, pÃncreas, estÃmago e jejuno) e avaliaÃÃo histolÃgica (pÃncreas) foram determinadas. O esvaziamento gÃstrico e trÃnsito gastrintestinal foram medidos pelo mÃtodo de centro geomÃtrico. A contratilidade gastrintestinal in vitro foi registrada atravÃs de transdutores de forÃa conectados a sistema computadorizado de aquisiÃÃo de dados. Carbacol (0,01 ÂM - 30 ÂM), KCl 60 mM e estimulaÃÃo elÃctrica (0,5-8,0 Hz; 1ms, 40 V), foram aplicados sobre o fundo gÃstrico e jejuno dos animais 24 horas apÃs a pancreatite induzida por TLC-S. Resultados: Os nÃveis de MDA pancreÃtico, amilase, lipase, nitrito, TNF-α e IL-1β sÃricos, bem como MPO pancreÃtica e pulmonar estavam aumentados tanto no modelo de pancreatite aguda induzida por TLCS quanto no modelo ceruleÃna quando comparado aos grupos controle correspondentes. Fucoidina reduziu significativamente os nÃveis aumentados de amilase, lipase, MPO pancreÃtica e pulmonar, MDA, TNF-α, IL-1β e nitrito em ambos os modelos de pancreatite aguda. As mudanÃas histolÃgicas observadas no pÃncreas em ambos os modelos foram significativamente atenuadas pela fucoidina. O modelo de pancreatite aguda induzida por TLC-S induziu retardo no esvaziamento gÃstrico e trÃnsito gastrointestinal, aumento de MPO no estÃmago e no jejuno, alÃm de hipercontratilidade de jejuno in vitro. Fucoidina reverteu significativamente os distÃrbios gastrintestinais in vivo e in vitro e os nÃveis aumentados de MPO gÃstrica e jejunal induzidos pela injeÃÃo de TLC-S. ConclusÃo: Fucoidina reduziu a gravidade da pancreatite aguda experimental atravÃs da diminuiÃÃo da infiltraÃÃo de neutrÃfilos, inflamaÃÃo sistÃmica e dismotilidades gastrintestinais, sugerindo que a modulaÃÃo das selectinas, pode constituir uma abordagem terapÃutica promissora para pancreatite aguda. / Background & Aims: Neutrophils play a critical role in severe acute pancreatitis. Tissue infiltration of neutrophils in the pancreas is a multistep process, coordinated by specific adhesion molecules, such as P-selectin. Fucoidin is a sulphated fucosylated polysaccharide that binds to and blocks the function of L- and P-selectins, and the present study has evaluated whether fucoidin treatment could prevent neutrophil infiltration, and thereby reverse the systemic inflammation and gastrointestinal dysmotility associated with severe acute pancreatitis. Methods: Acute pancreatitis was induced in Swiss mice either by the retrograde infusion of taurolithocholic acid (3.0%) (TLC-S) into the pancreatic duct or by intraperitoneal injections of cerulein (50 Âg/kg/h). The experimental groups received fucoidan (25 mg/kg, i.v.) before pancreatitis induction whist control groups received only saline. After 24 hours, pancreatic malondialdehyde (MDA), serum amylase, lipase, IL-1β, TNF- and nitrite were measured. In addition, myeloperoxidase (MPO) activity (lung, pancreas, stomach and jejunum) and histological assessment (pancreas) were determined. Gastric emptying and gastrointestinal transit (using the geometric center method) were also measured. Gastrointestinal contractility in vitro was recorded through force transducers coupled to a computerized data acquisition system, carbachol (0,01 ÂM â 30 ÂM), KCl 60mM and electrical field stimulation (0.5-8.0 Hz; 1ms; 40 V), was applied on gastric fundus and jejunum of mice 24 hours after TLC-S induced pancreatitis. Results: Pancreatic MDA, serum amylase, lipase, nitrite, TNF- and IL-1β, pancreatic and lung MPO, were increased in both TLCS- and cerulein acute pancreatitis compared with respective control groups. Fucoidan significantly decreased the augmented levels of amylase, lipase, pancreatic and lung MPO, MDA, TNF-, IL-1β and nitrite in both acute pancreatitis models. Pancreas histological changes observed in both models were significantly attenuated by fucoidan. The acute pancreatitis model induced by TLC-S caused delayed gastric emptying and gastrointestinal transit, incresead gastric and jejunum MPO, and jejunum hypercontractility in vitro. Fucoidan significantly reversed the gastrointestinal disorders in vivo and in vitro and augmented levels of gastric and jejunum MPO induced by TLC-S. Conclusion: Fucoidan reduced the severity of acute pancreatitis in mice by decreasing neutrophil infiltration, systemic inflammation and gastrointestinal dysmotility, suggesting that modulation of selectins may constitute a promising therapeutic approach for acute pancreatitis.

No description available.

Lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) express large numbers of P2X7 receptors for extracellular adenosine triphosphate (ATP). Activation of P2X7 receptors induces multiple downstream effects, of which the best documented is the opening of an ionic channel that is selective for divalent cations. Another effect of ATP is to induce the shedding of L-selectin (CD62L), a molecule which is involved in the adhesive interactions of lymphocytes on endothelial cells. High levels of soluble L-selectin and CD23 are found in the serum of patients with B-CLL, although the mechanisms involved in their production are poorly characterized. Because extracellular ATP causes shedding of L-selectin, we studied the effect of ATP on shedding of CD23, an adhesion molecule expressed on the surface of B-CLL lymphocytes. ATP induced the shedding of CD23 at an initial rate of 12% of that for L-selectin, while the EC50 of ATP (35 uM) and BzATP (10 uM) was identical for shedding of both molecules. Inactivation of the P2X7 receptor by pre-incubation with OxATP, an irreversible inhibitor of P2X7 purinoceptor, abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2X7 receptor inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nM). Ro 31-9790, a membrane permeant zinc chelator which inhibits the phorbol-ester stimulated shedding of L-selectin also inhibited shedding of CD23 from B-CLL lymphocytes, but the IC50 was different for the two shed molecules (25 versus 1 ug/ml respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester no CD23 was lost under these conditions. Also, Ca2+ inhibits ATP-induced CD23 shedding but not L-selectin shedding. Since soluble CD23 and L-selectin are found in the serum of normal subjects and B-CLL patients, the expression of these two adhesion molecules on lymphocytes before and after transendothelial migration was studied in an in vitro model of this process. In normal and B-CLL subjects, 71�b5% of L-selectin from both T and B cells and 90% of CD23 from B cells was lost following transmigration, while the expression of a range of other adhesion molecules such as VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with OxATP retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Ro 31-9790, which can protect ATP-induced both L-selectin and CD23 shedding, had no effect on inhibiting L-selectin and CD23 lost during transmigration. These data show the presence of a second pathway for the downregulation of L-selectin and CD23 from the lymphocyte surface. Data in vivo from 'knock-out' mice show that L-selectin is essential for the emigration of lymphocytes through high endothelial venules into lymph nodes. The migration of normal and B-CLL lymphocytes across confluent human umbilical vein endothelial monolayers was studied in an in vitro model of this process. Lymphocytes treated with ATP or BzATP showed 56�b25% or 67�b16% loss of L-selectin on the surface and 36�b24% or 64�b19% decrease of transmigration, respectively, while OxATP, which does not alter the L-selectin level, had no effect on lymphocyte transmigration. Further experiments examined this correlation between L-selectin expression and lymphocyte transendothelial migration in this model system. A quantitative assay for cell surface L-selectin showed that expression of L-selectin was lower on B-CLL lymphocytes (8,880�b5,700 molecules/cell) than on normal lymphocytes (29,500�b7,500 molecules/cell, p less than 0.001). Also the rate of transmigration of B-CLL lymphocytes (1.5�b0.9 migrated cells/HUVEC) was lower than normal peripheral lymphocytes (2.4�b0.9 migrated cells/HUVEC, p=0.04). Incubation of lymphocytes in complete medium for 24 hrs increased the expression of L-selectin on B-CLL lymphocytes by 1.5 to 2 fold while the normal lymphocyte L-selectin remained at the initial level. This upregulation of B-CLL L-selectin correlated with a 2 fold increased rate of transendothelial migration. A correlation was found between L-selectin expression on lymphocytes and their ability for transendothelial migration (r^2=0.6). This study shows that the adhesion molecules L-selectin and CD23 can be lost from lymphocytes by two different physiological pathways. One is by P2X7 receptor activation by extracellular ATP while the second is activated by transendothelial migration of these cells. A second finding is that B-CLL lymphocytes have lower level of L-selectin expression and an impaired ability for transendothelial migration compared with normal peripheral blood lymphocytes. Do these results explain the high serum levels of soluble L-selectin and CD23 observed in B-CLL? Although B-CLL lymphocytes do not recirculate as rapidly as normal peripheral blood lymphocytes, the greatly increased number of leukaemic cells in B-CLL ensures that much more soluble L-selectin and CD23 is generated during the recirculation of these cells through the body.

No description available.

Lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) express large numbers of P2X7 receptors for extracellular adenosine triphosphate (ATP). Activation of P2X7 receptors induces multiple downstream effects, of which the best documented is the opening of an ionic channel that is selective for divalent cations. Another effect of ATP is to induce the shedding of L-selectin (CD62L), a molecule which is involved in the adhesive interactions of lymphocytes on endothelial cells. High levels of soluble L-selectin and CD23 are found in the serum of patients with B-CLL, although the mechanisms involved in their production are poorly characterized. Because extracellular ATP causes shedding of L-selectin, we studied the effect of ATP on shedding of CD23, an adhesion molecule expressed on the surface of B-CLL lymphocytes. ATP induced the shedding of CD23 at an initial rate of 12% of that for L-selectin, while the EC50 of ATP (35 uM) and BzATP (10 uM) was identical for shedding of both molecules. Inactivation of the P2X7 receptor by pre-incubation with OxATP, an irreversible inhibitor of P2X7 purinoceptor, abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2X7 receptor inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nM). Ro 31-9790, a membrane permeant zinc chelator which inhibits the phorbol-ester stimulated shedding of L-selectin also inhibited shedding of CD23 from B-CLL lymphocytes, but the IC50 was different for the two shed molecules (25 versus 1 ug/ml respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester no CD23 was lost under these conditions. Also, Ca2+ inhibits ATP-induced CD23 shedding but not L-selectin shedding. Since soluble CD23 and L-selectin are found in the serum of normal subjects and B-CLL patients, the expression of these two adhesion molecules on lymphocytes before and after transendothelial migration was studied in an in vitro model of this process. In normal and B-CLL subjects, 71�b5% of L-selectin from both T and B cells and 90% of CD23 from B cells was lost following transmigration, while the expression of a range of other adhesion molecules such as VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with OxATP retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Ro 31-9790, which can protect ATP-induced both L-selectin and CD23 shedding, had no effect on inhibiting L-selectin and CD23 lost during transmigration. These data show the presence of a second pathway for the downregulation of L-selectin and CD23 from the lymphocyte surface. Data in vivo from 'knock-out' mice show that L-selectin is essential for the emigration of lymphocytes through high endothelial venules into lymph nodes. The migration of normal and B-CLL lymphocytes across confluent human umbilical vein endothelial monolayers was studied in an in vitro model of this process. Lymphocytes treated with ATP or BzATP showed 56�b25% or 67�b16% loss of L-selectin on the surface and 36�b24% or 64�b19% decrease of transmigration, respectively, while OxATP, which does not alter the L-selectin level, had no effect on lymphocyte transmigration. Further experiments examined this correlation between L-selectin expression and lymphocyte transendothelial migration in this model system. A quantitative assay for cell surface L-selectin showed that expression of L-selectin was lower on B-CLL lymphocytes (8,880�b5,700 molecules/cell) than on normal lymphocytes (29,500�b7,500 molecules/cell, p less than 0.001). Also the rate of transmigration of B-CLL lymphocytes (1.5�b0.9 migrated cells/HUVEC) was lower than normal peripheral lymphocytes (2.4�b0.9 migrated cells/HUVEC, p=0.04). Incubation of lymphocytes in complete medium for 24 hrs increased the expression of L-selectin on B-CLL lymphocytes by 1.5 to 2 fold while the normal lymphocyte L-selectin remained at the initial level. This upregulation of B-CLL L-selectin correlated with a 2 fold increased rate of transendothelial migration. A correlation was found between L-selectin expression on lymphocytes and their ability for transendothelial migration (r^2=0.6). This study shows that the adhesion molecules L-selectin and CD23 can be lost from lymphocytes by two different physiological pathways. One is by P2X7 receptor activation by extracellular ATP while the second is activated by transendothelial migration of these cells. A second finding is that B-CLL lymphocytes have lower level of L-selectin expression and an impaired ability for transendothelial migration compared with normal peripheral blood lymphocytes. Do these results explain the high serum levels of soluble L-selectin and CD23 observed in B-CLL? Although B-CLL lymphocytes do not recirculate as rapidly as normal peripheral blood lymphocytes, the greatly increased number of leukaemic cells in B-CLL ensures that much more soluble L-selectin and CD23 is generated during the recirculation of these cells through the body.

Recent development of super-resolution (SR) fluorescence microscopy techniques has provided a new tool for direct visualization of subcellular structures and their dynamics in cells. The homing of Hematopoietic stem/progenitor cells (HSPCs) to bone marrow is a multistep process that is initiated by tethering of HSPCs to endothelium and mediated by spatiotemporally organised ligand-receptor interactions of selectins expressed on endothelial cells to their ligands expressed on HSPCs which occurs against the shear stress exerted by blood flow. Although molecules and biological processes involved in this multi-step cellular interaction have been studied extensively, molecular mechanisms of the homing, in particular the nanoscale spatiotemporal behaviour of ligand-receptor interactions and their role in the cellular interaction, remain elusive. Using our new method of microfluidics-based super-resolution fluorescence imaging platform we can now characterize the correlation between both nanoscale ligand-receptor interactions and tethering/rolling of cells under external shear stress. We found that cell rolling on E-selectin caused significant reorganization of the nanoscale clustering behavior of CD44 and CD43, from a patchy clusters of ~ 200 nm in size to an elongated network-like structures where for PSGL-1 the clustering size did not change significantly as it was 85 nm and after cell rolling the PSGL-1 aggregated to one side or even exhibited an increase in the footprint. Furthermore, I have established the use of 3D SR images that indicated that the patchy clusters of CD44 localize to protruding structures of the cell surface. On the other hand, a significant amount of the network-like elongated CD44 clusters observed after the rolling were located in the close proximity to the E-selectin surface. The effect of the nanoscale reorganization of the clusters on the HSPC rolling over selectins is still an open question at this stage. Nevertheless, my results further demonstrate that this mechanical force-induced reorganisation is accompanied by a large structural reorganisation of actin cytoskeleton. Our microfluidics-based SR imaging also demonstrate an essential role of the nanoscale clustering of CD44 on stable rolling behaviours of cells. Our new experimental platform enhances understanding of the relationship between nanoscopic ligand-receptor interactions and macroscopic cellular interactions, providing a foundation for characterizing complicated HSPC homing

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. Their extracellular structure is composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e., E-selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. Furthermore, selectins play a role beyond fixing cells to a specific location by regulating important signaling pathways in the migrating cell during physiological and pathological processes. These interactions start mainly with the binding of the lectin domain of selectins and ligand on cells. Therefore, structural/functional studies to date have mainly focused on the direct interactions of the lectin domain of E-selectin with its ligands while other domains and conformational dynamics received less attention. For this purpose, we produced a number of different recombinant E-selectin proteins with and without artificial oligomerization and with changes in the SCR units in addition to proteins where strategic residues will be mutated to change the conformation of the selectin to an extended conformer. Moreover, double cysteine mutant candidates were produced for maleimide labeling for the real-time SM-FRET (single molecule fluorescence resonance energy transfer) studies to assess conformational dynamics of E-selectin. Using a comprehensive set of static- and flow-based assays, we concluded that SCR domains play a role by enhancing the interaction of recombinant E-selectin proteins with E-selectin ligand, while dimerization and extension of the lectin domain improve the binding. However, our double cysteine mutants purification and labeling requires further optimization to be utilized to study the conformational dynamics of E-selectin binding to its ligands using SM-FRET and force microscopy. Furthermore, our experiments extend to highlight the importance of phosphatases in regulating signaling pathways that are affected by E-selectin binding to migrating cells. Collectively, these studies are beneficial to understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic stem cells (HSCs) adhesion to the selectin expressing endothelial cells.