Rolling cell adhesion.

Rolling adhesion on vascular surfaces is the first step in recruiting circulating leukocytes, hematopoietic progenitors, or platelets to specific organs or to sites of infection or injury. Rolling requires the rapid yet balanced formation and dissociation of adhesive bonds in the challenging environment of blood flow. This review explores how structurally distinct adhesion receptors interact through mechanically regulated kinetics with their ligands to meet these challenges. Remarkably, increasing force applied to adhesive bonds first prolongs their lifetimes (catch bonds) and then shortens their lifetimes (slip bonds). Catch bonds mediate the counterintuitive phenomenon of flow-enhanced rolling adhesion. Force-regulated disruptions of receptor interdomain or intradomain interactions remote from the ligand-binding surface generate catch bonds. Adhesion receptor dimerization, clustering in membrane domains, and interactions with the cytoskeleton modulate the forces applied to bonds. Both inside-out and outside-in cell signals regulate these processes.

The kinetics of E-selectin- and P-selectin-induced intermediate activation of integrin αLß2 on neutrophils.

Selectins and integrins are key players in the adhesion and signaling cascade that recruits leukocytes to inflamed tissues. Selectin binding induces ß2 integrin binding to slow leukocyte rolling. Here, a micropipette was used to characterize neutrophil adhesion to E-selectin and intercellular adhesion molecule-1 (ICAM-1) at room temperature. The time-dependent adhesion frequency displayed two-stage kinetics, with an E-selectin-mediated fast increase to a low plateau followed by a slow increase to a high plateau mediated by intermediate-affinity binding of integrin αLß2 to ICAM-1. The αLß2 activation required more than 5 s contact to E-selectin and spleen tyrosine kinase (Syk) activity. A multi-zone channel was used to analyze αLß2 activation by P-selectin in separate zones of receptors or antibodies, finding an inverse relationship between the rolling velocity on ICAM-1 and P-selectin dose, and a P-selectin dose-dependent change from bent to extended conformations with a closed headpiece that was faster at 37°C than at room temperature. Activation of αLß2 exhibited different levels of cooperativity and persistent times depending on the strength and duration of selectin stimulation. These results define the precise timing and kinetics of intermediate activation of αLß2 by E- and P-selectins.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin.

During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

Th1 Cells Rolling on Selectins Trigger DAP12-Dependent Signals That Activate Integrin αLß2.

During inflammation, both neutrophils and effector T cells use selectins to roll and integrins to arrest in postcapillary venules. In both cell types, chemokines can transduce signals that convert integrin αLß2 to a high-affinity conformation, which interacts with ICAM-1 to mediate arrest. In neutrophils, selectins also trigger an immunoreceptor-like signaling cascade that converts integrin αLß2 to an intermediate-affinity conformation, which interacts with ICAM-1 to slow rolling. It is not known whether selectins induce similar signaling events in T cells. Ag engagement causes phosphorylation of ITAMs on the TCR; these motifs recruit kinases and adaptors that lead to the activation of αLß2. We found that mouse Th1 cells rolling on P- or E-selectin triggered signals that promoted αLß2-dependent slow rolling on ICAM-1 in vitro and in vivo. The selectin signaling cascade resembled that used by the TCR, except that unexpectedly, Th1 cells employed the ITAM-bearing protein DAP12, which was not known to be expressed in these cells. Importantly, outside-in signaling through ligand-occupied αLß2 also required DAP12. Cooperative selectin and chemokine signaling in Th1 cells promoted αLß2-dependent slow rolling and arrest in vitro and in vivo and migration into Ag-challenged tissues in vivo. Our findings reveal an important function for DAP12 in Th1 cells and a new mechanism to recruit effector T cells to sites of inflammation.

'It Makes You Sit Back and Think Where You Wanna Go': Veteran experiences in virtual whole health peer-led groups.

BACKGROUND: The Veterans Health Administration (VHA) is building a Whole Health system of care that aspires to empower and equip each Veteran to pursue a personally meaningful vision of health and well-being. As part of this effort, VHA has developed Taking Charge of My Life and Health (TCMLH), a peer-led, group-based programme that seeks to support Veterans in setting and pursuing health and well-being goals. Prior research showed TCMLH groups to positively impact Veteran outcomes; yet, little is known about Veterans' own experiences and perspectives. METHODS: We completed semi-structured telephone interviews with 15 Veterans across 8 sites who had participated in TCMLH groups offered by the VHA in the virtual format between Summer 2020 and Fall 2021. Inductive thematic analysis was applied to interview transcripts to generate themes. FINDINGS: We identified five themes regarding Veterans' experiences with TCMLH: (1) navigating the virtual format; (2) internalizing the value of health engagement; (3) making healthy lifestyle changes; (4) forging social connections; and (5) taking on a more active role in healthcare. CONCLUSION: Veterans perceived virtual TCMLH groups as meaningful and beneficial, yet also highlighted several challenges. Their perspectives speak to the need to supplement time-limited programmes like TCMLH with ongoing, community-based support. Virtual group-based well-being programmes are a promising innovation. Other healthcare systems may draw on VHA's experience while tailoring format and content to the needs of their patient populations. PATIENT OR PUBLIC CONTRIBUTION: Veterans were involved as evaluation participants. A Veteran consultant, who is a coauthor on this paper, was engaged through the conceptualization of the evaluation, development of data collection materials (interview guide) and writing.

Locust bean gum adsorption onto softwood kraft pulp fibres: isotherms, kinetics and paper strength.

The adsorption of locust bean gum (LBG) onto Northern Bleached Softwood Kraft (NBSK) pulp improved paper tensile and burst strength and lowered refining energy by strengthening inter-fibre bonding. Adsorption kinetics and isotherms were investigated to develop a fundamental understanding of the adsorption mechanism. The adsorption rate followed pseudo-second-order kinetics and the activation energy was 99.34 kJ·mol-1, suggesting chemisorption. The adsorption rate constant increased rapidly with temperature from 25 to 45 °C (k = 1.93 to 24.03 g·mg-1·min-1), but the amount adsorbed at equilibrium decreased (q e = 1.91 to 0.48 mg·g-1 o.d. fibre). LBG adsorption to NBSK at 25 °C was consistent with the Langmuir adsorption model for LBG < 2.1 wt% of o.d. fibre, suggesting reversible, homogenous adsorption to a finite number of sites on the fibre surface. Refining to 3000 rev increased the heterogeneity of the NBSK pulp surface leading to multi-layer Freundlich adsorption with adsorption constant n = 5.00, and the equilibrium constant K f = 2.57 mg·g-1·(mg·L-1)-1/n at 25 °C. Favorable adsorption conditions for negatively charged LBG were identified: 25 °C for 10 min, low dosage level (< 2 wt%), lightly refined (< 3000 rev) NBSK pulp at low fibre consistency (< 0.5 wt%), high agitation rate (> 150 r.p.m.), acidic or neutral conditions (pH 2-7) without salt addition. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10570-021-04133-w.

Cooperative PSGL-1 and CXCR2 signaling in neutrophils promotes deep vein thrombosis in mice.

Inflammation is a major contributor to deep vein thrombosis (DVT). Flow restriction of the inferior vena cava (IVC) in mice induces DVT like that in humans. In this model, P-selectin-dependent adhesion of neutrophils and monocytes leads to release of neutrophil extracellular traps (NETs) and expression of tissue factor. However, it is not known what signals cause myeloid cells to generate these procoagulant effectors. Using ultrasonography and spinning-disk intravital microscopy in genetically engineered mice, we found that engagement of P-selectin glycoprotein ligand-1 (PSGL-1) and the chemokine receptor CXCR2 on rolling neutrophils propagated signals that cooperated to induce ß2 integrin-dependent arrest in flow-restricted IVCs. Unlike previous reports, PSGL-1 signaling in neutrophils did not require L-selectin, and it used tyrosine 145 rather than tyrosines 112 and 128 on the adaptor Src homology domain-containing leukocyte phosphoprotein of 76 kDa. PSGL-1 and CXCR2 signaling cooperated to increase the frequency and size of thrombi, in part by stimulating release of NETs. Unlike in neutrophils, blocking PSGL-1 or CXCR2 signaling in monocytes did not affect their recruitment into thrombi or their expression of tissue factor. Our results demonstrate that neutrophils cooperatively signal through PSGL-1 and CXCR2 to promote DVT.

A classical view on nonclassical nucleation.

Understanding and controlling nucleation is important for many crystallization applications. Calcium carbonate (CaCO3) is often used as a model system to investigate nucleation mechanisms. Despite its great importance in geology, biology, and many industrial applications, CaCO3 nucleation is still a topic of intense discussion, with new pathways for its growth from ions in solution proposed in recent years. These new pathways include the so-called nonclassical nucleation mechanism via the assembly of thermodynamically stable prenucleation clusters, as well as the formation of a dense liquid precursor phase via liquid-liquid phase separation. Here, we present results from a combined experimental and computational investigation on the precipitation of CaCO3 in dilute aqueous solutions. We propose that a dense liquid phase (containing 4-7 H2O per CaCO3 unit) forms in supersaturated solutions through the association of ions and ion pairs without significant participation of larger ion clusters. This liquid acts as the precursor for the formation of solid CaCO3 in the form of vaterite, which grows via a net transfer of ions from solution according to z Ca2+ + z CO32- → z CaCO3 The results show that all steps in this process can be explained according to classical concepts of crystal nucleation and growth, and that long-standing physical concepts of nucleation can describe multistep, multiphase growth mechanisms.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1-/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1-/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1-/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1-/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1-/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice.

Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease.

Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2.

Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α) in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B). Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

Meningoencephalitis Following Endoscopic Dacryocystorhinostomy.

An 81-year-old man with unilateral nasolacrimal duct obstruction underwent routine endonasal dacryocystorhinostomy and 2 days postoperatively developed a severe headache and rigors. Subsequent CT and MRI imaging demonstrated a defect in the floor of the right anterior cranial fossa, which possibly was damaged during the operation, and meningoencephalitis of the adjacent frontal lobe gyrus. Following intravenous antibiotic treatment, the patient made a full recovery with no ongoing sequelae, and his epiphora completely resolved. To the authors' knowledge, this is the first report of meningoencephalitis following endonasal dacryocystorhinostomy. This complication should be considered in any patient with a febrile septic presentation or headache following endonasal dacryocystorhinostomy.

Glycan Bound to the Selectin Low Affinity State Engages Glu-88 to Stabilize the High Affinity State under Force.

Selectin interactions with fucosylated glycan ligands mediate leukocyte rolling in the vasculature under shear forces. Crystal structures of P- and E-selectin suggest a two-state model in which ligand binding to the lectin domain closes loop 83-89 around the Ca2+ coordination site, enabling Glu-88 to engage Ca2+ and fucose. This triggers further allostery that opens the lectin/EGF domain hinge. The model posits that force accelerates transition from the bent (low affinity) to the extended (high affinity) state. However, transition intermediates have not been described, and the role of Glu-88 in force-assisted allostery has not been examined. Here we report the structure of the lectin and EGF domains of L-selectin bound to a fucose mimetic; that is, a terminal mannose on an N-glycan attached to a symmetry-related molecule. The structure is a transition intermediate where loop 83-89 closes to engage Ca2+ and mannose without triggering allostery that opens the lectin/EGF domain hinge. We used three complementary assays to compare ligand binding to WT selectins and to E88D selectins that replaced Glu-88 with Asp. Soluble P-selectinE88D bound with an ∼9-fold lower affinity to PSGL-1, a physiological ligand, due to faster dissociation. Adhesion frequency experiments with a biomembrane force probe could not detect interactions of P-selectinE88D with PSGL-1. Cells expressing transmembrane P-selectinE88D or L-selectinE88D detached from immobilized ligands immediately after initiating flow. Cells expressing E-selectinE88D rolled but detached faster. Our data support a two-state model for selectins in which Glu-88 must engage ligand to trigger allostery that stabilizes the high affinity state under force.

O-glycans direct selectin ligands to lipid rafts on leukocytes.

Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1ß, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

P-Selectin Expressed by a Human SELP Transgene Is Atherogenic in Apolipoprotein E-Deficient Mice.

OBJECTIVE: During inflammation, P-selectin expressed on activated endothelial cells and platelets mediates rolling adhesion of leukocytes. Atherosclerosis-prone mice crossed with P-selectin-deficient (Selp(-/-)) mice develop smaller lesions. Cytokines, such as tumor necrosis factor-α, increase Selp transcripts and augment atherosclerosis in mice. However, they decrease SELP transcripts in humans, challenging assumptions that human P-selectin is atherogenic. We used mice expressing a human SELP transgene to examine the atherogenic role of P-selectin. APPROACH AND RESULTS: We crossed apolipoprotein E-deficient (Apoe(-/-)) mice with Selp(-/-) mice or transgenic mice expressing the entire human SELP gene (TgSELP(+/-)). Aortas developed larger, macrophage-rich atheromas in Apoe(-/-)Selp(-/-)TgSELP(+/-) mice than in Apoe(-/-)Selp(-/-) mice after 8 or 16 weeks on a Western diet. Confocal microscopy of Apoe(-/-)Selp(-/-)TgSELP(+/-) aortas revealed staining for human P-selectin in endothelial cells overlying atheromas but not in lesional macrophages. We also observed staining for human P-selectin in aortic endothelial cells of 3- to 4-week-old Apoe(-/-)Selp(-/-)TgSELP(+/-) weanlings before atheromas developed. Furthermore, human SELP transcripts were ≈3-fold higher in aortas of Apoe(-/-)Selp(+/-)TgSELP(+/-) weanlings than in Selp(+/-)TgSELP(+/-) weanlings, whereas murine Selp and Sele transcripts were equivalent in weanlings of both genotypes. Human SELP transcripts in aortas of Apoe(-/-)Selp(+/-)TgSELP(+/-) mice remained nearly constant during 16 weeks on a Western diet, whereas murine Selp and Sele transcripts progressively increased. Bone marrow transplantation in Apoe(-/-)Selp(-/-) and Apoe(-/-)Selp(-/-)TgSELP(+/-) mice demonstrated that both platelets and endothelial cells must express human P-selectin to promote atherogenesis. CONCLUSIONS: P-selectin expressed by human SELP is atherogenic in Apoe(-/-) mice, suggesting that P-selectin contributes to atherogenesis in humans.

Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, ß2 integrin-dependent slow rolling and chemokine-induced, ß2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced ß2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-ß2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and ß2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases.

Predictors of functional status at service entry and discharge among young people with first episode psychosis.

OBJECTIVE: Most patients with first episode psychosis (FEP) are neither studying nor employed (have a poor functional status) when first accessing care. Knowledge of the characteristics of patients with poor functioning and the features influencing functional status over time may pave the way to better treatment. METHOD: A medical file audit was used to collect data on premorbid, entry, treatment and 18-month outcome characteristics on 661 FEP patients who consecutively attended the Early Psychosis Prevention and Intervention Centre, Melbourne, Australia, between 1998 and 2000. Functional status was ascertained using the modified vocational status index and was rated at baseline (poor or good) and according to its evolution over the treatment period (stable good, stable poor, deteriorating or improved functional status). RESULTS: 52.0% of patients had a poor functional status at service entry. They were more likely to be male with a non-affective psychosis. They also had lower levels of premorbid global functioning and education, and were more likely to have self-reported histories of learning disability, forensic issues, traumatic experiences and substance use. At service entry, they had more severe symptoms and poorer global functioning. 37% of these patients maintained a poor functional status at discharge, and 18% of those with a good functional status at service entry experienced a decline. CONCLUSIONS: Although psychosocial interventions might assist a young person with FEP with working towards functional goals, for some, the impact of factors such as ongoing substance use and forensic issues on functional status needs to be addressed.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets.

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan-deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan-deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph.