Societal costs of illegal drug use in Sweden.

BACKGROUND: Illegal drug use is a public health concern with far-reaching consequences for people who use them and for society. In Sweden, the reported use of illegal drugs has been growing and the number of drug-induced deaths is among the highest in Europe. The aim of this study was to provide a comprehensive and up-to-date estimation of the societal costs of illegal drug use in Sweden, relying as much as possible on registry and administrative data. METHODS: A prevalence-based cost-of-illness study of illegal drug use in Sweden in 2020 was conducted. A societal approach was chosen and included direct costs (such as costs of health care, social services, and the criminal justice system), indirect costs (such as lost productivity due to unemployment and drug-induced death), and intangible costs (such as reduced quality of life among people who use drugs and their family members). Costs were estimated by combining registry, administrative, and survey data with unit cost data. RESULTS: The estimated societal costs of illegal drug use were 3.7 billion euros in 2020. This corresponded to 355 euros per capita and 0.78 % of the gross domestic product. The direct and intangible costs were of similar sizes, each contributing to approximately 40 % of total costs, whereas indirect costs contributed to approximately 20 %. The largest individual cost components were reduced quality of life among people who use drugs and costs of the criminal justice system. CONCLUSION: Illegal drug use has a negative impact on the societal aim to create good and equitable health in Sweden. The findings call for evidence-based prevention of drug use and treatment for those addicted. It is important to address the co-morbidity of mental ill-health and drug dependence, to develop low-threshold services and measures for early prevention among children and young adults, as well as to evaluate laws and regulations connected to illegal drug use.

Motivation 2 Quit (M2Q): A cluster randomized controlled trial evaluating the effectiveness of Tobacco Cessation on Prescription in Swedish primary healthcare.

OBJECTIVE: To evaluate the effectiveness of Tobacco Cessation on Prescription (TCP) compared to standard treatment in socioeconomically disadvantaged areas in Swedish primary healthcare (PHC). STUDY DESIGN: A pragmatic cluster randomized controlled trial, where randomization was conducted at the PHC center level using a computer-generated random allocation sequence. SETTING: 18 PHC centers in socioeconomically disadvantaged areas in Stockholm. PARTICIPANTS: 250 adult daily tobacco users (56% female, 41% foreign born) with Swedish social security numbers and permanent resident permits, fluent in Swedish or Arabic, of which 140 responded to the follow-up at 6 months and 139 to the follow-up at 12 months. No blinding was applied. INTERVENTIONS: TCP (tobacco cessation counseling for ≥10 minutes, an individualized prescription for tobacco cessation treatment and follow-up on ≥1 occasion) compared to standard treatment. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was self-reported 7-day abstinence at 6 months and the secondary outcomes included self-reported 7-day abstinence at 12 months and 3-month continued abstinence at 6 and 12 months follow-up. RESULTS: PHC centers were randomized to the intervention group (n = 8) and control group (n = 10). At the PHC centers, 250 patients (TCP n = 188, standard treatment n = 62) were recruited. There was a statistically significant effect of TCP compared to standard treatment for the outcomes 7-day abstinence at 6 months (OR adjusted 5.4, 95% CI 1.57 to 18.93) and 3-month continued abstinence at 6 (OR adjusted 6.4, 95% CI 1.30 to 31.27) and 12 months follow-up (OR adjusted 7.8, 95% CI 1.25 to 48.82). CONCLUSIONS: TCP may be effective in achieving abstinence from tobacco use compared to standard treatment in the given setting but due to several limitations, resulting in high attrition rates and a low statistical power in the study, more research is needed to evaluate this. TRIAL REGISTRATION: ISRCTN 11498135.

Examination of Whole-Cell Galectin Binding by Solid Phase and Flow Cytometric Analysis.

We have utilized simple flow cytometric and fluorescence-based solid phase assays to study the interaction of glycan binding proteins (GBP) to cell surface glycoconjugates. These methods utilize commonly employed flow cytometry techniques and commercially available streptavidin-coated microplates to immobilize various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells. Using this approach, fluorescently labeled GBPs, in particular, members of the galectin family, can be interrogated for potential interactions with cell surface carbohydrates, including elucidation of the potential impact of alterations in glycosylation on carbohydrate recognition. Using these approaches, we present examples of flow cytometric and fluorescence-based solid phase assays to study galectin-carbohydrate interactions.

Experiences of tobacco cessation including a prescription approach among patients in Swedish primary health care with a focus on socioeconomically disadvantaged areas.

BACKGROUND: Tobacco Cessation on Prescription (TCP) is a new intervention that is being evaluated in socioeconomically disadvantaged areas in Swedish primary health care (PHC). Patients' perceptions of TCP are important to understand as this may have implications for the acceptability and adherence to treatment and explain cessation outcomes. Patients' general experiences of tobacco cessation are also important to explore to improve cessation support in this setting. AIM: To explore experiences of tobacco cessation and TCP among patients in Swedish PHC focusing on socioeconomically disadvantaged areas. METHODS: Inductive content analysis of transcripts from eight semi-structured interviews with patients recruited from the intervention group in a randomized controlled trial evaluating the effectiveness of TCP in socioeconomically disadvantaged areas in PHC in Stockholm. RESULTS: Two themes were identified: needing individualized support to quit, taking differences in patients' experiences of tobacco use and cessation into account, acknowledging individual factors such as impact of health and wellbeing on tobacco use and differing attitudes towards tobacco and cessation and needing a supportive environment to facilitate tobacco cessation, taking contextual factors like professional support from the health care system, the importance of the social environment and supportive societal structures into account. Regarding TCP, the prescription form was perceived as useful for providers but did not appear to have a direct impact on tobacco cessation from the informants' perspective. However, individualized counseling from a tobacco cessation specialist, an empathetic approach in the treatment and long-term follow-up was considered important. CONCLUSION: A holistic approach may be needed in cessation treatment, combined with interventions outside the health care system, to facilitate tobacco cessation among patients in socioeconomically disadvantaged areas in Swedish PHC. The TCP prescription form may be helpful for PHC providers but counseling and follow-up appear to be the most important components of TCP for patients in this setting.

Tobacco Cessation on Prescription as a primary health care intervention targeting a context with socioeconomically disadvantaged groups in Sweden: A qualitative study of perceived implementation barriers and facilitators among providers.

BACKGROUND: A new intervention, Tobacco Cessation on Prescription (TCP), has been developed in the Swedish primary health care (PHC) setting to address inequalities in health caused by tobacco use. It consists of counseling for at least 10 minutes, an individualized prescription of tobacco cessation treatment and follow-up on at least one occasion. TCP is currently being tested in clinical practice for the first time but there is a lack of knowledge about how it is perceived by health care providers. AIM: To explore PHC provider's perceived barriers and facilitators of implementing TCP as an intervention targeting a context with socioeconomically disadvantaged groups in Sweden. METHODS: Directed content analysis of transcripts from eight semi-structured interviews and one focus group interview with PHC providers with personal experience of TCP as informants. Data collection and analysis was guided by The Consolidated Framework for Implementation Research. RESULTS: Perceived facilitators of implementing TCP were increased self-efficacy among the informants and involvement in the treatment among patients, which led to more intensive counseling and advice being taken more seriously by patients. Lack of resources, routines, and collaboration to work with tobacco cessation and lack of knowledge, motivation and self-efficacy among colleagues were perceived as barriers. Motivation and self-efficacy to quit was perceived as low among some patients, which was explained by low social support to quit, negative attitude and low adherence to treatment and tobacco being used as a coping strategy for life stress. Access to treatment for patients was limited by cost of treatment, long waiting times and focus on face-to-face counseling. CONCLUSION: TCP was perceived positively by the informants but access to treatment for patients was partly limited by how tobacco cessation services were organized. Lack of structural support, resources and differing attitudes among PHC providers need to be addressed to facilitate its implementation.

A Cluster-Randomized Controlled Trial Evaluating the Effectiveness and Cost-Effectiveness of Tobacco Cessation on Prescription in Swedish Primary Health Care: A Protocol of the Motivation 2 Quit (M2Q) Study.

BACKGROUND: In Sweden, the prevalence of tobacco use is disproportionately high among socioeconomically disadvantaged groups. Previous research and clinical experience suggest that prescribed lifestyle interventions in the primary health care (PHC) setting such as Physical Activity on Prescription are effective in changing behavior. However, there is a lack of evidence for if and how such a prescription approach could be effectively transferred into the tobacco cessation context. OBJECTIVE: The aim of this trial is to evaluate the effectiveness and cost-effectiveness of Tobacco Cessation on Prescription (TCP) compared to current practice for tobacco cessation targeting socioeconomically disadvantaged groups in the PHC setting in Sweden. METHODS: The design is a pragmatic cluster-randomized controlled trial. The sample will consist of 928 daily tobacco users with Swedish social security numbers and permanent resident permits, recruited from 14-20 PHC centers located in socioeconomically disadvantaged areas in Stockholm County. The primary outcome will be measured in self-reported 7-day abstinence at 6 and 12 months after the intervention. The secondary outcomes will be measured in daily tobacco consumption, number of quit attempts, and health-related quality of life at 6 and 12 months after the intervention. Data will be collected through questionnaires and review of electronic medical records. Cost-effectiveness will be estimated through decision analytic modeling and measured by the incremental cost per quality-adjusted life year. RESULTS: In the first set of PHC centers participating in the study, eight centers have been included. Recruitment of individual study participants is currently ongoing. Inclusion of a second set of PHC centers is ongoing with expected study start in September 2016. CONCLUSIONS: If TCP is found effective and cost-effective compared to standard treatment, the method could be implemented to facilitate tobacco cessation for socioeconomically disadvantaged groups in the PHC setting in Sweden. TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN): 11498135; http://www.isrctn.com/ISRCTN11498135 (Archived by WebCite at http://www.webcitation.org/6kTu6giYQ).

Perceived feasibility of a primary care intervention for Tobacco Cessation on Prescription targeting disadvantaged groups in Sweden: a qualitative study.

BACKGROUND: There is a lack of scientific evidence on how socioeconomically disadvantaged tobacco users can be reached with tobacco cessation interventions in Swedish primary healthcare (PHC). In this setting other lifestyle interventions are available by prescription, and there is the potential to develop a similar tool for tobacco cessation. The aim of this study was thus to explore the perceived feasibility and optimal design of Tobacco Cessation on Prescription (TCP) in PHC, targeting disadvantaged groups in Sweden. METHODS: This qualitative study is based on semi-structured interviews with 32 participants including (1) three experts in lifestyle interventions on prescription, (2) 14 healthcare providers and (3) 15 clients from three PHC centres in socioeconomically disadvantaged areas in Stockholm where tobacco use is high. The interviews were audio-recorded and transcribed verbatim. The manifest content of the transcripts was analysed according to a modified conventional approach to content analysis. RESULTS: The interviewees proposed that TCP should include a template comprising the client's information, evidence-based tobacco cessation options and choices for follow-up. They also suggested including information about the benefits of tobacco cessation, as well as empowerment and planning support tools. The participants also commented that other measures for tobacco cessation could be included on the prescription. From the clients' point of view, the perceived advantages of TCP were often linked to an emotional meaning (e.g. increased motivation to quit tobacco use, sign of support from the healthcare system to seek care for tobacco cessation). For providers, advantages with TCP were frequently related to a practical meaning (e.g. improved documentation and facilitation of tobacco cessation treatment). The disadvantages identified were mainly connected with the future implementation of TCP (e.g. low self-efficacy among clients and providers). CONCLUSIONS: TCP was perceived to be a useful tool for both clients and providers, potentially facilitating a structured and effective approach to tobacco cessation in PHC, and targeting disadvantaged groups. More research is needed to develop the prescription and investigate its effectiveness and cost-effectiveness compared to current strategies for tobacco cessation in a PHC setting.

Examination of whole cell galectin binding by solid phase and flow cytometric analysis.

We have utilized simple flow cytometric and fluorescence-based solid phase assays to study the interaction of glycan-binding proteins (GBP) to cell surface glycoconjugates. These methods utilize commonly employed flow cytometry techniques and commercially available streptavidin-coated microplates to immobilize various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells. Using this approach, fluorescently labeled GBPs, in particular, members of the galectin family, can be interrogated for potential interactions with cell surface carbohydrates, including elucidation of the potential impact of alterations in glycosylation on carbohydrate recognition. Using these approaches, we present examples of flow cytometric and fluorescence-based solid phase assays to study galectin-carbohydrate interactions.

Fluorescence-based solid-phase assays to study glycan-binding protein interactions with glycoconjugates.

Development of glycan microarray technologies have recently revealed many new features in the binding specificities of glycan-binding proteins (GBPs) including animal and plant lectins, antibodies, toxins, and pathogens, including viruses and bacteria. Printed glycan microarrays are very sensitive, robust, and require very small quantities of glycans and GBPs. However, glycan arrays have been limited mostly to chemoenzymatically synthesized oligosaccharides and N-glycans isolated from natural glycoproteins. O-Glycans and more complex glycoconjugates, such as glycopeptides or whole cells, are generally lacking from most types of glycan microarrays. Certain GBPs such as selectins, that have more complex binding specificity, require peptide components besides the glycan structure for high-affinity binding to the ligand. GBP binding assays on glycan microarrays will provide only partial information about the specificity and high-affinity ligands for those GBPs. Therefore, more "natural" glycoconjugate arrays are required to study more complex GBP-glycoconjugate interactions. We have utilized a simple fluorescence-based solid-phase assay on a microplate format to study GBP-glycoconjugate interactions. The method utilizes commercial streptavidin-coated microplates, where various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells, can be immobilized at a defined density. The binding of GBPs to immobilized ligands can be studied using fluorescently labeled GBPs or cells, or bound GBPs can be detected using fluorescently labeled anti-GBP antibodies. Our approach utilizing biotinylated and fixed cells in a solid-phase assay is a versatile method to study binding of GBPs to natural cell-surface glycoconjugates. Not only mammalian cells, but also microorganisms can be biotinylated and fixed, and adhesion of fluorescently labeled GBPs and antibodies to immobilized cells can be studied using standard streptavidin-coated microplates. Here, we present examples of fluorescence-based solid-phase assays to study P- and L-selectin and galectin-1 binding to immobilized glycopeptides, oligosaccharides, and cells. It should be noted that with the availability of complex glycoconjugates containing available primary amine groups, such as semisynthetic glycopeptides described here, that these could also be printed on covalent microarrays for interrogation by GBPs.

Human L-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl Lewis x in core 2 O-glycans.

Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.

Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.

Replacing a lectin domain residue in L-selectin enhances binding to P-selectin glycoprotein ligand-1 but not to 6-sulfo-sialyl Lewis x.

Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.

Dimeric galectin-1 binds with high affinity to alpha2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans.

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.

Structurally distinct requirements for binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to Anaplasma phagocytophilum and P-selectin.

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.

Model glycosulfopeptides from P-selectin glycoprotein ligand-1 require tyrosine sulfation and a core 2-branched O-glycan to bind to L-selectin.

L-selectin expressed on leukocytes is involved in lymphocyte homing to secondary lymphoid organs and leukocyte recruitment into inflamed tissue. L-selectin binds to the sulfated sialyl Lewis x (6-sulfo-sLex) epitope present on O-glycans of various glycoproteins in high endothelial venules. In addition, L-selectin interacts with the dimeric mucin P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. PSGL-1 lacks 6-sulfo-sLex but contains sulfated tyrosine residues (Tyr-SO3)at positions 46, 48, and 51 and sLex in a core 2-based O-glycan (C2-O-sLex) on Thr at position 57. The role of tyrosine sulfation and core 2 O-glycans in binding of PSGL-1 to L-selectin is not well defined. Here, we show that L-selectin binds to a glycosulfopeptide (GSP-6) modeled after the extreme N terminus of human PSGL-1, containing three Tyr-SO3 and a nearby Thr modified with C2-O-sLex. Leukocytes roll on immobilized GSP-6 in an L-selectin-dependent manner, and rolling is dependent on Tyr-SO3 and C2-O-sLex on GSP-6. The dissociation constant for binding of L-selectin to GSP-6, as measured by equilibrium gel filtration, is approximately 5 microm. Binding is dependent on Tyr-SO3 residues as well as the sialic acid and fucose residues of C2-O-sLex. Binding to an isomeric glycosulfopeptide containing three Tyr-SO3 residues and a core 1-based O-glycan expressing sLex was reduced by approximately 90%. All three Tyr-SO3 residues of GSP-6 are required for high affinity binding to L-selectin. Low affinity binding to mono- and disulfated GSPs is largely independent of the position of the Tyr-SO3 residues, except for some binding preference for an isomer sulfated on both Tyr-48 and -51. These results demonstrate that L-selectin binds with high affinity to the N-terminal region of PSGL-1 through cooperative interactions with three sulfated tyrosine residues and an appropriately positioned C2-O-sLex O-glycan.

Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells.

Murine leukocytes are thought to express alpha2-3-sialylated and alpha1-3-fucosylated selectin ligands such as sialyl Lewis x (sLe(x)), although monoclonal antibodies (mAbs) to sLe(x) or Le(x) reportedly do not bind to murine leukocytes. We observed that P- and E-selectin bound to pronase-sensitive ligands on murine monocytic WEHI-3 cells and murine neutrophils, indicating that the ligands for both selectins are glycoproteins. CSLEX-1, HECA-452, and other widely used mAbs to sLe(x) and Le(x) did not bind to WEHI-3 cells and bound at very low levels to murine neutrophils. Only the anti-sLe(x) mAbs 2H5 and KM93, which also recognize nonfucosylated glycans, bound to WEHI-3 cells. 2H5 and KM93 bound to pronase-resistant structures, indicating that the mAbs did not identify selectin ligands. Treatment of WEHI-3 cells with glycosidases or chlorate demonstrated that sialic acid modifications, alpha1-3-galactosylation, or sulfation did not mask epitopes for mAbs to sLe(x) or Le(x). Compared to human promyelocytic HL-60 cells, WEHI-3 cells and murine neutrophils expressed low alpha1-3-fucosyltransferase activities. Consistent with very low endogenous fucosylation, forced fucosylation of intact WEHI-3 cells or murine neutrophils by exogenous alpha1-3-fucosyltransferase FTVI and GDP-fucose created many new epitopes for anti-sLe(x) mAbs such as HECA-452 and CSLEX-1. Nevertheless, forced fucosylation of intact cells did not significantly augment their ability to bind to fluid-phase P- or E-selectin or to roll on immobilized P- or E-selectin under flow. These data suggest that murine myeloid leukocytes fucosylate only a few specific glycans, which interact preferentially with P- and E-selectin.

Glycosulfopeptides modeled on P-selectin glycoprotein ligand 1 inhibit P-selectin-dependent leukocyte rolling in vivo.

Leukocytic inflammation can be limited by inhibiting selectin-dependent leukocyte rolling. In spite of intensive efforts to develop small molecule selectin inhibitors with defined structure-activity profiles, inhibition of P-selectin-dependent leukocyte rolling in vivo by such a compound has yet to be described. We recently reported that glycosulfopeptides (GSP), modeled on the high affinity selectin ligand PSGL-1, inhibit leukocyte binding to P-selectin in vitro. Here, we have used intravital microscopy to investigate whether GSP can inhibit P-selectin-dependent leukocyte rolling in vivo. Surgical preparation of the mouse cremaster muscle for intravital microscopy induced P-selectin-dependent leukocyte rolling. Baseline rolling was recorded for 1 min followed by i.v. injection of GSP. 2-GSP-6 and 4-GSP-6 substantially reversed P-selectin-dependent leukocyte rolling, whereas control GSP, which are not fully glycosylated, did not. Inhibition of leukocyte rolling by 2- and 4-GSP-6 lasted 2-4 min. Clearance studies with 125I-labeled 4-GSP-6 demonstrated rapid reduction in its circulating levels concurrent with accumulation in urine. These data represent the first demonstration that a precisely defined structure based on a natural P-selectin ligand can inhibit P-selectin-dependent leukocyte rolling in vivo.

Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow.

Leukocytes roll on selectins at nearly constant velocities over a wide range of wall shear stresses. Ligand-coupled microspheres roll faster on selectins and detach quickly as wall shear stress is increased. To examine whether the superior performance of leukocytes reflects molecular features of native ligands or cellular properties that favor selectin-mediated rolling, we coupled structurally defined selectin ligands to microspheres or K562 cells and compared their rolling on P-selectin. Microspheres bearing soluble P-selectin glycoprotein ligand (sPSGL)-1 or 2-glycosulfopeptide (GSP)-6, a GSP modeled after the NH2-terminal P-selectin-binding region of PSGL-1, rolled equivalently but unstably on P-selectin. K562 cells displaying randomly coupled 2-GSP-6 also rolled unstably. In contrast, K562 cells bearing randomly coupled sPSGL-1 or 2-GSP-6 targeted to a membrane-distal region of the presumed glycocalyx rolled more like leukocytes: rolling steps were more uniform and shear resistant, and rolling velocities tended to plateau as wall shear stress was increased. K562 cells treated with paraformaldehyde or methyl-beta-cyclodextrin before ligand coupling were less deformable and rolled unstably like microspheres. Cells treated with cytochalasin D were more deformable, further resisted detachment, and rolled slowly despite increases in wall shear stress. Thus, stable, shear-resistant rolling requires cellular properties that optimize selectin-ligand interactions.

Glycosulfopeptides with O-glycans containing sialylated and polyfucosylated polylactosamine bind with low affinity to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO3-) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO3- residues and a short, monofucosylated C2-O-sLe(x) bound to P-selectin with high affinity (K(d) approximately 650 nm). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6' and GSP-6", with three TyrSO3- residues and either difucosylated polylactosamine (C2-O-Le(x)-sLe(x)) or trifucosylated polylactosamine (C2-O-Le(x)-Le(x)-sLe(x)). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6' and GSP-6" bound to P-selectin with low affinity (K(d) approximately 37 microm for GSP-6' and K(d) approximately 50 microm for GSP-6"). Binding of GSP-6' and GSP-6" to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6' and GSP-6". These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.

The monoclonal antibody CHO-131 binds to a core 2 O-glycan terminated with sialyl-Lewis x, which is a functional glycan ligand for P-selectin.

Core 2 O-glycans terminated with sialyl-Lewis x (sLe(X)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLe(X) extended from a core 2 branch (C2-O-sLe(X)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLe(X) was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(X) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(X)-related glycans. Unlike anti-sLe(X) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both.