Inhibition of two temporal phases of HIV-1 transfer from primary Langerhans cells to T cells: the role of langerin.

Epidermal Langerhans cells (eLCs) uniquely express the C-type lectin receptor langerin in addition to the HIV entry receptors CD4 and CCR5. They are among the first target cells to encounter HIV in the anogenital stratified squamous mucosa during sexual transmission. Previous reports on the mechanism of HIV transfer to T cells and the role of langerin have been contradictory. In this study, we examined HIV replication and langerin-mediated viral transfer by authentic immature eLCs and model Mutz-3 LCs. eLCs were productively infected with HIV, whereas Mutz-3 LCs were not susceptible because of a lack of CCR5 expression. Two successive phases of HIV viral transfer to T cells via cave/vesicular trafficking and de novo replication were observed with eLCs as previously described in monocyte-derived or blood dendritic cells, but only first phase transfer was observed with Mutz-3 LCs. Langerin was expressed as trimers after cross-linking on the cell surface of Mutz-3 LCs and in this form preferentially bound HIV envelope protein gp140 and whole HIV particles via the carbohydrate recognition domain (CRD). Both phases of HIV transfer from eLCs to T cells were inhibited when eLCs were pretreated with a mAb to langerin CRD or when HIV was pretreated with a soluble langerin trimeric extracellular domain or by a CRD homolog. However, the langerin homolog did not inhibit direct HIV infection of T cells. These two novel soluble langerin inhibitors could be developed to prevent HIV uptake, infection, and subsequent transfer to T cells during early stages of infection.

[Venous thrombosis as a complication of pacemaker implantation--own observations]. / Zakrzepica zylna jako powiklanie po wszczepieniu rozrusznika serca--wlasne obserwacje.

UNLABELLED: Thrombosis is a fairly rare, yet dangerous complication of pacemaker implantation. It may occur in various time intervals after the actual implantation procedure. The risk factors are as follows: inflammation or injury, vein anomalies, arrhythmia, venous stenosis. The aim of the study was to assess the risk factors for venous thrombosis, determine the interrelationship between venous thrombosis and the level of select inflammatory and pro-thrombotic factors, as well as to evaluate the actual impact of pacemaker implantation procedure within a group of patients with permanent heart stimulation system. MATERIAL AND METHODS: The study involved 48 patients with permanent heart stimulation system, divided into 2 groups. Group A (18 F, 20 M; mean age 71.7 +/- 13.6 years) consisted of patients who had up to 3 thrombotic risk factors, whereas group B (2 F, 8 M; mean age 71.6 +/- 7.5 years) comprised the patients with more than 5 thrombotic risk factors. All patients were subsequently followed up for 18 months. Prior to pacemaker implantation the following factors were assessed in each patient: specific indications for pacemaker implantation, overall clinical condition, coexisting diseases, history of tobacco smoking, medications used prior to the procedure (anti-platelet, anti-coagulation), history of thrombotic or infectious complications, previous temporary heart stimulation. A transthoracic echocardiogram (TTE) and an ultrasound examination of the veins in both upper extremities were carried out. The levels of: D-dimers, fibrinogen, interleukin-6 (IL-6), hsCRP, TF and PAI-1 were determined in the venous blood. During the pacemaker implantation procedure the actual access to the veins and possible venous anomalies were assessed. After the pacemaker implantation, the TTE and venous ultrasound examination were carried out after 6 and 12 months, blood analyses for up to 7 days after the implantation, and subsequently after 6 and 12 months. RESULTS: Patients in group B had significantly lower left ventricle ejection fraction and larger left ventricle end diastolic diameter than group A patients. In 3 patients in group B (which made up 6.25% of the study population), symptomatic venous thrombosis occurred after a 12-month observation period (mean 13.06 months). Initially, the levels of IL-6, hsCRP, TF, PAI-1, fibrinogen and D-dimers were considerably higher in group B, in comparison to group A. In all patients the levels of the above referenced factors kept on increasing for up to 7 days after the procedure. In group A they subsequently decreased, whereas in group B they kept on growing. In group B a significant negative correlation was observed between the left ventricle ejection fraction and the inflammatory and thrombotic factors under study. The lower the ejection fraction, the higher actually were their values. CONCLUSIONS: In patients with more than 5 thrombosis risk factors, the increased levels of IL-6, hsCRP, fibrinogen, D-dimers, TF and PAI-1 were observed. A pacemaker implantation procedure is believed to increase both thrombotic and inflammatory state in a patient for up to 7 days after the procedure. In patients prone to thrombosis, a negative correlation between the ejection fraction and the levels of hsCRP, D-dimers, TF and PAI-1 were observed.

The Associations of Perceived and Oxidative Stress with Hypertension in a Cohort of Police Officers.

PURPOSE: Associations between perceived stress and oxidative stress marker and metabolic syndrome (MetS) components were investigated in a cohort of police officers. METHODS: Cross-sectional data from a cohort of non-diabetic subjects (n=233; 19F), median [interquartile range] age 50 [37-44] years, were analysed. MetS was construed in line with International Diabetes Federation (IDF) criteria and perceived stress with Cohen's 10-item Perceived Stress Scale. Plasma oxidative stress marker (free 8-iso-prostaglandin F2α; 8-iso-PGF2α), presence of coronary plaque, carotid artery intima-media thickness (cIMT), and physical activity level were also determined. RESULTS: Obesity was established in 100 (42.92%), hypertension in 111 (47.64), whereas MetS was identified in 104 (44.63%) of the study subjects. A significant difference (p=0.003) in plasma 8-iso-PGF2α level, depending on the MetS components status, was noted. The associations of perceived stress with plasma 8-iso-PGF2α level and the select study variables were gender-specific. In multivariate analysis (adjusted for age and current smoking), positive associations of plasma 8-iso-PGF2α levels with PSS score (B=0.108, 95% CI [0.008, 0.209], p=0.03) and systolic blood pressure (B=0.029, 95% CI [0.003, 0.057], p=0.02) in men only were established. Both the perceived stress (OR 1.101, 95% CI [1.001-1.202], p=0.03) and plasma 8-iso-PGF2α levels (OR 1.223, 95% CI [1.046-1.432], p=0.01) impacted the prevalence of hypertension. Out of the MetS components, the effect of waist circumference (OR=1.138, 95% CI [1.064-1.218], p=0.0001) and glucose (B=2.696, 95% CI [1.081-6.725], p=0.03) were also encountered. No such associations were noted in women, though, neither in univariate nor in multivariate analyses. The prevalence of coronary plaque (0.001), obesity (p<0.001), hypertension (p<0.001) and median cIMT value (p=0.005), as well as leisure-time (p=0.04) and total walking physical activity (p=0.03), differed significantly between the subgroups stratified by MetS components status. CONCLUSION: Both the perceived and oxidative stress were found instrumental in promoting hypertension in a cohort of police officers under study, whereas all study outcomes were conclusively gender-related.

Potential roles of psychological and oxidative stress in insulin resistance: a cohort-based study.

BACKGROUND: The present study investigated the relationships between psychological stress indices and oxidative stress marker, also when combined with emergent insulin resistance (IR), in the non-diabetic, middle-aged subjects, exposed to frequent/chronic psychological stressors. METHODS: Cross-sectional data from a cohort of non-diabetic police officers (n = 234; 19F), aged 27-56 years, were used. Plasma inflammatory (CRP, TNF-α), oxidative stress (free 8-iso-prostaglandin F2α; 8-iso-PGF2α) markers, and insulin were measured. The value of homeostasis model assessment of IR index (HOMA-IR) was assumed the threshold value of IR, i.e. 2.04. Free cortisol in urine and perceived stress (psychological stress indices) were also measured. RESULTS: In the IR subjects, most biochemical variables, inflammatory markers and urine cortisol were significantly higher, as compared to the non-IR ones. Psychological stress indices were associated with plasma 8-iso-PGF2α [B = 0.139, 95% CI (0.048, 0.230), p = 0.002, and B = 0.007, 95% CI (0.0006, 0.014), p = 0.03; for perceived stress level and cortisol, respectively]. Positive associations were established between plasma 8-iso-PGF2α [B = 0.069, 95% CI (0.016-0.120), p = 0.01] and urine cortisol [B = 0.003, 95% CI (0.0003, 0.005), p = 0.02] with HOMA-IR. Metabolic syndrome, as defined by IDF criteria, was established in 110 study subjects, whereas 136 of them were hypertensive. Waist circumference [B = 0.056, 95% CI (0.039, 0.074), p < 0.0001], and systolic blood pressure [B = 0.009, 95% CI (0.00003, 0.018), p = 0.04] were positively associated with HOMA-IR, whereas the association of HDL cholesterol [B = - 0.597, 95% CI (- 1.139, - 0.055), p = 0.03] was a negative one. Cortisol [OR = 1.007, 95% CI (1.002, 1.012), p = 0.006], and 8-iso-PGF2α [OR = 1.103, 95% CI (1.010, 1.201), p = 0.02] affected the incidence of IR. After adjustment for metabolic syndrome (or its components), age, sex, and current smoking, the effects became non-significant. Out of metabolic syndrome components, waist circumference [OR 4.966, 95% CI (2.29, 10.751), p = 0.00004] and hypertriglyceridemia [OR 1.993, 95% CI (1.063, 3.736), p = 0.03] increased the chance of IR incidence. CONCLUSIONS: Both psychological stress indices were associated with oxidative stress, but only cortisol with HOMA-IR. In the subjects exposed to frequent/chronic psychological stressors, cortisol and oxidative stress marker affected IR incidence, being statistically attenuated, though, following adjustment for metabolic syndrome, or its components.

Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibalpha.

Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbalpha is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbalpha, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbalpha. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561-Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbalpha binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbalpha in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbalpha Trp570-->Ala and Phe568-->Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbalpha, which is critically dependent on both Trp570 and Phe568.

Early antithrombotic and anti-inflammatory effects of simvastatin versus fenofibrate in patients with hypercholesterolemia.

The aim of the study was to determine whether a short-term treatment with simvastatin or fenofibrate may result in beneficial anti-inflammatory and antithrombotic effects in patients with high risk of coronary artery disease. In a randomized, double-blind study, we compared markers of inflammation, thrombin formation and platelet activation in patients with LDL cholesterol >130 mg/dl assigned to receive simvastatin (40 mg/d; n=20) or micronised fenofibrate (160 mg/d; n=22) for 28 days. Simvastatin, but not fenofibrate, lowered C-reactive protein (CRP) by 32% on day 3 (p<0.001), while both drugs reduced CRP significantly on day 28. Interleukin-6, soluble CD40 ligand, and monocyte chemoattractant protein-1 levels decreased significantly (by 20 to 50%) in both treatment groups on days 3 and 28. Soluble cell adhesion molecules remained unchanged in both groups. Simvastatin and fenofibrate significantly lowered plasma concentrations of thrombin-antithrombin complexes on days 3 and 28, but not platelet beta-thromboglobulin (betaTG) levels. Soluble P-selectin was lowered only in the simvastatin group. The total amount of thrombin generated at the site of microvascular injury also declined (by about 30%) as early as after 3 days of fenofibrate or simvastatin therapy, whereas beta TG release was reduced only in the simvastatin group on days 3 and 28. All the effects were independent of the changes in lipid profiles. Our results suggest that statins and fibrates can exert antithrombotic and anti-inflammatory effects as early as after 3 days of therapy. However, in contrast to statins, fibrates have no influence on platelet function within one month of therapy.

The Relationship of Metabolic Syndrome with Stress, Coronary Heart Disease and Pulmonary Function--An Occupational Cohort-Based Study.

BACKGROUND AND AIMS: Higher levels of stress impact the prevalence of metabolic syndrome (MetS) and coronary heart disease. The association between MetS, impaired pulmonary function and low level of physical activity is still pending assessment in the subjects exposed to stress. The study aimed to examine whether higher levels of stress might be related to MetS and the plaque presence, as well as whether MetS might affect pulmonary function. DESIGN AND METHODS: The study embraced 235 police officers (mean age 40.97 years) from the south of Poland. The anthropometrics and biochemical variables were measured; MetS was diagnosed using the International Diabetes Federation criteria. Computed tomography coronary angiography of coronary arteries, exercise ECG, measurements of brachial flow-mediated dilation, and carotid artery intima-media thickness were completed. In order to measure the self-perception of stress, 10-item Perceived Stress Scale (PSS-10) was applied. Pulmonary function and physical activity levels were also addressed. Multivariate logistic regression analyses were applied to determine the relationships between: 1/ incidence of coronary plaque and MetS per se, MetS components and the number of classical cardiovascular risk factors, 2/ perceived stress and MetS, 3/ MetS and pulmonary function parameters. RESULTS: Coronary artery atherosclerosis was less associated with MetS (OR = 2.62, 95%CI 1.24-5.52; p = 0.011) than with a co-existence of classical cardiovascular risk factors (OR = 5.67, 95% CI 1.07-29.85, p = 0.03; for 3 risk factors and OR = 9.05; 95% CI 1.24-66.23, p = 0.02; for 6 risk factors, respectively). Perceived stress increased MetS prevalence (OR = 1.07, 95% CI 1.03-1.13; p = 0.03), and impacted coronary plaque prevalence (OR = 1.05, 95% CI 1.001-1.10; p = 0.04). Leisure-time physical activity reduced the chances of developing MetS (OR = 0.98 95% CI 0.96-0.99; p = 0.02). MetS subjects had significantly lower values of certain pulmonary function parameters. CONCLUSIONS: Exposure to job-specific stress among police officers increased the prevalence of MetS and impacted coronary plaque presence. MetS subjects had worse pulmonary function parameters. Early-stage, comprehensive therapeutic intervention may reduce overall risk of cardiovascular events and prevent pulmonary function impairment in this specific occupational population.

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model.

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor ß-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor ß-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1ß, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

Effect of selected prothrombotic and proinflammatory factors on the incidence of venous thrombosis after pacemaker implantation.

BACKGROUND: Venous thrombosis (VT), a major cause of venous obstruction, is a rather rare but dangerous complication of pacemaker (PM) implantation. AIM: To assess the prognostic value of selected proinflammatory and prothrombotic markers in predicting symptomatic venous obstruction after PM implantation. METHODS: The study involved 81 patients (31 females; mean age 71 ± 8 years) divided into 2 groups depending on the occurrence of venous obstruction. Group I included 71 patients (29 females; mean age 71 ± 2 years) without this complication and group II included 10 patients (2 females, mean age 71.6 ± 2) with venous obstruction. All patients were followed up for 19 months. Transthoracic echocardiography and venous ultrasonography were performed before PM implantation and at the time of incident venous obstruction. Interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), D-dimer, fibrinogen, tissue factor (TF), factor VII and plasminogen activator inhibitor-1 (PAI-1) levels were measured at baseline and within 7 days after PM implantation. RESULTS: Patients in group II had a significantly lower left ventricular ejection fraction (LVEF), higher left ventricular end--diastolic diameter (LVEDD) and impaired left ventricular filling (Vp) compared to group I. Patients in group II developed VT on average at 13.06 (range 7-18) months following PM implantation. At baseline, IL-6, hsCRP, D-dimer, fibrinogen, TF, factor VII, and PAI-1 levels were significantly higher in group II compared to group I. In all patients, levels of prothrombotic factors were higher following PM implantation compared to baseline values, with the exception of fibrinogen level in group I. Cut-off values indicating increased thrombosis risk were determined for the examined parameters (LVEF, LVEDD, Vp, IL-6, hsCRP, D-dimer, fibrinogen, factor VII, TF, PAI-1) based on the ROC curves. Major predictors of thrombotic risk included LVEF, LVEDD, and D-dimer, fibrinogen and TF levels. Highest predictive values were noted for LVEDD > 58 mm (OR = 52.8) and D-dimer level > 498 mg/L (OR = 3003). CONCLUSIONS: 1. Patients who developed VT after PM implantation had elevated baseline levels of IL-6, hsCRP, fibrinogen, D-dimer, TF, factor VII, and PAI-1. 2. Levels of pro-inflammatory markers increased after the implantation procedure in all patients. 3. Parameters with the highest diagnostic value for predicting incident VT included decreased LVEF, increased LVEDD and elevated D-dimer, fibrinogen and TF levels.

Relationship between changes in selected thrombotic and inflammatory factors, echocardiographic parameters and the incidence of venous thrombosis after pacemaker implantation based on our own observations.

INTRODUCTION: Thrombosis (VTh) is a rare dangerous complication of pacemaker implantation (PM). The aim of the study was to determine the dynamics of change in selected thrombotic and inflammatory factors after PM. MATERIAL AND METHODS: The study involved 81 patients (30 female, mean age: 71.1 years) with PM, divided into two groups. Group A (71 patients) consisted of patients without VTh, whereas group B (10 patients) comprised the patients with VTh. A transthoracic echocardiogram (TTE) and a venous ultrasound (VU) examination were performed. The levels of D-dimers, fibrinogen, tissue factor (TF), factor VII, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) were determined in the venous blood. After PM, the TTE and VU examinations were repeated at 6 and 12 months, and blood analyses were performed within 7 days after PM, and subsequently at 6 and 12 months. RESULTS: In 10 patients of group B, symptomatic VTh occurred at a mean time of 13.06 months after PM. Initially, the levels of IL-6, hsCRP, D-dimers, fibrinogen, TF, VII factor and PAI-1 were considerably higher in group B than in group A. In all patients the levels of these factors kept on increasing for up to 7 days after the procedure. In group A they subsequently decreased, whereas in group B they continued to rise. CONCLUSIONS: Increased levels of inflammatory and thrombotic factors were observed in patients with VTh before and after PM. The factors of highest risk of VTh occurrence were D-dimers, fibrinogen and TF.

Nitric oxide production and endothelium-dependent vasorelaxation ameliorated by N1-methylnicotinamide in human blood vessels.

N(1)-methylnicotinamide (MNA(+)) has until recently been thought to be a biologically inactive product of nicotinamide metabolism in the pyridine nucleotides pathway. However, the latest observations imply that MNA(+) may exert antithrombotic and anti-inflammatory effects through direct action on the endothelium. We examined both in vivo and in vitro whether the compound might induce vasorelaxation in human blood vessels through the improvement of nitric oxide (NO) bioavailability and a reduction of oxidative stress mediated by endothelial NO synthase (eNOS) function. MNA(+) treatment (100 mg/m(2) orally) in healthy normocholesterolemic and hypercholesterolemic subjects increased the l-arginine (l-NMMA)-inhibitable flow-mediated dilation (FMD) of brachial artery responses that also positively correlated with MNA(+) plasma concentrations (r=0.73 for normocholesterolemics and r=0.78 for hypercholesterolemics; P<0.0001). MNA(+) increased FMD at the same concentration range at which it enhanced NO release from cultured human endothelial cells after stimulation with either the receptor-dependent (acetylcholine) or the receptor-independent endothelial NO synthase agonists (calcium ionophore A23187). MNA(+) restored the endothelial NO synthase agonist-stimulated NO release after the exposure of the cells to oxidized low-density lipoprotein. This effect was also associated with the normalization of the [NO]/[superoxide] balance in the endothelial cells. Taken together, the increased NO bioavailability in the endothelium contributes to the vasorelaxating properties of MNA(+). Targeting eNOS with MNA(+) might be therapeutically relevant for functional disorders of the endothelium, such as hypercholesterolemia and atherosclerosis.

Association between selected risk factors and the incidence of venous obstruction after pacemaker implantation: demographic and clinical factors.

BACKGROUND: Venous obstruction and subsequent pulmonary embolism belong to the most common and dangerous complications of pacemaker implantation. Thus, identification of patients at risk of venous obstruction seems to be of critical importance. AIM: To determine risk factors of venous obstruction following pacemaker implantation. METHODS: Eighty one patients with permanent cardiac pacing (31 F, 50 M; mean age 71.1 ± 7.6 years) were included. Prior to pacemaker implantation, the following factors were evaluated in each patient: indications for pacemaker implantation, heart failure severity assessed using the NYHA classification, coexisting diseases, a history of tobacco smoking, medications used before the procedure (antiplatelet drugs, anticoagulants, antibiotics), a history of thrombotic or infectious complications, and previous temporary cardiac pacing. Type of venous access and procedure time were also assessed. Venous ultrasound examination to evaluate veins in both upper extremities, shoulder areas and the neck was performed before pacemaker implantation and 6 and 12 months following the procedure. Computed tomography and conventional digital subtraction angiography were performed to confirm the diagnosis of venous obstruction. RESULTS: The patients were divided into two groups based on the occurrence of venous obstruction after pacemaker implantation. Group I (n = 71, 29 F, 42 M; mean age 71.0 ± 7.7 years) included patients without venous obstruction, and group II (n = 10, 2 F, 8 M; mean age 71.6 ± 7.0 years) included patients diagnosed with venous obstruction. Each patient was followed for 19 months. In group II (12.3% of the study population), venous obstruction developed mean 13 months after pacemaker implantation. In this group, symptomatic venous obstruction was observed in 3 patients (3.7% of the study population), mean 15 months after pacemaker implantation. Risk factors for venous obstruction included a history of myocardial infarction, temporary cardiac pacing, arrhythmia, venous anomalies, NYHA class III and IV heart failure, a history of infection, and tobacco smoking. Depending on the number of risk factors, the probability of development of venous obstruction was described by the following equation: e(-14.6 + 3.19x)/1 + e(-14.6 + 3.19x), where x is the number of risk factors. In patients who had more than 6 risk factors, almost a 100% probability of the occurrence of venous obstruction was observed. CONCLUSIONS: 1. Risk factors for venous obstruction include a history of myocardial infarction, temporary cardiac pacing, arrhythmia, venous anomalies, infections, NYHA class III and IV heart failure, and tobacco smoking. 2. In patients who had more than 6 risk factors, almost a 100% risk of venous obstruction was observed.

Anti-TNFα domain antibody construct CEP-37247: Full antibody functionality at half the size.

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 - 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising VL dAb, truncated CH1, hinge, CH2 and CH3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact - CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.

A novel class of anti-IL-12p40 antibodies: potent neutralization via inhibition of IL-12-IL-12Rß2 and IL-23-IL-23R.

While current therapeutic antibodies bind to IL-12 and IL-23 and inhibit their binding to IL-12Rß1, we describe a novel antibody, termed 6F6, that binds to IL-12 and IL-23 and inhibits the interaction of IL-12 and IL-23 with their cognate signalling receptors IL-12Rß2 and IL23R. This antibody does not affect the natural inhibition of the IL-12/23 pathway by the antagonists monomeric IL-12p40 and IL-12p80, which suggests that a dual antagonist system is possible. We have mapped the epitope of 6F6 to domain 3 of the p40 chain common to IL-12 and IL-23 and demonstrate that an antibody bound to this epitope is sufficient to inhibit engagement of the signalling receptors. Antibodies with this unique mechanism of inhibition are potent inhibitors of IL-12 induced IFN-γ production and IL-23 induced IL-17 production in vitro, and in an in vivo model of psoriasis, treatment with a humanized variant of this antibody, h6F6, reduced the inflammatory response, resulting in decreased epidermal hyperplasia. We believe that this new class of IL-12/23 neutralising antibodies has the potential to provide improved potency and efficacy as anti-inflammatory agents, particularly in diseases characterized by an overproduction of IL-12.

The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF).

A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric a-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and (1)H-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblastsAs we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.

Interaction between platelet glycoprotein Ibalpha and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear.

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIbalpha and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIbalpha intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569-579 of the GPIbalpha cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant Delta557-568 and Delta569-579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIbalpha for interaction with filamin-1.