Shear stress-induced changes in atherosclerotic plaque composition are modulated by chemokines

We previously found that low shear stress (LSS) induces atherosclerotic plaques in mice with increased lipid and matrix metalloproteinase content and decreased vascular smooth muscle and collagen content. Here, we evaluated the role of chemokines in this process, using an extravascular device inducing regions of LSS, high shear stress, and oscillatory shear stress (OSS) in the carotid artery. One week of shear stress alterations induced expression of IFN-gamma-inducible protein-10 (IP-10) exclusively in the LSS region, whereas monocyte chemoattractant protein-1 (MCP-1) and the mouse homolog of growth-regulated oncogene alpha (GRO-alpha) were equally upregulated in both LSS and OSS regions. After 3 weeks, GRO-alpha and IP-10 were specifically upregulated in LSS regions. After 9 weeks, lesions with thinner fibrous caps and larger necrotic cores were found in the LSS region compared with the OSS region. Equal levels of MCP-1 expression were observed in both regions, while expression of fractalkine was found in the LSS region only. Blockage of fractalkine inhibited plaque growth and resulted in striking differences in plaque composition in the LSS region. We conclude that LSS or OSS triggers expression of chemokines involved in atherogenesis. Fractalkine upregulation is critically important for the composition of LSS-induced atherosclerotic lesions.     

Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5)

Background and Purpose

A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors – a subgroup of 7TM receptors – it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity.

Experimental Approach

The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein-and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation.

Key Results

[L203F]-CCR5 increased the basal level of G-protein coupling (20–70% of E_max) and β-arrestin recruitment (50% of E_max) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr²⁴⁴ in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist.

Conclusions and Implications

The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile¹¹⁶ acts as a gate for the movement of Tyr²⁴⁴ towards TM-5 in the active state, a mechanism proposed previously for the β₂-adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists.     

CYP2C19*17 increases clopidogrel‐mediated platelet inhibition but does not alter the pharmacokinetics of the active metabolite of clopidogrel

The aim of the present study was to determine the impact of CYP2C19*17 on the pharmacokinetics and pharmacodynamics of the active metabolite of clopidogrel and the pharmacokinetics of proguanil. Thus, we conducted an open‐label two‐phase cross‐over study in 31 healthy male volunteers (11 CYP2C19*1/*1, 11 CYP2C19*1/*17 and nine CYP2C19*17/*17). In Phase A, the pharmacokinetics of the derivatized active metabolite of clopidogrel (CAMD) and platelet function were determined after administration of a single oral dose of 600 mg clopidogrel (Plavix; Sanofi‐Avensis, Horsholm, Denmark). In Phase B, the pharmacokinetics of proguanil and its metabolites cycloguanil and 4‐chlorphenylbiguanide (4‐CPB) were determined in 29 of 31 subjects after a single oral dose of 200 mg proguanil given as the combination drug Malarone (GlaxoSmithKline Pharma, Brondby, Denmark). Significant correlations were found between the area under the time–concentration curve (AUC_0–∞) of CAMD and both the absolute ADP‐induced P2Y₁₂ receptor‐activated platelet aggregation (r = ?0.60, P = 0.0007) and the percentage inhibition of aggregation (r = 0.59, P = 0.0009). In addition, the CYP2C19*17/*17 and CYP2C19*1/*17 genotype groups had significantly higher percentage inhibition of platelet aggregation compared with the CYP2C19*1/*1 subjects (geometric mean percentage inhibition of 84%, 73% and 63%, respectively; P = 0.014). Neither the absolute ADP‐induced P2Y₁₂ receptor‐activated platelet aggregation, exposure to CAMD nor the pharmacokinetic parameters of proguanil, cycloguanil and 4‐CPB exhibited any significant differences among the genotype groups. In conclusion, carriers of CYP2C19*17 exhibit higher percentage inhibition of platelet aggregation, but do not have significantly lower absolute P2Y₁₂ receptor‐activated platelet aggregation or higher exposure to the active metabolite after a single oral administration of 600 mg clopidogrel.     

Expression of CD18 (integrin β2 chain) correlates with prognosis in malignant B cell lymphomas

Summary. Several studies have shown that cell cycle related parameters including DNA synthesis and activation antigen expression can predict patient survival in lymphoma patients. In this study of 69 malignant B cell lymphomas we have examined the cell surface expression of several cell interaction and activation molecules by flow cytometry. Expression of CD18 (integrin β₂ chain) was found to correlate strongly with patient survival (median follow up 50 months) even when adjusting for other important prognostic factors ( P = 0.0001). The percentage of cells positive for CDw75 proved important both as a single parameter and in the multivariate analysis. Histology, classified as low versus high grade malignancy, bulky versus not bulky disease and high versus low thymidine incorporation, was also found to correlate with prognosis in this study.     

Auto-inflammatory challenge of the endolymphatic sac – Cochlear damage measured by distortion product oto-acoustic emissions

Conclusion: Twenty-five rats were challenged by an immunologic attack of the endolymphatic sac. After 6 months, distortion product oto-acoustic emissions (DPOAE) revealed a dysfunction of the outer hair cells and immunological active cells were observed in the endolymphatic sac. This information could contribute to the understanding of Ménière’s disease. Objectives: This study investigated if an autoimmune challenge of the endolymphatic sac could affect DPOAE output measurements in rats. Also, a potential autoimmune cell infiltration of the endolymphatic sac was investigated. Methods: Eighteen Lewis rats were immunized with a crude endolymphatic sac extract in complete Freund’s adjuvant. Seven control animals were injected with Freund’s adjuvant in saline. Cochlear damage was estimated by DPOAE dynamics 3 weeks and 6 months after the immunization. Infiltrative cells in the endolymphatic sac were investigated with transmission electron microscopy. Results: The hearing assessment 6 months after immunization revealed a reduction of the DPOAE, on the full range of frequencies (2–63 kHz) in an average of the mean, of 2 dB ± 1.1 in the immunized group compared to the controls (p < 0.05). The same test showed a 2.5 dB decrease from 2 to 5 kHz (p < 0.01). Immunological active cells were observed in the endolymphatic sac in most of the immunized rats.     

Daytime Glibenclamide and Bedtime NPH Insulin Compared to Intensive Insulin Treatment in Secondary Sulphonylurea Failure: a 1-Year Follow-up

The aim of this study was to compare the metabolic effects of a combination of daytime glibenclamide and evening NPH insulin with intensive insulin treatment (rapid acting insulin before meals and NPH insulin at bedtime) in patients exhibiting secondary failure to sulphonylurea treatment. Thirty-nine mildly obese NIDDM patients (BMI 25.6 ± 0.5) were randomized after 6 weeks of intensive insulin treatment to either a combination treatment (CT, n = 20) or continued intensive insulin treatment (IT, n = 19). There were no differences between the two groups in age, diabetes duration, BMI, HbA_1c, or basal and glucagon stimulated C-peptide. The patients were followed for 1 year and the findings were analysed on an intent to treat basis. Two patients in the CT group were excluded after 2 and 6 months, respectively, due to unacceptably high postprandial glucose values. There was a significant difference in HbA_1c between the CT and IT groups at 6 months (8.2 ± 0.2, n = 19, vs 6.8 ± 0.4%, n = 19, p < 0.001)), but not at 12 months (7.8 ± 0.3, n = 18, vs 7.5 ± 0.4%, n = 19). After the initial intensive insulin treatment, BMI was constant in the CT group but increased significantly at 6 and 12 months in the IT group. We conclude that both treatments are associated with a marked and long-term improvement of glycaemic control. The intensive insulin treatment leads to a more pronounced weight increase which in the long run might have negative effect on overall metabolic control. Therefore, the combination treatment together with intensified education and dietary advice should be regarded as the initial treatment of choice for oral agent failure in moderately obese NIDDM patients.