Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

Long-term results after proximal thoracic aortic redo surgery

Objective: To evaluate early and mid-term results in patients undergoing proximal thoracic aortic redo surgery. Methods: We analyzed 60 patients (median age 60 years, median logistic EuroSCORE 40) who underwent proximal thoracic aortic redo surgery between January 2005 and April 2012. Outcome and risk factors were analyzed. Results: In hospital mortality was 13%, perioperative neurologic injury was 7%. Fifty percent of patients underwent redo surgery in an urgent or emergency setting. In 65%, partial or total arch replacement with or without conventional or frozen elephant trunk extension was performed. The preoperative logistic EuroSCORE I confirmed to be a reliable predictor of adverse outcome- (ROC 0.786, 95%CI 0.64–0.93) as did the new EuroSCORE II model: ROC 0.882 95%CI 0.78–0.98. Extensive individual logistic EuroSCORE I levels more than 67 showed an OR of 7.01, 95%CI 1.43–34.27. A EuroSCORE II larger than 28 showed an OR of 4.44 (95%CI 1.4–14.06). Multivariate logistic regression analysis identified a critical preoperative state (OR 7.96, 95%CI 1.51–38.79) but not advanced age (OR 2.46, 95%CI 0.48–12.66) as the strongest independent predictor of in-hospital mortality. Median follow-up was 23 months (1–52 months). One year and five year actuarial survival rates were 83% and 69% respectively. Freedom from reoperation during follow-up was 100%. Conclusions: Despite a substantial early attrition rate in patients presenting with a critical preoperative state, proximal thoracic aortic redo surgery provides excellent early and mid-term results. Higher EuroSCORE I and II levels and a critical preoperative state but not advanced age are independent predictors of in-hospital mortality. As a consequence, age alone should no longer be regarded as a contraindication for surgical treatment in this particular group of patient

Long-lasting protection of activity of nucleoside reverse transcriptase inhibitors and protease inhibitors (PIs) by boosted PI containing regimens

Background The accumulation of mutations after long-lasting exposure to a failing combination antiretroviral therapy (cART) is problematic and severely reduces the options for further successful treatments. Methods We studied patients from the Swiss HIV Cohort Study who failed cART with nucleoside reverse transcriptase inhibitors (NRTIs) and either a ritonavir-boosted PI (PI/r) or a non-nucleoside reverse transcriptase inhibitor (NNRTI). The loss of genotypic activity <3, 3–6, >6 months after virological failure was analyzed with Stanford algorithm. Risk factors associated with early emergence of drug resistance mutations (<6 months after failure) were identified with multivariable logistic regression. Results Ninety-nine genotypic resistance tests from PI/r-treated and 129 from NNRTI-treated patients were analyzed. The risk of losing the activity of ≥1 NRTIs was lower among PI/r- compared to NNRTI-treated individuals <3, 3–6, and >6 months after failure: 8.8% vs. 38.2% (p = 0.009), 7.1% vs. 46.9% (p<0.001) and 18.9% vs. 60.9% (p<0.001). The percentages of patients who have lost PI/r activity were 2.9%, 3.6% and 5.4% <3, 3–6, >6 months after failure compared to 41.2%, 49.0% and 63.0% of those who have lost NNRTI activity (all p<0.001). The risk to accumulate an early NRTI mutation was strongly associated with NNRTI-containing cART (adjusted odds ratio: 13.3 (95% CI: 4.1–42.8), p<0.001). Conclusions The loss of activity of PIs and NRTIs was low among patients treated with PI/r, even after long-lasting exposure to a failing cART. Thus, more options remain for second-line therapy. This finding is potentially of high relevance, in particular for settings with poor or lacking virological monitoring.

Minor protease inhibitor mutations at baseline do not increase the risk for a virological failure in HIV-1 subtype B infected patients

Background Minor protease inhibitor (PI) mutations often exist as polymorphisms in HIV-1 sequences from treatment-naïve patients. Previous studies showed that their presence impairs the antiretroviral treatment (ART) response. Evaluating these findings in a larger cohort is essential. Methods To study the impact of minor PI mutations on time to viral suppression and time to virological failure, we included patients from the Swiss HIV Cohort Study infected with HIV-1 subtype B who started first-line ART with a PI and two nucleoside reverse transcriptase inhibitors. Cox regression models were performed to compare the outcomes among patients with 0 and ≥1 minor PI mutation. Models were adjusted for baseline HIV-1 RNA, CD4 cell count, sex, transmission category, age, ethnicity, year of ART start, the presence of nucleoside reverse transcriptase inhibitor mutations, and stratified for the administered PIs. Results We included 1199 patients of whom 944 (78.7%) received a boosted PI. Minor PI mutations associated with the administered PI were common: 41.7%, 16.1%, 4.7% and 1.9% had 1, 2, 3 or ≥4 mutations, respectively. The time to viral suppression was similar between patients with 0 (reference) and ≥1 minor PI mutation (multivariable hazard ratio (HR): 1.1 [95% confidence interval (CI): 1.0–1.3], P = .196). The time to virological failure was also similar (multivariable HR:.9 [95% CI:.5–1.6], P = .765). In addition, the impact of each single minor PI mutation was analyzed separately: none was significantly associated with the treatment outcome. Conclusions The presence of minor PI mutations at baseline has no effect on the therapy outcome in HIV infected individuals.

Is 7-day event triggered ECG recording equivalent to 7-day Holter ECG recording for atrial fibrillation screening?

Prolonged ECG monitoring is standard for atrial fibrillation (AF) screening. This study investigated whether 7-day event triggered (tECG) ECG recording is equivalent to 7-day continuous Holter (cECG) ECG recording for AF screening.

Prevalence of asymptomatic and electrically undetectable intracardiac inside-out abrasion in silicon-coated Riata® and Riata® ST implantable cardioverter-defibrillator leads

BACKGROUND: Recently, several cases of symptomatic and/or electrically detectable intracardiac inside-out abrasions in silicon-coated Riata® and Riata® ST leads have been described. However, the prevalence in asymptomatic patients with unremarkable implantable cardioverter defibrillator (ICD) interrogation is unknown. The aim of this study was to determine the prevalence of asymptomatic and electrically undetectable intracardiac inside-out abrasion in silicon-coated Riata® and Riata® ST leads. METHODS: All 52 patients with an active silicone-coated Riata® and Riata® ST lead followed up in our outpatient clinic were scheduled for a premature ICD interrogation and a biplane chest radiograph. When an intracardiac inside-out abrasion was suspected, this finding was confirmed by fluoroscopy. RESULTS: Mean time since implantation was 71±18months. An intracardiac inside-out abrasion was confirmed by fluoroscopy in 6 patients (11.5%). Mean time from lead implantation to detection of intracardiac inside-out abrasion was 79±14months. In all patients with an intracardiac inside-out abrasion, ICD interrogation showed normal and stable electrical parameters. Retrospectively, in 4 of these 6 patients, a coronary angiography performed 25±18months before diagnosis of intracardiac inside-out abrasion already showed the defect. Despite undetected intracardiac inside-out abrasion, 2 of these 4 patients experienced adequate antitachycardia pacing and ICD-shocks. ICD leads were replaced in all 6 patients. CONCLUSIONS: The prevalence of asymptomatic intracardiac inside-out abrasion in silicon-coated Riata® and Riata® ST leads is higher than 10% when assessed by fluoroscopy, and most intracardiac inside-out abrasions are not detectable by ICD interrogation.

"In vivo" imaging of atherosclerosis

Atherosclerosis is a systemic and multifocal disease, which starts early in life, and that usually takes decades before overt disease eventually appears as a consequence of progressive obstruction or abrupt thrombotic occlusion. This silent course makes necessary to develop predictors of disease long before symptomatic lesions develop. Besides several classical risk factors and new emerging humoral risk predictors, imaging may constitute a formidable diagnostic and prognostic tool in order to identify presence, extension, progression (or regression) of disease as well as vulnerability of atherosclerotic lesions. This review summarizes the rapidly growing clinical and research field in imaging atherosclerosis from different perspectives opening important opportunities for timely detection and treatment of atherosclerosis.

Cannabinoid receptor trafficking in peripheral cells is dynamically regulated by a binary biochemical switch

The cannabinoid G protein-coupled receptors (GPCRs) CB₁ and CB₂ are expressed in different peripheral cells. Localization of GPCRs in the cell membrane determines signaling via G protein pathways. Here we show that unlike in transfected cells, CB receptors in cell lines and primary human cells are not internalized upon agonist interaction, but move between cytoplasm and cell membranes by ligand-independent trafficking mechanisms. Even though CB receptors are expressed in many cells of peripheral origin they are not always localized in the cell membrane and in most cancer cell lines the ratios between CB₁ and CB₂ receptor gene and surface expression vary significantly. In contrast, CB receptor cell surface expression in HL60 cells is subject to significant oscillations and CB₂ receptors form oligomers and heterodimers with CB₁ receptors, showing synchronized surface expression, localization and trafficking. We show that hydrogen peroxide and other nonspecific protein tyrosine phosphatase inhibitors (TPIs) such as phenylarsine oxide trigger both CB₂ receptor internalization and externalization, depending on receptor localization. Phorbol ester-mediated internalization of CB receptors can be inhibited via this switch. In primary human immune cells hydrogen peroxide and other TPIs lead to a robust internalization of CB receptors in monocytes and an externalization in T cells. This study describes, for the first time, the dynamic nature of CB receptor trafficking in the context of a biochemical switch, which may have implications for studies on the cell-type specific effects of cannabinoids and our understanding of the regulation of CB receptor cell surface expression.

Total synthesis of (-)-zampanolide and structure-activity relationship studies on (-)-dactylolide derivatives

A new total synthesis of the marine macrolide (-)-zampanolide (1) and the structurally and stereochemically related non-natural levorotatory enantiomer of (+)-dactylolide (2), that is, ent-2, has been developed. The synthesis features a high-yielding, selective intramolecular Horner-Wadsworth-Emmons (HWE) reaction to close the 20-membered macrolactone ring of 1 and ent-2. The β-keto phosphonate/aldehyde precursor for the ring-closure reaction was obtained by esterification of a ω-diethylphosphono carboxylic acid fragment and a secondary alcohol fragment incorporating the THP ring that is embedded in the macrocyclic core structure of 1 and ent-2. THP ring formation was accomplished through a segment coupling Prins-type cyclization. Employing the same overall strategy, 13-desmethylene-ent-2 as well as the monocyclic desTHP derivatives of 1 and ent-2 were prepared. Synthetic 1 inhibited human cancer cell growth in vitro with nM IC(50) values, while ent-2, which lacks the diene-containing hemiaminal-linked side chain of 1, is 25- to 260-fold less active. 13-Desmethylene-ent-2 as well as the reduced versions of ent-2 and 13-desmethylene-ent-2 all showed similar cellular activity as ent-2 itself. The same activity level was attained by the monocyclic desTHP derivative of 1. Oxidation of the aldehyde functionality of ent-2 gave a carboxylic acid that was converted into the corresponding N-hexyl amide. The latter showed only μM antiproliferative activity, thus being several hundred-fold less potent than 1.

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Synthesis and cytotoxic activity of non-naturally substituted 4-oxycoumarin derivatives

Coumarins are a large family of natural and synthetic compounds exerting different pharmacological effects, including cytotoxic, anti-inflammatory or antimicrobial. In the present communication we report the synthesis of a series of 12 diversely substituted 4-oxycoumarin derivatives including methoxy substituted 4-hydroxycoumarins, methyl, methoxy or unsubstituted 3-aryl-4-hydroxycoumarins and 4-benzyloxycoumarins and their anti-proliferative effects on breast adenocarcinoma cells (MCF-7), human promyelocytic leukemia cells (HL-60), human histiocytic lymphoma cells (U937) and mouse neuroblastoma cells (Neuro2a). The most potent bioactive molecule was the 4-hydroxy-5,7-dimethoxycoumarin (compound 1) which showed similar potency (IC(50) 0.2-2 μM) in all cancer cell lines tested. This non-natural product reveals a simple bioactive scaffold which may be exploited in further studies.

Evidence for bidirectional endocannabinoid transport across cell membranes

Despite extensive research on the trafficking of anandamide (AEA) across cell membranes, little is known about the membrane transport of other endocannabinoids, such as 2-arachidonoylglycerol (2-AG). Previous studies have provided data both in favor and against a cell membrane carrier-mediated transport of endocannabinoids, using different methodological approaches. Because AEA and 2-AG undergo rapid and almost complete intracellular hydrolysis, we employed a combination of radioligand assays and absolute quantification of cellular and extracellular endocannabinoid levels. In human U937 leukemia cells, 100 nm AEA and 1 μm 2-AG were taken up through a fast and saturable process, reaching a plateau after 5 min. Employing differential pharmacological blockage of endocannabinoid uptake, breakdown, and interaction with intracellular binding proteins, we show that eicosanoid endocannabinoids harboring an arachidonoyl chain compete for a common membrane target that regulates their transport, whereas other N-acylethanolamines did not interfere with AEA and 2-AG uptake. By combining fatty acid amide hydrolase or monoacyl glycerol lipase inhibitors with hydrolase-inactive concentrations of the AEA transport inhibitors UCM707 (1 μm) and OMDM-2 (5 μm), a functional synergism on cellular AEA and 2-AG uptake was observed. Intriguingly, structurally unrelated AEA uptake inhibitors also blocked the cellular release of AEA and 2-AG. We show, for the first time, that UCM707 and OMDM-2 inhibit the bidirectional movement of AEA and 2-AG across cell membranes. Our findings suggest that a putative endocannabinoid cell membrane transporter controls the cellular AEA and 2-AG trafficking and metabolism.