Lysosomal membrane permeabilization causes secretion of IL-1β in human vascular smooth muscle cells

Objective

IL-1β secretion by the inflammasome is strictly controlled and requires two sequential signals: a priming signal and an activating signal. Lysosomal membrane permeabilization (LMP) plays a critical role in the regulation of NLRP3 inflammasome, and generally acts as an activating signal. However, the role of LMP controlling NLRP3 inflammasome activation in human vascular smooth muscle cells (hVSMCs) is not well defined.

Methods

LMP was induced in hVSMCs by Leu-Leu-O-methyl ester. Cathepsin B was inhibited by CA-074 Me. Cytokine release, mRNA, and protein were quantified by enzyme-linked immunosorbent assay, quantitative PCR, and Western blot, respectively. NF-κB activity was analyzed by immunostaining of the NF-κB p65 nuclear translocation and using the dual-luciferase reporter assay system.

Results

LMP had both priming and activating roles, causing an upregulation of proIL-1β and NLRP3 and the secretion of mature IL-1β from unprimed hVSMCs. LMP activated the canonical NF-κB pathway. The priming effect of LMP was inhibited by CA-074 Me, indicating an upstream role of cathepsin B.

Conclusions

These data support a novel role of LMP as a single stimulus for the secretion of IL-1β from hVSMCs, implying the possibility that hVSMCs are an important initiator of the sterile inflammatory response caused by lysosomal disintegration.

     

Synthesis of Novel Monomers and Copolymers from 1‐Vinylpyrrolidin‐2‐one: Attractive Materials for Drug Delivery Systems?

Summary: Polyvinylpyrrolidone (PVP) is a synthetic, non‐toxic, water‐soluble polymer commonly used in a wide range of applications including several pharmaceutical applications. One example of an important application is the controlled release and delivery of therapeutic agents into sites of inflammation or tumours. However, PVP lacks reactive groups, which limits the possibility of adding new functions to the polymer in order to modify its physical and chemical properties. Furthermore, large differences in radical reactivity between 1‐vinylpyrrolidin‐2‐one (NVP) and most other monomers lead to compositional drift during copolymerization. This complicates the introduction of reactive groups into the polymer using this method. Monomers that are derivatives of NVP itself are expected to show smaller differences in radical reactivity and therefore provide a way of preparing PVP with adjustable properties. Here we present the synthesis of five NVP‐based monomers and their use in the preparation of functional PVP with adjustable properties in terms of solubility, loading of functional groups, and molar mass. The results show the possibility of tailoring PVP for different biomedical applications e.g. drug delivery systems.

Copolymers from 1‐vinylpyrrolidin‐2‐one.     

Share or compete? Load‐dependent recruitment of prefrontal cortex during dual‐task performance

Dual-task performance requires flexible attention allocation to two or more streams of information. Dorsolateral prefrontal cortex (DLPFC) is considered important for executive function, and recent modeling work proposes that attention control may arise from selective activation and inhibition of different processing units within this region. Here, we used a tone discrimination task and a visual letter memory task to examine whether this type of competition could be measurable using a neuroimaging technique, the event-related optical signal, with high spatial and temporal resolution. Left and right DLPFC structures were differentially affected by task priority and load, with the left middle frontal gyrus (MFG) being preferentially recruited by the visual memory task, whereas the two tasks competed for recruitment, in a spatially segregated manner, in right MFG. The data provide support for a competition view of dual-task processing.     

Is fungal α‐amylase in bread an allergen?

BACKGROUND: The enzyme alpha-amylase from Aspergillus oryzae used in bakeries to improve the bread quality has been identified as an inhalative allergen in baker's asthma. It is doubtful whether this enzyme can induce allergic sensitization in regular bread consumers. OBJECTIVE: To find out whether fungal alpha-amylase in bread and rolls retains its antibody-binding capacity and allergenicity after the baking procedure. METHODS: Rabbit antibodies directed to fungal alpha-amylase were used for the development of a two-site enzyme-linked immunosorbent assay (ELISA). This assay was used to analyse different fractions of bread and rolls baked with the usual amounts of alpha-amylase in comparison with control products without added enzyme. Competitive experiments between bakers' sera containing specific immunoglobulin (Ig) E to alpha-amylase and the rabbit antibodies were performed. Additionally, specific IgE binding to fungal alpha-amylase was inhibited by native or heated alpha-amylase. RESULTS: With the highly specific two-site ELISA for native alpha-amylase in the crust of bread bottom and sides, 2.3-7 ng antigenic alpha-amylase per gram crust were measured. No alpha-amylase could be detected in the crumb fractions. Rabbit antibodies to native alpha-amylase completely inhibited human IgE binding to alpha-amylase allergen disks. CONCLUSION: The results prove residual antibody-binding capacity of alpha-amylase in bread crusts and in the crust of some rolls. In comparison with the content of alpha-amylase in dough, between 0.1 and 20% of the antibody-binding capacity remained.     

Post‐CPAP sleepiness – a specific syndrome?

Following treatment with continuous positive airway pressure (CPAP), some patients with obstructive sleep apnoea (OSA) remain sleepy despite effective CPAP and attention to other diagnoses that can provoke sleepiness. It is unclear if this residual sleepiness is an irreversible result of their previous OSA and merits consideration for pharmacological treatment or simply because of the many and varied causes of sleepiness normally found in the community. We have measured levels of sleepiness, using the Epworth Sleepiness Score (ESS), in 572 patients on CPAP and compared them with a control group of 525 subjects from a community survey, which would have included the usual lifestyle reasons for sleepiness as well as any undiagnosed sleep disorders. There was no difference in the percentage of patients with an ESS >10 in the CPAP group compared with the controls (16.1 versus 14.3, P = 0.54). Thus, although there clearly are sleepy patients within the CPAP group, the prevalence is no higher than in the community. We question whether so-called 'post-CPAP sleepiness' should be regarded as any more abnormal and worthy of treatment than a 'normal' population. Post-CPAP sleepiness as a specific disorder may not exist.     

Are 6‐month‐old foals sensitive to dam's influence?

A recent study has shown that gently handling dams in front of their few days old foals may strongly influence the development of human-foal relationships. In the present study, we test whether 6-month-old foals remain sensitive to their dams' influence. The study was performed on 16 foal-mare dyads, with half of the mares receiving positive contacts from the experimenter in presence of their 6-month-old foals (n = 8) whereas the other mares were not handled (n = 8). All foals were tested 15 and 30-35 days later under various conditions (reaction to a motionless human, approach test, saddle-pad tolerance test). We observe a positive effect of mare' handling on foals' reactions to humans but with a high interindividual variability, suggesting a higher effect of the foals' own behavioral characteristics at this age than at earlier stages.     

Syndiotactic Poly(propylene) from [Me2Si(3,6‐di‐tert‐butyl‐9‐fluorenyl)(N‐tert‐butyl)]TiCl2–Based Catalysts: Chain‐End or Enantiotopic‐Sites Stereocontrol?

The origin of the syndiotactic selectivity in propene polymerization of a typical bridged fluorenyl‐amido catalyst, namely [Me₂Si(3,6‐di‐tert‐butyl‐9‐fluorenyl)(N‐tert‐butyl)]TiCl₂ ( 1 ), activated with methylaluminoxane or [HMe₂N(C₆H₅)][B(C₆F₅)₄]/Al(i‐butyl)₃, was investigated by means of 150 MHz ¹³C NMR spectroscopic microstructural polymer analysis. The asymmetric induction was traced unambiguously to enantiotopic‐sites control. Compared with the better‐known cyclopentadienyl‐fluorenyl ansa‐zirconocenes, 1 turned out to be almost identically enantioselective (in agreement with computer modeling), but less stereoselective because of a higher propensity to undergo site epimerization. This results in a chain microstructure with large predominance of m over mm stereodefects, deceptively similar to that of syndiotactic poly(propylene) produced under chain‐end control.

Methyl region of the 150 MHz ¹³C NMR spectrum of a poly(propylene) sample prepared with catalyst system 1 /borate/TIBAl.     

Alternative splicing in the first α‐helical region of the Rab‐binding domain of Rim regulates Rab3A binding activity: is Rim a Rab3 effector protein during evolution?

Rim1 and Rim2 were originally described as specific Rab3A-effector proteins involved in the regulation of secretory vesicle exocytosis. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs. Although alternative splicing in the RBD1 of Rim is known to produce long and short forms of RBD (named Rim1 and Rim1Delta56-105, and Rim2(+40A) and Rim2, respectively), with the long form of Rim1 and short form of Rim2 being dominant in mouse brain, the physiological significance of the alternative splicing in RBD1 has never been elucidated. In the present study I discovered that alternative splicing in Rim RBD1 alters Rab3A binding affinity to Rims, and found that insertion of 40 amino acids into the RBD1 of Rim2 (i.e. Rim2(+40A)) dramatically reduced its Rab3A binding activity (more than a 50-fold decrease in affinity). Similarly, Rim1Delta56-105 exhibited higher affinity binding to Rab3A than the long form of Rim1. Expression of the short forms of the Rim RBD in PC12 cells co-localized well with endogenous Rab3A, whereas expression of the long forms of the Rim RBD in PC12 cells resulted in cytoplasimc and nuclear localization. Moreover, I found that Caenorhabditis elegans Rim/UNC-10 (ce-Rim) and Drosophila Rim (dm-Rim) do not interact with ce-Rab3 and dm-Rab3, respectively, indicating that the Rab3-effector function of Rim has not been retained during evolution. Based on these findings, I propose that the Rab3A-effector function of Rim during secretory vesicle exocytosis is limited to the short form of the mammalian Rim RBD alone.     

A 7‐year follow‐up study of the Mindfulness‐Based Program for Infertility: Are there long‐term effects?

The Mindfulness‐Based Program for Infertility (MBPI) was developed for people facing infertility and proved effective in cultivating mindfulness skills, improving infertility self‐efficacy, and decreasing depression, shame, entrapment, and defeat feelings. Fifty‐five women attended the MBPI sessions and completed self‐report measures of depression, anxiety, mindfulness, and experiential avoidance at post‐MBPI (T1), 6‐month follow‐up (T2), and 7‐year follow‐up (T3). There were significant direct time effects regarding experiential avoidance (F = 3.81; p < 0.033; η_p² = 0.08), the mindfulness facets describing (F = 3.54; p = 0.037; η_p² = 0.13), acting with awareness (F = 6.87; p = 0.002; η_p² = 0.22), nonjudging of inner experience (F = 10.66; p < 0.001; η_p² = 0.31), and depressive symptoms (F = 4.85; p = 0.020; η_p² = 0.10). There was an increase in the describing facet from T1 to T3 (p = 0.036). The act with awareness facet increased from T1 to T2 (p = 0.010) and from T1 to T3 (p = 0.007), as well as the nonjudging of inner experience facet (T1 to T2 [p = 0.030] and T1 to T3 [p = 0.002]). Experiential avoidance decreased from T1 to T3 (p = 0.022) and depressive symptoms from T1 to T2 (p = 0.019). Post‐MBPI scores were maintained at T2 and T3 concerning anxiety symptoms and the observing and no‐reactivity mindfulness facets. There were long‐term effects of MBPI on mindfulness and experiential avoidance. Moreover, therapeutic gains were maintained regarding depression and anxiety symptoms, independently of the reproductive outcome.     

Radical Cation,Dimer Radical Cation and Neutral Radical of 2,3‐Dihydropyran – Possible Initiators of its Polymerisation?

The radical cation, distonic dimer radical cation, and radical of 2,3‐dihydropyran (DHP), which may play an important role in mechanism of radical polymerisations of DHP, were radiolytically generated at low temperatures in freon matrices and investigated by electron paramagnetic resonance (EPR) spectroscopy, as well as by quantum chemical methods. DHP radical cation with a half‐chair conformation and six coupling protons (0.88, 0.18, 4.60, 1.88, 1.27, 0.56 mT) was found to be stable in CF₃CCl₃ between 77 and 145 K. The same radical cation was observed at 77 K also in CF₂ClCFCl₂, but on increasing temperature, transformation into the neutral 4‐dihydropyryl radical occurred; the latter also shows the half‐chair conformation at 140 K and is characterised by six coupling protons (0.14, 2.31, 0.92, 1.42, 0.28, 1.35 mT). At a high DHP concentration in CF₂ClCFCl₂ a distonic DHP dimer radical cation, with strongly separated spin and charge and only two coupling protons (1.8 and 3.2 mT) was identified at 100 K, also yielding, at higher temperatures, the 4‐dihydropyryl radical.

The radiolytically generated species from DHP and their observed EPR spectra.     

Nuclear translocation of β‐catenin and decreased expression of epithelial cadherin in human papillomavirus‐positive tonsillar cancer: an early event in human papillomavirus‐related tumour progression?

Stenner M, Yosef B, Huebbers C U, Preuss S F, Dienes H‐P, Speel E‐J M, Odenthal M & Klussmann J P (2011) Histopathology 58 , 1117–1126 Nuclear translocation of β‐catenin and decreased expression of epithelial cadherin in human papillomavirus‐positive tonsillar cancer: an early event in human papillomavirus‐related tumour progression? Aims: High‐risk human papillomaviruses (HPVs) constitute an important risk factor for tonsillar cancer. This study describes changes in cell adhesion molecules during metastasis of HPV‐related and HPV‐unrelated tonsillar carcinomas. Methods and results: We examined 48 primary tonsillar carcinoma samples (25 HPV‐16 DNA‐positive, 23 HPV‐16 DNA‐negative) and their respective lymph node metastases for their HPV status and for the expression of p16, epithelial cadherin (E‐cadherin), β‐catenin, and vimentin. A positive HPV‐specific polymerase chain reaction finding correlated significantly with p16 overexpression in both primary tumours and their metastases (P < 0.0001 for both). In HPV‐unrelated carcinomas, the expression of E‐cadherin was significantly lower in metastases than in primary tumours (P < 0.001). In contrast, the expression of nuclear β‐catenin was significantly higher in metastases than in primary tumours (P = 0.016). In HPV‐related carcinomas, nuclear localization of β‐catenin expression was already apparent in primary tumours (P = 0.030). The expression of vimentin significantly correlated with the grading of the primary tumour (P = 0.021). Conclusions: Our data indicate that the down‐regulation of E‐cadherin and the up‐regulation of nuclear β‐catenin expression might be crucial steps during tumour progression of tonsillar carcinomas, being already present in primary tumours in HPV‐driven carcinomas, but becoming apparent in HPV‐unrelated tumours later in the process of metastasis.     

γδ T‐cell lymphomas: a homogeneous entity?

gammadelta T-cells comprise an immunologically distinct lymphoid population, characterized by specific morphological, phenotypical and functional properties. Therefore it seems reasonable to speculate that neoplasms derived from this particular T-cell subset display distinct features. Indeed, the prototype gammadelta T-cell lymphoma, hepatosplenic T-cell lymphoma constitutes a unique clinicopathological entitity which is intimately associated with a gammadelta T-cell phenotype. However, gammadelta T-cell lymphomas have also been described in other extranodal sites where, unlike reactive gammadelta T-cells and hepatosplenic gammadelta T-cell lymphomas, they display an important morphological heterogeneity. Moreover, these nonhepatosplenic gammadelta T-cell lymphomas are essentially not that different from their alphabeta T-cell receptor for antigen (TCR)-expressing counterparts and thus may be incorporated in the established T-cell lymphoma subclasses. However, subtle differences regarding their histopathological appearance as well as their biological behaviour indicate that further studies to determine the exact significance of TCR expression are required. Such inquiries may contribute to the general understanding of T-cell lymphomagenesis in general, which is still obscure.