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Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca 2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca 2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.

Mast cells, in addition to endocrine cells and neurons, are typical secretory cells. Their function in allergic inflammation is to secrete inflammatory mediators from secretory vesicles. Intracellular synthesized inflammatory mediators are transported by vesicular monoamine transporters (VMATs) to vesicles where they are stored. After stimulation, the contents of the secretory vesicles are released via exocytosis. This study established a high throughput imaging screening system to monitor the functions of secretory vesicles in mast cells, including molecular uptake via VMAT2 and the exocytotic process, by using a novel fluorescent probe, FFN206, which was developed as a VMAT2 substrate. After loading with FFN206, the rapid uptake of FFN206 was observed and secretory vesicles in mouse bone marrow derived mast cells and a cultured mast cell line were clearly visualized. FFN206 uptake by secretory vesicles was time-dependent and was blocked by reserpine. Furthermore, exocytotic trafficking was monitored dynamically by real-time high-throughput fluorescence quantitation. In the present study, we verified the application of FFN206 for the monitoring of functional vesicles. This high-throughput screening system may benefit instinctive drug evaluation.

Background To promote understanding of the pathogenesis of cognitive impairment or dementia, we explored the potential interaction between transient cerebral ischemia and amyloid-β (Aβ) infusion in mediating cognitive decline and examined the possible ameliorative effect of angiotensin II type 2 (AT 2 ) receptor activation in vascular smooth muscle cells (VSMC) on this cognitive deficit. Methods Adult male wild-type mice (WT) and mice with VSMC-specific AT 2 receptor overexpression (smAT 2 ) were subjected to intracerebroventricular (ICV) injection of Aβ1-40. Transient cerebral ischemia was induced by 15 min of bilateral common carotid artery occlusion (BCCAO) 24 h after Aβ injection. Results Aβ injection in WT induced a cognitive decline, whereas BCCAO did not cause a significant cognitive deficit. In contrast, WT with BCCAO following Aβ injection exhibited more marked cognitive decline compared to Aβ injection alone, in concert with increases in superoxide anion production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and expression of p22phox, p40phox, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1β in the hippocampus, and upregulation of RAGE (receptor for advanced glycation end product), an Aβ transporter. BCCAO following Aβ injection further enhanced neuronal pyknosis in the hippocampus, compared with BCCAO or Aβ injection alone. In contrast, smAT 2 did not show a cognitive decline, increase in oxidative stress, inflammation, and RAGE level or neuronal pyknosis, which were induced by BCCAO with/without Aβ injection in WT. Conclusions Transient cerebral ischemia might worsen Aβ infusion-mediated cognitive decline and vice versa, with possible involvement of amplified oxidative stress and inflammation and impairment of the RAGE-mediated Aβ clearance system, contributing to exaggerated neuronal degeneration. AT 2 receptor activation in VSMC could play an inhibitory role in this cognitive deficit.

Systemic lupus erythematosus (SLE) is characterized by production of a variety of autoantibodies. Although anti-double-stranded DNA (anti-dsDNA) antibodies contribute to the pathogenesis of lupus nephritis (LN), they are not sufficient for diagnosis and evaluation of disease activity. To obtain other autoantibodies associated with LN, we screened autoantigens reacting with the sera of LN patients by using an N-terminal biotinylated protein library created from a wheat cell-free protein production system. We screened 17 proteins that showed higher positive signals in the active phase than in the inactive phase of SLE, and higher positive signals in the serum of SLE patient with nephritis than in that of patient without nephritis. Of these, two LN-associated autoantigens, ribosomal RNA-processing protein 8 (RRP8) and spermatid nuclear transition protein 1 (TNP1) were identified by immunoprecipitation and immunofluorescence of renal tissues. Circulating anti-RRP8 and anti-TNP1 autoantibodies were recognized and deposited as an immune complex (IC) in glomeruli. IC was deposited preferentially in glomeruli rather than in other organs in C57BL/6 mice injected with RRP8 or TNP1. ELISA analysis of sera from patients with various rheumatic diseases demonstrated reactivity for RRP8 and TNP1 in 20% and 14.7% of SLE patients, respectively, whereas there was little or no reactivity in patients with other rheumatic diseases. Among SLE patients, 63.6% and 45.5% of those with LN were positive for anti-RRP8 and anti-TNP1 antibodies, compared with 12.5% and 9.4% of SLE patients without nephritis, respectively. Both proteins are cationic, and their respective antibodies did not cross-react with dsDNA. These proteins released from apoptotic cells form ICs with each autoantibody, and their ICs may become trapped at anionic sites in the glomerular basement membrane, leading to deposition in glomeruli. These autoantibodies may be useful for prediction of LN in subsets of SLE patients who are negative for anti-dsDNA antibodies.

Although neurons and glia inevitably undergo degeneration in the core of ischemic lesions, many cells, particularly immune cells, infiltrate the core and survive in it. Such infiltrating cells may play certain roles in the regeneration and repair of damaged brain tissues. In this study, we characterized macrophage-like cells that accumulated in the ischemic core of a rat brain whose right middle cerebral artery was transiently occluded for 90 mins. Many of the accumulated macrophage-like cells expressed Iba1, a marker of macrophages/microglia, as well as NG2 chondroitin sulfate proteoglycan (NG2), which has been recognized as a marker of oligodendrocyte progenitor cells. Such macrophage-like cells were termed BINCs (brain Iba1+/NG2+ cells) to distinguish them from NG2/Iba1+ or NG2+/Iba1− cells that were also present in the perilesion and the contralateral hemisphere. Electron microscopy showed the localization of NG2 along the plasma membrane of cells that had many phagosomes and irregular-shaped or reniform heterochromatin-rich nuclei, which are characteristics of monocytes/macrophages. Brain Iba1+/NG2+ cells were highly proliferative and their number peaked at 7 days post-reperfusion. An immunoblot analysis of NG2 revealed the presence of two NG2s: one expressed by BINCs with a molecular weight of 300 kDa, and the other found in the contralateral hemisphere with a molecular weight of 290 kDa. Taken the various functions of NG2, BINCs may be involved in not only phagocytosis of degenerated cells but also the healing and regeneration of lesion cores.

Mucosal mast cells (MMCs) have an important role in allergic inflammation, and effective antagonists are required for their regulation. To discover a possible mechanism of controlling the activation of MMCs, we investigated the expression and function of syntaxin4, one of the soluble membrane N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins, in RBL‐2H3 cells, which is a rat mucosal mast cell line. Syntaxin4 silencing was induced by transfection of short interfering RNAs (siRNAs). Syntaxin4 was knocked down in mast cells at both the mRNA and protein levels. The release of granule contents that are involved in inflammation, such as histamine and hexosaminidase, was significantly suppressed by the gene silencing of syntaxin4. Silencing of this gene was also induced in the trachea and bronchi of rats by intratracheal application of the siRNAs using an atelocollagen delivery system. The activation of MMCs, which was monitored by the level of rat mast cell protease‐II (RMCPII) in the bronchoalveolar lavage fluid (BALF), was inhibited, and asthmatic airway constriction was prevented by administration of the syntaxin/atelocollagen complex. These results indicate that siRNAs targeting syntaxin4 can stabilize mucosal mast cells and may have beneficial therapeutic effects on the asthmatic response. Autoimmunity occurs when an organism develops an immune response against itself, resulting in an inflammatory reaction which damages organs such as brain, joints or pancreas. This results in diseases such as Type 1 diabetes, vasculitis, or rheumatoid arthritis. A fine balance exists in order to accommodate the control of microbial pathogens and commensals, and immune self‐tolerance. The March 2012 issue will include a review series on Autoimmune Disease, particularly featuring articles on clinical translation, and the current state of research in this area. Articles include reasons for the increased incidence of certain autoimmune diseases and allergic diseases in Western society and the advances made by the application of novel and high throughput technologies to the analysis of diseased tissues. The accompanying web focus presents links to related articles from across Nature Publishing Group.

The presence of pre-existing basement membrane (BM) components improves the morphogenesis of epidermis and BM in constructing a human living skin-equivalent (LSE). De-epithelialized amniotic membrane (AM) retains key BM components. We have therefore investigated the usefulness of AM for constructing LSE. De-epithelialized AM was overlaid on type I collagen gel embedded with fibroblasts. Normal human keratinocytes (NHKs) were then seeded onto the epithelial side of the AM to construct an AM-LSE. A conventional LSE was constructed by seeding NHKs on a fibroblast-populated type I collagen gel. When the keratinocytes reached confluence, the LSE was lifted to the air-liquid interface and cultured for up to 3 weeks. Samples were harvested at various times and investigated morphologically, immunohistochemically, and ultrastructurally. In AM-LSE, the epidermis was better stratified, with more compact, polarized, columnar basal cells, and the expression of differentiation and proliferation markers was more similar to that of normal human skin than was that of LSE without AM. A more continuous BM and better-developed hemidesmosomes were found in AM-LSE. The epidermis of AM-LSE outgrew much faster than that of LSE without AM. When transplanted onto nude mice, both LSEs took well; however, the AM-LSE graft showed better morphogenesis of the epidermis, BM, and hemidesmosomes. The better epidermal morphology and better-developed BM in AM-LSE in vitro and in vivo indicates its superiority over LSE without AM for clinical applications.

During an allergic inflammatory response in the airway, if a failure of the epithelial cell barrier occurs before the systemic immune response is triggered by allergens, more allergens can invade. Using a rat model of asthma, we previously found that mucosal mast cells, which localise to the epithelial layer of the airways, are activated to promote a pro‐asthmatic immune response. In this study, we developed a neonatal rat model of allergic airway hypersensitivity that mimics some features of childhood asthma. Airway hypersensitivity was measured using unrestrained whole‐body plethysmography after analysis of the serum IgE titre. Inflammatory cells and inflammatory mediators in bronchoalveolar lavage fluid samples were examined. Two mast cell‐specific proteases were detected using PCR. In addition, we analysed the phenotype and the number of mast cells in the airways by immunohistochemistry, and we found that the number of mucosal mast cells and the expression level of the proteases increased 2 weeks after sensitisation. Changes in the IgE titre, airway hypersensitivity and the activation of other inflammatory cells were delayed, appearing during the 4 weeks after sensitisation. Our results indicate that the activation of mucosal mast cells contributes to the pro‐asthmatic immune response. This activation may be a biomarker allowing early intervention that could help prevent allergic airway inflammation.

The regulated control of Ca(2+) influx is essential for the activation and function of the adaptive immune response, as Ca(2+) is a key regulator of important transcription factors. To determine whether Ca(2+) release-activated Ca(2+) (CRAC) channels contribute to the abnormal behaviour of T cells in patients with rheumatoid arthritis (RA), we performed a cross-sectional study to characterize the expression and functional status of CRACM1 channels in RA patients. Peripheral blood was obtained from 50 RA patients, 50 osteoarthritis (OA) patients and healthy donors. We measured Ca(2+) influx and CRAC currents in naïve and memory CD4(+) T cells. CRACM1 expression was evaluated in T cells from each of the three groups. These cells were further characterized by flow cytometric analysis of interleukin-4 (IL-4), IL-17, interferon-γ and tumour necrosis factor-α. These cytokines were also measured in naïve CD4(+) T cells following the lentivirus-mediated silencing of CRACM1.There was a significant positive correlation between Ca(2+) influx in naïve T cells and RA activity. Functionally aberrant naïve CD4(+) T cells from patients with active RA showed the different cytokine release pattern and exhibited increased Ca(2+) influx as well as increased CRACM1 protein expression and function. Specific lentiviral-induced gene silencing of CRACM1 reversed the alterations in T-cell cytokine production. The data presented here indicate that an upregulation of CRACM1 expression and function may be responsible for the abnormal cytokine release of naïve CD4(+) T cells in RA patients. CRACM1 might therefore represent a new molecular target for RA therapies.