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Chèdiak-Higashi syndrome is a rare autosomal recessive disease that causes hypopigmentation, recurrent infections, mild coagulation defects and neurological problems. Beige mice carry a mutation in the lysosome trafficking regulator (LYST) gene and display some of the key characteristics of human Chèdiak-Higashi syndrome, in particular, a high susceptibility to infection due to aberrant natural killer (NK) cell and polymorphonuclear leucocyte function. Morphological analysis of beige mice reveals the presence of enlarged lysosomes in a variety of cell types, including leucocytes, hepatocytes, fibroblasts and renal tubule cells. To examine the process of granule maturation and degranulation in beige mice mast cells, morphological studies have been conducted using a combination of electrophysiological techniques; however, few functional studies have been conducted with mast cells, such as mediator release. The aim of the present study was to determine the morphological and functional characteristics of skin and peritoneal mast cells and bone marrow-derived mast cells of homozygous (bg/bg) and heterozygous (bg/+) beige mice and wild-type (+/+) mice. The histamine concentration was lower in the peritoneal and bone marrow-derived mast cells of bg/bg mice compared with those of bg/+ and +/+ mice, but the histamine release response was potentiated. In vivo studies of passive cutaneous anaphylaxis showed no differences between bg/bg mice and either bg/+ or +/+ mice. Although bg/bg mast cells with enlarged granules display specific exocytotic processes in vitro, the consequences of mast cell activation in beige mice were similar to those of wild-type mice in vivo.

Objective Hydrogen-rich water, which is a potent antioxidant agent, was investigated for its protective effects against ischemic damage of the cochlea in gerbils. Methods The animals were subjected to transient cochlear ischemia by occluding the bilateral vertebral arteries for l5 min. Five milliliters of hydrogen-rich saline was then intravenously administered immediately after the insult. Saline without hydrogen was used as a control. Effects of hydrogen were evaluated using the auditory brainstem response (ABR) and histological studies of the inner ear. Results In non-ischemia animals, ABR thresholds and histological findings of the cochlea did not change by administration of saline or hydrogen-rich saline. In contrast, transient cochlear ischemia caused a 24.2 ± 3.8 dB increase in the ABR threshold at 8 kHz, and a decrease of 14.1% ± 1.8% in the number of inner hair cells (IHCs) at the basal turn on day 7. Ischemic damage was more severe at 16 and 32 kHz. When the animals were treated with hydrogen-rich saline, cochlear damage was significantly reduced: the increase in ABR threshold was 11.7 ± 2.6 dB at 8 kHz and the IHC loss was 7.5% ± 2.1% at the basal turn on day 7. The effects of hydrogen-rich saline were more prominent at higher frequencies. Conclusions Intravenous administration of hydrogen-rich saline was effective in preventing acute hearing loss due to transient cochlear ischemia.

Additional file 1: Supplemental Figure S1. Transmission electron microscopy of Aβ1-40 solution after incubation at 37 °C for two days. Aβ1-40 solution represents a mixture of more the soluble oligomers, monomers and less the insoluble fibrils. Only parts of the aggregation exhibits long and straight fibrils with some intertwining fibrils. Upper photos, X 100,000 magnification. Lower photos, X 200,000 magnification. Supplemental Figure S2. Mean swimming speed of Morris Water Maze Test (MWMT). Three weeks after injection, MWMT was performed for 5 days. Time courses of mean swimming speed in each group in WT and smAT2 mice are show. n=12 in WT-PBS, BCCAO (-); n=12 in WT-PBS, BCCAO (+); n=15 in WT-Aβ, BCCAO (-); n=14 in WT-Aβ, BCCAO (+); n=8 in smAT2-PBS, BCCAO (-); n=9 in smAT2-PBS, BCCAO (+); n=14 in smAT2-Aβ, BCCAO (-); n=14 in smAT2-Aβ, BCCAO (+). Supplemental Figure S3. Effect of BCCAO with/without Aβ1-40 on cerebrovascular function markers. mRNA expression of collagen IV and VEGF-A in the hippocampus 3 weeks after injection by real-time RT-PCR in WT and smAT2 mice are shown. *P

Estrogen deficiency caused by bilateral ovariectomy (OVX) has been reported to lead to morphological changes in otoconia. Thus, we examined the morphological changes in the otoconial layer after OVX. We also investigated whether micro-computed tomography (µCT) is useful for the detection of morphological changes in the otoconial layer.The otic capsules of C57BL/6 J mice were removed and evaluated using histological techniques and µCT at 2, 4, and 8 weeks after OVX or sham surgery. The volume of the utricle otoconial layer was measured and compared between the OVX and sham groups. The µCT scan and histological study results were also compared.The volume of the utricle otoconial layer was significantly increased 4 weeks after OVX compared to the sham group in both histological and µCT studies (p 0.05). The volume of the otoconial layer measured using µCT was significantly correlated with the histological study results (p 0.05).The volume of the utricle otoconial layer increased after OVX. These morphological changes could be detected by µCT.

We conducted photo-activated delivery of drugs based on the fusion of liposomes with endocytic membranes, thus allowing the direct release of encapsulated drugs inside the cytoplasm. As described in our earlier works, liposomes can be photoresponsive and fusogenic following the incorporation of a malachite green derivative carrying a long alkyl chain (MGL) into the lipid membrane. We prepared MGL liposomes using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine and encapsulated doxorubicin (DOX). Though the shape of MGL liposomes became elliptical after encapsulating DOX, UV irradiation did not enhance DOX leakage from MGL liposomes. We demonstrated the cellular uptake of MGL liposomes into murine cells derived from colon cancer (Colon 26 cells) using flow cytometry, and we found that the uptake was governed by a clathrin-dependent endocytosis pathway. Confocal fluorescence microscopic observations of Colon 26 cells treated with MGL liposomes encapsulating DOX revealed that DOX was localized in endosomes under dark conditions, while DOX was observed in the cytosol and nucleus after UV irradiation. The viability of Colon 26 cells treated with MGL liposomes encapsulating DOX was reduced by UV irradiation, indicating photo-induced enhancement of anti-cancer efficacy.

The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells.We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1.We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content.The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is required for the formation and maintenance of neuromuscular junctions (NMJs). Although autoantibodies against MuSK have been demonstrated to cause myasthenia gravis (MG), the underlying pathogenic mechanism remains unclear because a major subclass of these antibodies is functionally monovalent. We investigated the pathogenic role of MuSK antibodies in the onset of MG in vivo and in vitro. Ultrastructural visualization of NMJs in paretic rabbits with MuSK antibodies indicated that postsynaptic membranes were preserved, despite a significant loss of complexity in the convoluted synaptic folds. In addition, an in vitro assay indicated that both divalent and monovalent antibodies from paretic rabbits could interfere with agrin-induced acetylcholine receptor (AChR) clustering in cultured myotubes. Furthermore, in the absence of agrin, divalent antibodies induced MuSK phosphorylation and accelerated downregulation of Dok-7, an essential intracellular MuSK binding protein, while monovalent antibodies inhibited agrin-induced phosphorylation of MuSK, thus demonstrating distinct molecular mechanisms underlying the MuSK dysfunction induced by these two types of antibodies. Taken together, these findings suggest that complement activation is not necessary for the MG onset and that both divalent and monovalent antibodies may cause MG in vivo by inducing MuSK dysfunction.

Angiogenesis is required for physiological tissue repair processes, such as cutaneous wound healing. However, recent studies indicate that endogenous angiogenic factors may enhance photo-induced skin alterations in response to experimental ultraviolet (UV)-B exposure. Angiopoietin-related growth factor (AGF), also known as angiopoietin-like protein 6 (Angptl6), is known to promote new blood vessel formation and vascular hyperpermeability. Importantly, epidermal overexpression of Angptl6/AGF in mice promotes wound healing in the skin. However, it remains unclear whether overexpression of Angptl6/AGF facilitates tissue repair processes in response to UV-B irradiation. To test this hypothesis, we subjected Angptl6/AGF transgenic mice to acute or chronic UV-B exposure. Surprisingly, transgenic mice showed enhanced photosensitivity to subthreshold doses of UV-B that did not induce skin alterations in wild-type littermates. Marked enlargement of blood vessels was observed after a single exposure to UV-B in Angptl6/AGF transgenic mice, although no epidermal changes were observed. Chronic UV-B exposure over 14 weeks promoted cutaneous skin damage in Angptl6/AGF transgenic mice, whereas wild-type mice showed little or no macroscopic skin alteration. In addition to pronounced angiogenesis and epidermal hyperplasia, marked enlargement of dermal lymphatic vessels was observed in UV-B-exposed Angptl6/AGF transgenic mice. Electron microscopy analysis further revealed that the number and size of collagen bundles in the dermis was markedly reduced after chronic UV-B exposure in Angptl6/AGF transgenic mice. Taken together, these results indicate that ectopic expression of Angptl6/AGF in mice likely promotes UV-B-induced skin alterations, and that angiogenesis could be a therapeutic target in prevention of skin photo-aging.

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.

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.

Amphiregulin (AR), a member of the EGF family, is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR yields a transmembrane-cytoplasmic fragment (AR-CTF), as well as a soluble AR. Here we demonstrate that the proAR-shedding stimuli trigger endocytosis of both AR-CTF and un-shed proAR. ProAR translocates from the plasma membrane to the inner nuclear membrane, whereas AR-CTF is translocated to the lysosome via retrograde membrane trafficking. Nuclear envelope localization of proAR involves truncation of the C-terminus, which subsequently activates the ER-retrieval signal. The truncated form of proAR interacts with A-type lamin and is retained at the inner nuclear membrane. Heterochromatin formation is then induced and global transcription is transiently suppressed. This study gives new insight into epigenetic chromatin organization in mammalian cells: a plasma-membrane-anchored growth factor is targeted to the inner nuclear membrane where it participates in dynamic chromatin organization and control of transcription.

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

Objective: Aquaporins (AQPs) function as selective pores allowing water, glycerol and other small solutes to pass through the cell membrane. The present study investigated the expression and localization of AQP3 in the human prostate. Methods: Three human prostate cancer cell lines (DU-145, LNCaP and PC-3) and one normal human prostate cell line (PNT1A) were used in this study. Clinical materials from patients with prostate cancer were also used. AQP3 mRNA and protein expression were examined using reverse transcription-polymerase chain reaction and immunohistochemical methods. Results: Aquaporin 3 mRNA was expressed in normal and cancerous prostate cells. Moreover, the expression of AQP3 mRNA was seen in both normal and cancerous epithelia of human prostate tissues, but not in the mesenchyme. In the normal epithelia of the prostate, localization was limited to cell membranes, particularly the basolateral membranes. However, the expression of AQP3 protein in the cancer epithelia was not observed on the cell membranes. Conclusion: The results suggest that water-channel protein may be involved in the preservation of cellular character in the human prostate and prostate cancer is associated with an alteration of water-transporting mechanisms. Changes in the localization of AQP3 in cancer cells may result from tumorigenesis.

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.

The effects of transient cochlear ischemia on spiral ganglion cells (SGCs) were studied in Mongolian gerbils. Ischemic insult was induced by occluding the bilateral vertebral arteries of gerbils for 15 min. Seven days after ischemia, the percentage of SGCs decreased to 67.5% from the preischemic baseline in the basal turn. Evaluation with immunohistochemical staining showed TUNEL-positive reactions in the SGCs with fragmented nuclei. In addition, we investigated the protective effects of ginsenoside Rb1 (gRb1) against ischemic injury to SGCs. Seven days after ischemia, the auditory brainstem response threshold shift was significantly reduced and the percentage of SGCs decreased to 90.2% from the preischemic baseline in the basal turn in the gRb1-treated group. These findings suggest that gRb1 prevented hearing loss caused by ischemic injury to SGCs in Mongolian gerbils.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.

The pathology of non-immunological airway contraction is not well understood. To define the activation of different phenotypes of mast cells, a rat non-immunological asthmatic model was prepared. Airway contraction in rats was measured by an unrestrained whole-body plethysmographic system following a 10-min inhalation challenge with a 5% solution of compound 48/80. Histamine, leukotrein C(4) (LTC(4)) and tumor necrosis factor (TNF)-alpha levels in bronchoalveolar lavage fluid, as well as tissue histamine content were quantified. Mast cells and eosinophils were detected by histology. Both the early and late phase of airway responses were induced by inhalation of compound 48/80. Histamine and TNF-alpha levels increased significantly 30 min after challenge, but no increases were detected at either 8 or 24 h after challenge. A high LTC(4) level was detected in 30 min and 8 h after challenge. Tissue histamine content decreased at 30 min after challenge and returned to the unstimulated level by 8 h. Connective tissue mast cells in rat trachea showed a degranulation response. Along with the increase in numbers of mucosal mast cells, rat mast cell protease II at both mRNA and protein levels in the trachea epithelial layer was also increased significantly at 30 min after challenge. We conclude that compound 48/80 inhalation causes both the early and late phase of airway contraction in rats. Mast cell degranulation is responsible for the early phase of airway response, which subsequently triggers the late phase of airway response.

The effects of transient cochlear ischemia on the stria vascularis were studied. Fifteen minutes of ischemia decreased the endocochlear potential by up to 17.5 mV on day 1; it returned to normal on day 7. Immunostaining for Na+,K+-ATPase, a marker for the Na+/K+-pump, and for connexin 26, a marker for gap junctions, was inhibited on days 1 and 4, and returned to normal on day 7. Electron microscopy showed expansion of the intercellular space with abundant vacuolar formation in the stria vascularis. These morphological changes disappeared completely by day 7. The results indicate that transient ischemia causes a reversible functional disorder of the stria vascularis with fine structural changes, which may be owing to dysfunction of Na+/K+-pump or gap junctions.

To elucidate whether ischemia-reperfusion can cause delayed cell death in the cochlea, the effects of transient cochlear ischemia on hearing and on neuronal structures in the cochlea were studied in Mongolian gerbils. Ischemia was induced by bilaterally occluding the vertebral arteries for 5 minutes in gerbils, which lack posterior cerebral communicating arteries. In gerbils, the labyrinthine arteries are fed solely by the vertebral arteries. Occlusion of the vertebral arteries caused a remarkable increase in the threshold of compound action potentials (CAPs), which recovered over the following day. However, 7 days after the onset of reperfusion, the threshold began to increase again. Morphologic changes in the hair cell stereocilia were revealed by electron microscopy. The number of nuclear collapses was counted in cells stained for DNA and F-actin to evaluate the degree of cell death in the organ of Corti. Changes in spiral ganglion cell (SGC) neuron number were detected, whether or not progressive neuronal death occurred in the SGC. These studies showed that sporadic fusion of hair cells and the disappearance of hair cell stereocilia did not begin until 4 days after ischemia. On subsequent days, the loss of hair cells, especially inner hair cells (IHCs), and the degeneration of SGC neurons became apparent. Ten days after ischemia, the mean percentage cell loss of IHCs was 6.4% in the basal turn, 6.4% in the second turn, and 0.8% in the apical turn, respectively, and the number of SGC neurons had decreased to 89% of preischemic status. These results indicate that transient ischemia causes delayed hearing loss and cell death in the cochlea by day 7 after ischemia.

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.

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.

We reported previously that an angiotensin II type 1 receptor blocker, telmisartan, improved cognitive decline with peroxisome proliferator-activated receptor-γ activation; however, the detailed mechanisms are unclear. Enhanced blood-brain barrier (BBB) permeability with alteration of tight junctions is suggested to be related to diabetes mellitus. Therefore, we examined the possibility that telmisartan could attenuate BBB impairment with peroxisome proliferator-activated receptor-γ activation to improve diabetes mellitus–induced cognitive decline. Type 2 diabetic mice KKA y exhibited impairment of cognitive function, and telmisartan treatment attenuated this. Cotreatment with GW9662, a peroxisome proliferator-activated receptor-γ antagonist, interfered with these protective effects of telmisartan against cognitive function. BBB permeability was increased in both the cortex and hippocampus in KKA y mice. Administration of telmisartan attenuated this increased BBB permeability. Coadministration of GW9662 reduced this effect of telmisartan. Significant decreases in expression of tight junction proteins and increases in matrix metalloproteinase expression, oxidative stress, and proinflammatory cytokine production were observed in the brain, and treatment with telmisartan restored these changes. Swollen astroglial end-feet in BBB were observed in KKA y mice, and this change in BBB ultrastructure was decreased in telmisartan. These effects of telmisartan were weakened by cotreatment with GW9662. In contrast, administration of another angiotensin II type 1 receptor blocker, losartan, was less effective compared with telmisartan in terms of preventing BBB permeability and astroglial end-foot swelling, and coadministration of GW9662 did not affect the effects of losartan. These findings are consistent with the possibility that, in type 2 diabetic mice, angiotensin II type 1 receptor blockade with peroxisome proliferator-activated receptor-γ activation by telmisartan may help with protection against cognitive decline by preserving the integrity of the BBB.

Different types of nonionic vesicles were prepared from commercial Span 80 (also called sorbitan monooleate), as an inexpensive, biocompatible alternative to conventional phospholipid-based vesicles (liposomes). The vesicles were characterized by different techniques and comparison was made with vesicles formed from POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine) or DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine). Dynamic light scattering measurements, electron microscopy analyses, and two types of fusion assays indicate that Span 80 vesicles are stable for at least 7 days at 4 or 25 degrees C, while storage at 42 degrees C causes irreversible vesicle fusion. This indicates that Span 80 vesicles are thermoresponsive with vesicle fusion occurring at elevated temperature. This property may be related to headgroup dehydration and is certainly not directly linked to the phase transition temperature (Tm) of the vesicles, since the Tm is below -30 degrees C, as determined by differential scanning calorimetry (DSC). The measured Tm value for Span 80 vesicles is lower than in the case of DOPC or POPC, correlating with a higher fluidity of Span 80 vesicles as compared to POPC or DOPC vesicles, as determined with DPH (1,6-diphenyl-1,3,5-hexatriene) as fluorescent membrane probe. High fluidity correlates with increased leakage of entrapped water-soluble dye molecules. Addition of cholesterol and soybean phosphatidylcholine lowers the extent of leakage, allowing a tuning of the bilayer permeability.

Aquaporins (AQPs) function as selective pores allowing water, glycerol and other small solutes to pass through the cell membrane. The present study investigated the expression and localization of AQP3 in the human prostate.Three human prostate cancer cell lines (DU-145, LNCaP and PC-3) and one normal human prostate cell line (PNT1A) were used in this study. Clinical materials from patients with prostate cancer were also used. AQP3 mRNA and protein expression were examined using reverse transcription-polymerase chain reaction and immunohistochemical methods.Aquaporin 3 mRNA was expressed in normal and cancerous prostate cells. Moreover, the expression of AQP3 mRNA was seen in both normal and cancerous epithelia of human prostate tissues, but not in the mesenchyme. In the normal epithelia of the prostate, localization was limited to cell membranes, particularly the basolateral membranes. However, the expression of AQP3 protein in the cancer epithelia was not observed on the cell membranes.The results suggest that water-channel protein may be involved in the preservation of cellular character in the human prostate and prostate cancer is associated with an alteration of water-transporting mechanisms. Changes in the localization of AQP3 in cancer cells may result from tumorigenesis.

Resident quiescent microglia have been thought to respond rapidly to various pathologic events in the brain by proliferating and producing many bioactive substances, including proinflammatory cytokines and nitric oxide (NO). In this study, we investigated the reaction of microglia in traumatic and ischemic lesions caused by stab wounds and the transient 90-min occlusion of middle cerebral artery in a mature rat brain. Although many Iba1+ resident microglia underwent apoptotic degeneration in the lesion core within 24 hr after the onset of the brain insult as revealed by TUNEL staining, numerous small, round, isolectin B4+/CD11b+/CD68+ cells were localized in the lesion core. These small, round cells with diameters of 7–9 μm and polymorph nuclei expressed neutrophil-specific elastase, alkaline phosphatase, and platelet-activating factor receptor. Accordingly, they were not activated microglia but neutrophils. Immunohistochemical staining with antibodies to inducible NO synthase (iNOS) showed that most iNOS+ cells were neutrophils. The results from spatial and kinetic analyses using RT-PCR and immunoblotting were consistent with the immunohistochemical observations. These results suggest the necessity of reevaluating the traditional view on the roles of activated microglia in severe neuropathologic events. Note that the traditional microglial markers isolectin B4, CD11b, and CD68 are not specific for microglia, particularly in a pathologic brain. © 2007 Wiley-Liss, Inc.

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.

We investigated the effect of glutamate receptor antagonists on progressive inner hair cell (IHC) loss following transient cochlear ischemia in gerbils. Transient cochlear ischemia was induced by 15-min bilateral vertebral artery occlusion. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist, 6-7-dinitroquinoxaline-2,3-dione (DNQX), or an N-methyl-D-aspartate (NMDA)-type receptor antagonist, MK-801, was administered 10 min before the ischemic insult. Hearing was assessed by sequentially recording compound action potentials (CAPs) before, during, and after the ischemia. The degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day after ischemia, the increases in the CAP threshold and the progressive IHC loss were significantly reduced in cochleae treated with DNQX, while MK-801 was ineffective. These results suggest that the AMPA receptor plays a critical role in the development of the progressive IHC loss induced by ischemia/reperfusion injury in the cochlea.

The effect of hypothermia on ischemic injury of the cochlea in gerbils was studied with particular regard to glutamate efflux in the perilymph. Under normothermic conditions interruption of the blood supply to the cochlea for 15 min caused a remarkable elevation of the compound action potential (CAP) threshold, and an increase in perilymphatic glutamate. The CAP threshold recovered to some extent with reperfusion, but not to preischemic levels. CAP thresholds, under hypothermic conditions and with reperfusion, recovered promptly to near pre-ischemic levels, while glutamate concentration did not change. These results, together with electron microscopy studies, suggest that hypothermia prevents hearing loss primarily through reduction of glutamate efflux at the synopses between inner hair cells and primary afferent auditory neurons.

The mechanism underlying ischemia-induced hearing loss was studied in gerbils with transient hindbrain ischemia. Occlusion of the vertebral arteries caused an increase in the concentration of glutamate in the perilymph and elevated the compound action potential (CAP) threshold to 24.6 dB at 5 minutes. the CAP threshold subsequently recovered on reperfusion, gradually reaching 8.3 dB 120 minutes after reperfusion. Under electron microscopy, afferent dendrites of the cochlear nerve in contact with inner hair cells exhibited abnormal swelling 5 minutes after ischemia/reperfusion. These morphological changes were not observed in cochleas treated with an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist, 6-7-dinitroquinoxaline-2,3-dione (DNQX), before hindbrain ischemia; an N-methyl-D-aspartate (NMDA)-type receptor antagonist, D-2-amino-5-phosphonopentanoate (D-AP5), was ineffective. Moreover, the histopathological alterations noted 5 minutes after reperfusion were spontaneously ameliorated 120 minutes after ischemia/reperfusion. These findings suggest that the ischemia-induced increase in extracellular glutamate concentration with subsequent activation of AMPA/kainate receptors is responsible for neurite degeneration and hearing loss in the early stages following transient hindbrain ischemia.