Combination Treatment with the Vimentin-Targeting Antibody hzVSF and Tenofovir Suppresses Woodchuck Hepatitis Virus Infection in Woodchucks.

Current treatment options for patients infected with hepatitis B virus (HBV) are suboptimal, because the approved drugs rarely induce cure due to the persistence of the viral DNA genome in the nucleus of infected hepatocytes, and are associated with either severe side effects (pegylated interferon-alpha) or require life-long administration (nucleos(t)ide analogs). We report here the evaluation of the safety and therapeutic efficacy of a novel, humanized antibody (hzVSF) in the woodchuck model of HBV infection. hzVSF has been shown to act as a viral entry inhibitor, most likely by suppressing vimentin-mediated endocytosis of virions. Targeting the increased vimentin expression on liver cells by hzVSF after infection with HBV or woodchuck hepatitis virus (WHV) was demonstrated initially. Thereafter, hzVSF safety was assessed in eight woodchucks naïve for WHV infection. Antiviral efficacy of hzVSF was evaluated subsequently in 24 chronic WHV carrier woodchucks by monotreatment with three ascending doses and in combination with tenofovir alafenamide fumarate (TAF). Consistent with the proposed blocking of WHV reinfection, intravenous hzVSF administration for 12 weeks resulted in a modest but transient reduction of viral replication and associated liver inflammation. In combination with oral TAF dosing, the antiviral effect of hzVSF was enhanced and sustained in half of the woodchucks with an antibody response to viral proteins. Thus, hzVSF safely but modestly alters chronic WHV infection in woodchucks; however, as a combination partner to TAF, its antiviral efficacy is markedly increased. The results of this preclinical study support future evaluation of this novel anti-HBV drug in patients.

A soft and transparent contact lens for the wireless quantitative monitoring of intraocular pressure.

Continuous detection of raised intraocular pressure (IOP) could benefit the monitoring of patients with glaucoma. Current contact lenses with embedded sensors for measuring IOP are rigid, bulky, partially block vision or are insufficiently sensitive. Here, we report the design and testing in volunteers of a soft and transparent contact lens for the quantitative monitoring of IOP in real time using a smartphone. The contact lens incorporates a strain sensor, a wireless antenna, capacitors, resistors, stretchable metal interconnects and an integrated circuit for wireless communication. In rabbits, the lens provided measurements that match those of a commercial tonometer. In ten human participants, the lens proved to be safe, and reliably provided accurate quantitative measurements of IOP without inducing inflammation.

Simplified Classification of Tear Film Break-Up Patterns and Their Clinicopathological Correlations in Patients With Dry Eye Disease.

PURPOSE: To analyze the pathophysiological differences between patients with dry eye disease (DED) having different tear film break-up patterns (TBUPs). METHODS: This investigative analysis involved 91 eyes of 91 patients with DED who were divided into two groups: those with "dot" break-up pattern (group I) and those with "random" break-up pattern (group II). Clinical severity was evaluated using the Ocular Surface Disease Index (OSDI), Oxford stain score system (OSS) score, and tear film break-up time (TF-BUT). Eighteen patients in group I and 17 patients in group II were selected for sampling of tears and the conjunctiva, and the concentrations of inflammatory cytokines and mucin in the tears and conjunctival tissue were measured. RESULTS: Thirty-seven patients were classified as group I and 54 patients as group II. Patients in group I had a statistically lower TF-BUT and a higher OSS score than those in group II, whereas the OSDI was not statistically different between the groups. The concentrations of interleukin (IL)-6 and IL-8 were statistically higher in group I than those in group II. Impression cytology showed that the expression of IL-1ß and IL-8 was higher in group I, whereas that of other genes was not statistically different. CONCLUSIONS: We were able to clearly classify patients with DED with different TBUPs into two groups, and each group had different clinical and pathophysiological characteristics. In patients with the dot break-up pattern, the disease was strongly associated with ocular surface inflammation, as opposed to that in patients without this pattern.

Collagen immobilization on ultra-thin nanofiber membrane to promote in vitro endothelial monolayer formation.

The endothelialization on the poly (ε-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (ε-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (ε-caprolactone) nanofiber using hydrolysis by NaOH treatment and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide reaction as a cost-effective and stable approach. NaOH was first applied to render the poly (ε-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (ε-caprolactone) nanofibers using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide. Scanning electron microscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and fluorescence microscopy were used to verify stable collagen immobilization on the surface of the poly (ε-caprolactone) nanofibers and the maintenance of the original structure of poly (ε-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (ε-caprolactone) nanofiber membrane and expressed tight junction proteins with the increase in transendothelial electrical resistance, which demonstrated the maintenance of the endothelial cell homeostasis on the collagen-immobilized-poly (ε-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (ε-caprolactone) nanofiber scaffolds.

Development of an in vitro 3D choroidal neovascularization model using chemically induced hypoxia through an ultra-thin, free-standing nanofiber membrane.

Choroidal neovascularization (CNV) is the pathological growth of new blood vessels in the sub-retinal pigment epithelial (RPE) space from the choroid through a break in the Bruch's membrane (BM). Despite its importance in studying biological processes and drug discovery, the development of an in vitro CNV model that achieves the physiological structures of native RPE-BM-choroidal capillaries (CC) is still challenging. Here, we develop a novel 3D RPE-BM-CC complex biomimetic system on an ultra-thin, free-standing nanofiber membrane. The thickness of the pristine nanofiber membrane is 2.17 ±â€¯0.81 µm, and the Matrigel-coated nanofiber membrane attains a permeability coefficient of 2.95 ±â€¯0.25 × 10-6 cm/s by 40 kDa FITC-dextran, which is similar to the physiological value of the native BM. On the in vitro 3D RPE-BM-CC complex system, we demonstrate endothelial cell invasion across the 3D RPE-BM-CC complex and the mechanism of the invasion by imposing a hypoxic condition, which is thought to be the major pathological cause of CNV. Furthermore, alleviation of the invasion is achieved by treating with chrysin and anti-VEGF antibody. Thus, the in vitro 3D RPE-BM-CC complex biomimetic system can recapitulate essential features of the pathophysiological environment and be employed for the screening of drug candidates to reduce the number of costly and time-consuming in vivo tests or clinical trials.

Biocompatibility evaluation of bioprinted decellularized collagen sheet implanted in vivo cornea using swept-source optical coherence tomography.

Corneal transplantation by full-thickness penetrating keratoplasty with human donor tissue is a widely accepted treatment for damaged or diseased corneas. Although corneal transplantation has a high success rate, a shortage of high-quality donor tissue is a considerable limitation. Therefore, bioengineered corneas could be an effective solution for this limitation, and a decellularized extracellular matrix comprises a promising scaffold for their fabrication. In this study, three-dimensional bioprinted decellularized collagen sheets were implanted into the stromal layer of the cornea of five rabbits. We performed in vivo noninvasive monitoring of the rabbit corneas using swept-source optical coherence tomography (OCT) after implanting the collagen sheets. Anterior segment OCT images and averaged amplitude-scans were acquired biweekly to monitor corneal thickness after implantation for 1 month. The averaged cornea thickness in the control images was 430.3 ± 5.9 µm, while the averaged thickness after corneal implantation was 598.5 ± 11.8 µm and 564.5 ± 12.5 µm at 2 and 4 weeks, respectively. The corneal thickness reduction of 34 µm confirmed the biocompatibility through the image analysis of the depth-intensity profile base. Moreover, hematoxylin and eosin staining supported the biocompatibility evaluation of the bioprinted decellularized collagen sheet implantation. Hence, the developed bioprinted decellularized collagen sheets could become an alternative solution to human corneal donor tissue, and the proposed image analysis procedure could be beneficial to confirm the success of the surgery.

Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering.

The microenvironments of tissues or organs are complex architectures comprised of structural proteins including collagen. Particularly, the cornea is organized in a lattice pattern of collagen fibrils which play a significant role in its transparency. This paper introduces a transparent bioengineered corneal structure for transplantation. The structure is fabricated by inducing shear stress to a corneal stroma-derived decellularized extracellular matrix bioink based on a 3D cell printing technique. The printed structure recapitulates the native macrostructure of the cornea with aligned collagen fibrils which results in the construction of a highly matured and transparent cornea stroma analog. The level of shear stress, controlled by the various size of the printing nozzle, manipulates the arrangement of the fibrillar structure. With proper parameter selection, the printed cornea exhibits high cellular alignment capability, indicating a tissue-specific structural organization of collagen fibrils. In addition, this structural regulation enhances critical cellular events in the assembly of collagen over time. Interestingly, the collagen fibrils that remodeled along with the printing path create a lattice pattern similar to the structure of native human cornea after 4 weeks in vivo. Taken together, these results establish the possibilities and versatility of fabricating aligned collagen fibrils; this represents significant advances in corneal tissue engineering.

Decellularized corneal lenticule embedded compressed collagen: toward a suturable collagenous construct for limbal reconstruction.

Recently, compressed collagen has attracted much attention as a potential alternative for a limbal epithelial stem cell (LESC) carrier to treat limbal stem cell deficiency (LSCD), in that it can provide mechanically improved collagen fibrillar structures compared to conventional collagen hydrogel. However, its clinical efficacy as an LESC carrier has not yet been studied through in vivo transplantation due to limited mechanical strength that cannot withstand a force induced by surgical suturing and low resistance to enzymatic degradation. This study firstly presents a suturable LESC carrier based on compressed collagen in the form of a biocomposite. The biocomposite was achieved by integrating a decellularized corneal lenticule, which is a decellularized stromal tissue obtained from corneal refractive surgery, inside a compressed collagen to form a sandwich structure. A suture retention test verified that the biocomposite has a much higher suture retention strength (0.56 ± 0.12 N) compared to the compressed collagen (0.02 ± 0.01 N). The biocomposite also exhibited more than 3 times higher resistance to enzymatic degradation, indicating long-term stability after transplantation. In vitro cell culture results revealed that the biocomposite effectively supported the expansion and stratification of the LESCs with expressions of putative stem cell and differentiated corneal epithelial cell markers. Finally, the biocomposite verified its clinical efficacy by stably delivering the LESCs onto an eye of a rabbit model of LSCD and effectively reconstructing the ocular surface.

Generation of Femtosecond Laser-Cut Decellularized Corneal Lenticule Using Hypotonic Trypsin-EDTA Solution for Corneal Tissue Engineering.

PURPOSE: To establish an optimized and standardized protocol for the development of optimal scaffold for bioengineering corneal substitutes, we used femtosecond laser to process human corneal tissue into stromal lenticules and studied to find the most efficient decellularization method among various reagents with different tonicities. METHODS: The decellularization efficacy of several agents (0.1%, 0.25%, and 0.5% of Triton X-100, SDS, and trypsin-EDTA (TE), resp.) with different tonicities was evaluated. Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and glycosaminoglycan (GAG) contents, recellularization efficacy, and biocompatibilities. RESULTS: 0.5% SDS in hypertonic and isotonic buffer, 0.25% TE in hypotonic buffer, and 0.5% TE in all tonicities completely decellularized the corneal lenticules. Of the protocols, decellularization with hypotonic 0.25 and 0.5% TE showed the lowest DNA contents, while the GAG content was the highest. Furthermore, the recellularization efficacy of the hypotonic TE method was better than that of the SDS-based method. Hypotonic TE-treated decellularized corneal lenticules (DCLs) were sufficiently transparent and biocompatible. CONCLUSION: We generated an ideal protocol for DCLs using a novel method. Furthermore, it is possible to create a scaffold using a bioengineered corneal substitute.

ω-3 Polyunsaturated fatty acids accelerate airway repair by activating FFA4 in club cells.

A G protein-coupled receptor (GPCR) named free fatty acid receptor 4 (FFA4, also known as GPR120) was found to act as a GPCR for ω-3 polyunsaturated fatty acids. Its expression has been reported in lung epithelial club cells. We investigated whether supplementation of the ω-3 fatty acids benefits lung health. Omacor (7.75 mg/kg), clinically prescribed preparation of ω-3 fatty acids, and FFA4-knockout mice were utilized in a naphthalene-induced mouse model of acute airway injury (1 injection of 30 mg/kg ip). Naphthalene injection induced complete destruction of bronchiolar epithelial cells within a day. Appearance of bronchiolar epithelial cells was observed after 21 days in control mice. It was found, however, that supplementation of Omacor accelerated the recovery. The appearance of bronchiolar epithelial cells was observed between 7 and 14 days after naphthalene injury in Omacor-treated mice. In isolated club cells, ω-3 fatty acids were found to stimulate cell proliferation and migration but to inhibit cell differentiation. With the use of pharmacological tools and FFA4-knockout mice, FFA4 was found to be responsible for ω-3 fatty acids-induced proliferation in vitro in club cells. Furthermore, accelerated recovery from naphthalene-induced airway injury in Omacor-treated mice was not observed in FFA4-knockout mice in vivo. Present findings indicate that ω-3 fatty acids-induced proliferation of bronchiole epithelial cells through FFA4 is responsible for Omacor-induced accelerated recovery from airway injury. Therefore, intermittent administration of Omacor needs to be tested for acute airway injury because ω-3 fatty acids stimulate proliferation but inhibit differentiation of club cells.

Petatewalide B, a novel compound from Petasites japonicus with anti-allergic activity.

ETHNOPHARMACOLOGICAL RELEVANCE: The giant butterbur Petasites japonicus is used to treat asthma and allergic diseases in traditional Korean, Japanese, and Chinese medicine. AIM OF THE STUDY: To elucidate the anti-allergic effect of Petasites genus, we studied effects of several compounds from Petasites japonicus leaves and found a novel bakkenolide-type sesquiterpine. In the present study, anti-allergic and anti-inflammatory effects of the new compound was examined using in vivo and in vitro experiments. MATERIALS AND METHODS: The novel compound was isolated from Petasites japonicus leaves and named petatewalide B. Antigen-induced degranulation and Ca(2+) mobilization were measured in RBL-2H3 mast cells by measuring ß-hexosaminidase activity and fluorescence change of Ca(2+) probe, fura-2. Induction of inducible nitric oxide synthase and cyclooxygenase 2 was measured by Western blotting in peritoneal macrophages. In addition, ovalbumin-induced asthma model was used for in vivo efficacy test of petatewalide B. Membrane potential was estimated by measuring fluorescence change of DiBAC in C6 glioma cells. RESULTS: Petatewalide B inhibited the antigen-induced degranulation of ß-hexosaminidase in RBL-2H3 mast cells, but did not affect antigen-induced Ca(2+) increase in the cells. Petatewalide B also showed inhibition of the LPS-induced induction of iNOS, but not COX-2 in mouse peritoneal macrophages. Nitric oxide production was also inhibited by petatewalide B in macrophages. In the ovalbumin-induced asthma model, petatewalide B strongly inhibited accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar lavage fluid. Petatewalide B increased the membrane potential of C6 glioma cells in a concentration-dependent manner. CONCLUSION: Petatewalide B from Petasites genus not only has anti-allergic and anti-inflammatory effects but also induces a transient increase of membrane potential in C6 glioma cells.

Immunocytochemical localization of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin in bat visual cortex.

It is a common misconception that bats are blind, and various studies have suggested that bats have visual abilities. The purpose of this study was to investigate the cytoarchitecture of calbindin D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunoreactive (IR) neurons in the bat visual cortex using immunocytochemistry. The highest density of CB- and PV-IR neurons was located in layer IV of the visual cortex. The majority of CB- and PV-IR neurons were characterized by a stellate or round/oval shape. CR-IR neurons were predominantly located in layers II/III, and the cells were principally round/oval in shape. Two-color immunofluorescence revealed that 65.96%, 24.24%, and 77.00% of the CB-, CR-, and PV-IR neurons, respectively, contained gamma-aminobutyric acid (GABA). We observed calcium-binding protein (CBP)-IR neurons in specific layers of the bat visual cortex and in specific cell types. Many of the CBP-IR neurons were GABAergic interneurons. These data provide useful clues to aid in understanding the functional aspects of the bat visual system.

Anti-allergic effect of α-cubebenoate isolated from Schisandra chinensis using in vivo and in vitro experiments.

AIM OF THE STUDY: In Oriental countries, the dried fruits of Schisandra chinensis are extensively used in traditional medicine to treat asthma, gonorrhea, and other diseases. Recently, α-cubebenoate was isolated as an anti-inflammatory component from Schisandra chinensis. In the present study, the authors examined the anti-allergic effect of α-cubebenoate using in vivo and in vitro experiments. MATERIALS AND METHODS: α-Cubebenoate was isolated from an extract of Schisandra chinensis fruits. Antigen-induced degranulation and Ca(2+) mobilization were measured in RBL-2H3 mast cells. In addition, BALB/c mice were sensitized with ovalbumin and aluminum hydroxide, and then challenged with ovalbumin for three consecutive days. α-Cubebenoate (1mg/kg) was administered intraperitoneally 30min before each ovalbumin challenge. RESULTS: In RBL-2H3 mast cells, α-cubebenoate inhibited antigen-induced degranulation and increase of intracellular Ca(2+) concentrations. In the ovalbumin-induced asthma model, α-cubebenoate suppressed bronchiolar structural changes induced by ovalbumin challenge. Furthermore, α-cubebenoate strongly inhibited accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar lavage fluid. α-Cubebenoate also suppressed Th2 cytokines (IL-4 and IL-13) and TGF-ß1 in lung tissues and in immune cells at the mRNA and protein levels. CONCLUSION: α-Cubebenoate has an inhibitory effect on allergic inflammation and could be utilized as an agent for the treatment of asthma.

Omega-3 fatty acids induce Ca(2+) mobilization responses in human colon epithelial cell lines endogenously expressing FFA4.

AIM: Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro. METHODS: HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4. RESULTS: Ten to 100 µmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca(2+)]i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca(2+)]i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca(2+)]i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca(2+)ATPase inhibitor thapsigargin, but was not affected by Gi/o protein inhibitor PTX or IP3R inhibitor 2-APB. CONCLUSION: Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca(2+) mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive Gi/o protein, followed by Ca(2+) release from thapsigargin-sensitive Ca(2+) stores and Ca(2+) influx across the plasma membrane.

Therapeutic effects of s-petasin on disease models of asthma and peritonitis.

To explore the anti-allergic and anti-inflammatory effects of extracts of Petasites genus, we studied the effects of s-petasin, a major sesquiterpene from Petasites formosanus (a butterbur species) on asthma and peritonitis models. In an ovalbumin-induced mouse asthma model, s-petasin significantly inhibited the accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar fluids. S-petasin inhibited the antigen-induced degranulation of ß-hexosamidase but did not inhibit intracellular Ca(2+) increase in RBL-2H3 mast cells. S-petasin inhibited the LPS induction of iNOS at the RNA and protein levels in mouse peritoneal macrophages. Furthermore, s-petasin inhibited the production of NO (the product of iNOS) in a concentration-dependent manner in the macrophages. Furthermore, in an LPS-induced mouse model of peritonitis, s-petasin significantly inhibited the accumulation of polymorpho nuclear and mononuclear leukocytes in peritoneal cavity. This study shows that s-petasin in Petasites genus has therapeutic effects on allergic and inflammatory diseases, such as, asthma and peritonitis through degranulation inhibition in mast cells, suppression of iNOS induction and production of NO in macrophages, and suppression of inflammatory cell accumulation.

Sphingosine 1-phosphate induced anti-atherogenic and atheroprotective M2 macrophage polarization through IL-4.

Sphingosine 1-phosphate (S1P) has been implicated in anti-atherogenic properties of high-density lipoproteins. However, the roles and signaling of S1P in macrophages, the main contributor to atherosclerosis, have not been well studied. Furthermore, pro-inflammatory M1 and anti-inflammatory M2 macrophage phenotypes may influence the development of atherosclerosis. Therefore, we investigated the effects of S1P on macrophage phenotypes, especially on M2 polarization and its signaling in relation to the anti-atherogenic properties of S1P. It was found that S1P induced anti-inflammatory M2 polarization via IL-4 secretion and its signaling, and induced IL-4Rα and IL-2Rγ. In addition, down-stream signalings, such as, stat-6 phosphorylation, SOCS1 induction, and SOCS3 suppression were also observed in macrophages in response to S1P. Furthermore, S1P-induced ERK activation, and the inhibitions of p38 MAPK and JNK were found to be key signals for IL-4 induction. Moreover, the anti-atherogenic effect of S1P in HDL was confirmed by the observation that oxidized LDL-induced lipid accumulation was attenuated in S1P-treated M2 macrophages. Furthermore, the atheroprotective effect of S1P was demonstrated by its anti-apoptotic effect on S1P-treated macrophages. The present study shows that S1P-induced M2 polarization of macrophages could be mediated via IL-4 signaling, and suggests that M2 polarization by S1P is responsible for the anti-atherogenic and atheroprotective properties of high-density lipoproteins in vivo.

Action and Signaling of Lysophosphatidylethanolamine in MDA-MB-231 Breast Cancer Cells.

Previously, we reported that lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, can increase intracellular Ca(2+) ([Ca(2+)]i) via type 1 lysophosphatidic acid (LPA) receptor (LPA1) and CD97, an adhesion G-protein-coupled receptor (GPCR), in MDA-MB-231 breast cancer cells. Furthermore, LPE signaling was suggested as like LPA1/CD97-Gi/o proteins-phospholipase C-IP3-Ca(2+) increase in these cells. In the present study, we further investigated actions of LPE not only in the [Ca(2+)]i increasing effect but also in cell proliferation and migration in MDA-MB-231 breast cancer cells. We utilized chemically different LPEs and a specific inhibitor of LPA1, AM-095 in comparison with responses in SK-OV3 ovarian cancer cells. It was found that LPE-induced Ca(2+) response in MDA-MB-231 cells was evoked in a different manner to that in SK-OV3 cells in terms of structural requirements. AM-095 inhibited LPE-induced Ca(2+) response and cell proliferation in MDA-MB-231 cells, but not in SK-OV3 cells, supporting LPA1 involvement only in MDA-MB-231 cells. LPA had significant effects on cell proliferation and migration in MDA-MB-231 cells, whereas LPE had less or no significant effect. However, LPE modulations of MAPKs (ERK1/2, JNK and p38 MAPK) was not different to those by LPA in the cells. These data support the involvement of LPA1 in LPE-induced Ca(2+) response and cell proliferation in breast MDA-MB-231 cells but unknown GPCRs (not LPA1) in LPE-induced responses in SK-OV3 cells. Furthermore, although LPE and LPA utilized LPA1, LPA utilized more signaling cascades than LPE, resulting in stronger responses by LPA in proliferation and migration than LPE in MDA-MB-231 cells.

Identification of a novel anti-inflammatory compound, α-cubebenoate from Schisandra chinensis.

AIMS OF THE STUDY: Extracts of Schisandra chinensis have been used as an anti-fatigue and tonic agent. Because chronic fatigue syndrome is related to inflammatory and oxidative stress, we assessed whether Schisandra chinensis has anti-inflammatory constituents and studied the effect of a novel α-cubebenoate isolated from Schisandra chinensis. MATERIALS AND METHODS: α-Cubebenoate was isolated from an extract of Schisandra chinensis fruits. The inductions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) by lipopolysaccharide (LPS) were quantified by RT-PCR and Western blotting in mouse peritoneal macrophages. Nitric oxide (NO) and prostaglandin E2 (PGE2) were also measured in the media by Griess reagent and EIA method. A mouse model of LPS-induced peritonitis was used to test the in vivo efficacy of α-cubebenoate. RESULTS: α-Cubebenoate (5-10µg/ml) inhibited the inductions of iNOS and COX-2 in mouse peritoneal macrophages at the mRNA and protein levels. LPS-induced productions of NO and PGE2 were inhibited by α-cubebenoate (5-10µg/ml). In addition, α-cubebenoate inhibited the LPS-induced activation of JNK, but not those of ERK and p38 MAPK in mouse peritoneal macrophages. Furthermore, in the LPS-induced in vivo peritonitis model, α-cubebenoate (1mg/kg) strongly inhibited the accumulation of polymorph nuclear lymphocytes in the peritoneal cavity. CONCLUSION: α-Cubebenoate inhibited LPS-induced expression of iNOS and COX-2 in a concentration-dependent manner, thereby suppressing productions of NO and PGE2 in vitro in peritoneal macrophages. α-Cubebenoate also inhibited LPS-induced accumulation of polymorph nuclear lymphocytes in LPS-induced peritonitis model in vivo. α-Cubebenoate may act as an anti-fatigue constituent of Schisandra chinensis through anti-inflammation and could be of therapeutic use as a treatment for inflammatory diseases.

Lysophosphatidylethanolamine utilizes LPA(1) and CD97 in MDA-MB-231 breast cancer cells.

Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca(2+) ([Ca(2+)]i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca(2+)]i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA1 and LPA3) inhibited LPE-induced [Ca(2+)]i increases, and knockdown of LPA1 by transfection with LPA1 siRNA completely inhibited LPE-induced [Ca(2+)]i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA1 in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of Gi/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP3 receptor) completely inhibited LPE-induced [Ca(2+)]i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA1-CD97/Gi/o proteins/phospholipase C/IP3/Ca(2+) rise in MDA-MB-231 breast cancer cells.

Anti-allergic and anti-inflammatory effects of bakkenolide B isolated from Petasites japonicus leaves.

AIMS OF THE STUDY: To elucidate the anti-allergic and anti-inflammatory effects of Petasites genus, we studied the effects of several compounds isolated from Petasites japonicas leaves. MATERIALS AND METHODS: Bakkenolide B was isolated from Petasites japonicus leaves. Antigen-induced degranulation was measured in RBL-2H3 mast cells by measuring ß-hexosamidase activity. Induction of inducible nitric oxide synthase and cyclooxygenase 2 was measured by Western blotting in peritoneal macrophages. Ovalbumin-induced asthma model was used for in vivo efficacy test of bakkanolide B. RESULTS: We found that bakkenolide B, a major component of the leaves, concentration-dependently inhibited RBL-2H3 mast cell degranulation. Bakkenolide B also inhibited the gene inductions of inducible nitric oxide synthase and cyclooxygenase 2 in mouse peritoneal macrophages. Furthermore, in an ovalbumin-induced asthma model, bakkenolide B strongly inhibited the accumulation of eosinophils, macrophages, and lymphocytes to bronchoalveolar lavage fluid. CONCLUSION: Bakkenolide B has suppressive properties for allergic and inflammatory responses and may be utilized as a potent agent for the treatment of asthma.