Using Curriculum Learning in Pattern Recognition of 3-dimensional Cryo-electron Microscopy Density Maps.

Although Cryo-electron microscopy (cryo-EM) has been successfully used to derive atomic structures for many proteins, it is still challenging to derive atomic structure when the resolution of cryo-EM density maps is in the medium range, e.g., 5-10 Å. Studies have attempted to utilize machine learning methods, especially deep neural networks to build predictive models for the detection of protein secondary structures from cryo-EM images, which ultimately helps to derive the atomic structure of proteins. However, the large variation in data quality makes it challenging to train a deep neural network with high prediction accuracy. Curriculum learning has been shown as an effective learning paradigm in machine learning. In this paper, we present a study using curriculum learning as a more effective way to utilize cryo-EM density maps with varying quality. We investigated three distinct training curricula that differ in whether/how images used for training in past are reused while the network was continually trained using new images. A total of 1,382 3-dimensional cryo-EM images were extracted from density maps of Electron Microscopy Data Bank in our study. Our results indicate learning with curriculum significantly improves the performance of the final trained network when the forgetting problem is properly addressed.

IL-1ß dominates the promucin secretory cytokine profile in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1ß, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1ß alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1ß induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1ß are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1ß-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.

Role of Spdef in the Regulation of Muc5b Expression in the Airways of Naive and Mucoobstructed Mice.

Understanding how expression of airway secretory mucins MUC5B and MUC5AC is regulated in health and disease is important to elucidating the pathogenesis of mucoobstructive respiratory diseases. The transcription factor SPDEF (sterile α-motif pointed domain epithelial specific transcription factor) is a key regulator of MUC5AC, but its role in regulating MUC5B in health and in mucoobstructive lung diseases is unknown. Characterization of Spdef-deficient mice upper and lower airways demonstrated region-specific, Spdef-dependent regulation of basal Muc5b expression. Neonatal Spdef-deficient mice exhibited reductions in BAL Muc5ac and Muc5b. Adult Spdef-deficient mice partially phenocopied Muc5b-deficient mice as they exhibited reduced Muc5b in nasopharyngeal and airway epithelia but not in olfactory Bowman glands, 75% incidence of nasopharyngeal hair/mucus plugs, and mild bacterial otitis media, without defective mucociliary clearance in the nasopharynx. In contrast, tracheal mucociliary clearance was reduced in Spdef-deficient mice in the absence of lung disease. To evaluate the role of Spdef in the development and persistence of Muc5b-predominant mucoobstructive lung disease, Spdef-deficient mice were crossed with Scnn1b-transgenic (Scnn1b-Tg) mice, which exhibit airway surface dehydration-induced airway mucus obstruction and inflammation. Spdef-deficient Scnn1b-Tg mice exhibited reduced Muc5ac, but not Muc5b, expression and BAL content. Airway mucus obstruction was not decreased in Spdef-deficient Scnn1b-Tg mice, consistent with Muc5b-dominant Scnn1b disease, but increased airway neutrophilia was observed compared with Spdef-sufficient Scnn1b-Tg mice. Collectively, these results indicate that Spdef regulates baseline Muc5b expression in respiratory epithelia but does not contribute to Muc5b regulation in a mouse model of Muc5b-predominant mucus obstruction caused by airway dehydration.

Metformin Treatment Does Not Inhibit Growth of Pancreatic Cancer Patient-Derived Xenografts.

There is currently tremendous interest in developing anti-cancer therapeutics targeting cell signaling pathways important for both cancer cell metabolism and growth. Several epidemiological studies have shown that diabetic patients taking metformin have a decreased incidence of pancreatic cancer. This has prompted efforts to evaluate metformin, a drug with negligible toxicity, as a therapeutic modality in pancreatic cancer. Preclinical studies in cell line xenografts and one study in patient-derived xenograft (PDX) models were promising, while recently published clinical trials showed no benefit to adding metformin to combination therapy regimens for locally advanced and metastatic pancreatic cancer. PDX models in which patient tumors are directly engrafted into immunocompromised mice have been shown to be excellent preclinical models for biomarker discovery and therapeutic development. We evaluated the response of four PDX tumor lines to metformin treatment and found that all four of our PDX lines were resistant to metformin. We found that the mechanisms of resistance may occur through lack of sustained activation of adenosine monophosphate-activated protein kinase (AMPK) or downstream reactivation of the mammalian target of rapamycin (mTOR). Moreover, combined treatment with metformin and mTOR inhibitors failed to improve responses in cell lines, which further indicates that metformin alone or in combination with mTOR inhibitors will be ineffective in patients, and that resistance to metformin may occur through multiple pathways. Further studies are required to better understand these mechanisms of resistance and inform potential combination therapies with metformin and existing or novel therapeutics.

Cardiomyocyte-specific role of miR-24 in promoting cell survival.

Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell-derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non-coding RNA, microRNA-24 (miR-24), as a pro-survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR-24 via viral transduction or chemical transfection, where the observed protective role of miR-24 in cardiomyocytes might have partially resulted from its effect on non-cardiomyocyte cells. To elucidate the cardiomyocyte-specific effects of miR-24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR-24 expression is driven by the cardiac-specific Myh6 promoter. The Myh6-miR-24 transgenic mice did not exhibit apparent difference from their wild-type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post-MI compared to their wild-type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non-genetic approach, raising the possibility that non-genetic approaches of introducing miR-24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte-specific role of miR-24 in the adult heart, and may provide insight to strategy design when considering miRNA-based therapies for cardiovascular diseases.

Dual modulation of cyclooxygenase and CYP epoxygenase metabolism and acute vascular inflammation in mice.

Cyclooxygenase (COX)-derived prostaglandins and cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids are important regulators of inflammation; however, functional interactions between these pathways in the regulation of vascular inflammation in vivo have not been studied. We investigated the relative and additive effects of endothelial CYP2J2 overexpression (Tie2-CYP2J2-Tr), global sEH disruption (Ephx2(-/-)), and pharmacologic COX inhibition with indomethacin on the acute vascular inflammatory response to endotoxin in mice. Compared to vehicle-treated wild-type C57BL/6 controls, induction of myeloperoxidase (MPO) activity in lung and liver was similarly attenuated in Tie2-CYP2J2-Tr mice, Ephx2(-/-) mice and wild-type mice treated with moderate dose indomethacin. Dual modulation of both pathways, however, did not produce an additive anti-inflammatory effect. These findings demonstrate that both COX and CYP epoxygenase-mediated eicosanoid metabolism are important regulators of the acute vascular inflammatory response in vivo, and suggest that the anti-inflammatory effects of modulating each pathway may be mediated, at least in part, by overlapping mechanisms.

Enalapril reverses high-fat diet-induced alterations in cytochrome P450-mediated eicosanoid metabolism.

Metabolism of arachidonic acid by cytochrome P450 (CYP) to biologically active eicosanoids has been recognized increasingly as an integral mediator in the pathogenesis of cardiovascular and metabolic disease. CYP epoxygenase-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET + DHET) and CYP ω-hydroxylase-derived 20-hydroxyeicosatetraenoic acid (20-HETE) exhibit divergent effects in the regulation of vascular tone and inflammation; thus, alterations in the functional balance between these parallel pathways in liver and kidney may contribute to the pathogenesis and progression of metabolic syndrome. However, the impact of metabolic dysfunction on CYP-mediated formation of endogenous eicosanoids has not been well characterized. Therefore, we evaluated CYP epoxygenase (EET + DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver and kidney in apoE(-/-) and wild-type mice fed a high-fat diet, which promoted weight gain and increased plasma insulin levels significantly. Hepatic CYP epoxygenase metabolic activity was significantly suppressed, whereas renal CYP ω-hydroxylase metabolic activity was induced significantly in high-fat diet-fed mice regardless of genotype, resulting in a significantly higher 20-HETE/EET + DHET formation rate ratio in both tissues. Treatment with enalapril, but not metformin or losartan, reversed the suppression of hepatic CYP epoxygenase metabolic activity and induction of renal CYP ω-hydroxylase metabolic activity, thereby restoring the functional balance between the pathways. Collectively, these findings suggest that the kinin-kallikrein system and angiotensin II type 2 receptor are key regulators of hepatic and renal CYP-mediated eicosanoid metabolism in the presence of metabolic syndrome. Future studies delineating the underlying mechanisms and evaluating the therapeutic potential of modulating CYP-derived EETs and 20-HETE in metabolic diseases are warranted.

M(3) muscarinic receptor antagonist bencycloquidium bromide attenuates allergic airway inflammation, hyperresponsiveness and remodeling in mice.

M(3) muscarinic receptors are localized on inflammatory cells, airway smooth muscle, and submucosal glands, known to mediate bronchoconstriction, mucus secretion, and airway remodeling. It is hypothesized bencycloquidium bromide (BCQB), a novel M(3) receptor antagonist, might have potential effects on airway hyperresponsiveness, inflammation and airway remodeling in a murine model of asthma. Mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was examined to determine the total and differential cell counts, and cytokine levels. Lung tissues were evaluated for cell infiltration, mucus hypersecretion, airway remodeling, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Inhalation administration of BCQB significantly not only reduced ovalbumin-induced airway hyperresponsiveness comparing to methacholine, and prevented the ovalbumin-induced increase in total cell counts and eosinophil counts. Reverse transcriptase polymerase chain reaction analysis of whole lung lysates revealed that BCQB markedly suppressed ovalbumin-induced mRNA expression of eotaxin, IL-5, IL-4 and MMP-9, and increased mRNA expression of IFN-γ and TIMP-1 in a dose-dependent manner. Substantial IFN-γ/IL-4 (Th1/Th2) levels were recovered in bronchoalveolar lavage fluid after BCQB treatment. In addition, histological studies showed that BCQB dramatically inhibited ovalbumin-induced lung tissue eosinophil infiltration, airway mucus production and collagen deposition in lung tissues. Results reported in current paper suggest that M(3) receptors antagonist may provide a novel therapeutic approach to treat airway inflammation, hyperresponsiveness and remodeling.

Activation of the acute inflammatory response alters cytochrome P450 expression and eicosanoid metabolism.

Cytochrome P450 (P450)-mediated metabolism of arachidonic acid regulates inflammation in hepatic and extrahepatic tissue. CYP2C/CYP2J-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET+DHET) elicit anti-inflammatory effects, whereas CYP4A/CYP4F-derived 20-hydroxyeicosatetraenoic acid (20-HETE) is proinflammatory. Because the impact of inflammation on P450-mediated formation of endogenous eicosanoids is unclear, we evaluated P450 mRNA levels and P450 epoxygenase (EET+DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver, kidney, lung, and heart in mice 3, 6, 24, and 48 h after intraperitoneal lipopolysaccharide (LPS) (1 mg/kg) or saline administration. Hepatic Cyp2c29, Cyp2c44, and Cyp2j5 mRNA levels and EET+DHET formation were significantly lower 24 and 48 h after LPS administration. Hepatic Cyp4a12a, Cyp4a12b, and Cyp4f13 mRNA levels and 20-HETE formation were also significantly lower at 24 h, but recovered to baseline at 48 h, resulting in a significantly higher 20-HETE/EET+DHET formation rate ratio compared with that for saline-treated mice. Renal P450 mRNA levels and P450-mediated eicosanoid metabolism were similarly suppressed 24 h after LPS treatment. Pulmonary EET+DHET formation was lower at all time points after LPS administration, whereas 20-HETE formation was suppressed in a time-dependent manner, with the lowest formation rate observed at 24 h. No differences in EET+DHET or 20-HETE formation were observed in heart. Collectively, these data demonstrate that acute activation of the innate immune response alters P450 expression and eicosanoid metabolism in mice in an isoform-, tissue-, and time-dependent manner. Further study is necessary to determine whether therapeutic restoration of the functional balance between the P450 epoxygenase and ω-hydroxylase pathways is an effective anti-inflammatory strategy.

Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice.

Cytochrome P-450 (CYP)-derived epoxyeicosatrienoic acids (EETs) possess potent anti-inflammatory effects in vitro. However, the effect of increased CYP-mediated EET biosynthesis and decreased soluble epoxide hydrolase (sEH, Ephx2)-mediated EET hydrolysis on vascular inflammation in vivo has not been rigorously investigated. Consequently, we characterized acute vascular inflammatory responses to endotoxin in transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases and mice with targeted disruption of Ephx2. Compared to wild-type controls, CYP2J2 transgenic, CYP2C8 transgenic, and Ephx2(-/-) mice each exhibited a significant attenuation of endotoxin-induced activation of nuclear factor (NF)-κB signaling, cellular adhesion molecule, chemokine and cytokine expression, and neutrophil infiltration in lung in vivo. Furthermore, attenuation of endotoxin-induced NF-κB activation and cellular adhesion molecule and chemokine expression was observed in primary pulmonary endothelial cells isolated from CYP2J2 and CYP2C8 transgenic mice. This attenuation was inhibited by a putative EET receptor antagonist and CYP epoxygenase inhibitor, directly implicating CYP epoxygenase-derived EETs with the observed anti-inflammatory phenotype. Collectively, these data demonstrate that potentiation of the CYP epoxygenase pathway by either increased endothelial EET biosynthesis or globally decreased EET hydrolysis attenuates NF-κB-dependent vascular inflammatory responses in vivo and may serve as a viable anti-inflammatory therapeutic strategy.

Intracerebroventricular injection of leukotriene B4 attenuates antigen-induced asthmatic response via BLT1 receptor stimulating HPA-axis in sensitized rats.

BACKGROUND: Basic and clinical studies suggest that hypothalamic-pituitary-adrenal (HPA) axis is the neuroendocrine-immune pathway that functionally regulates the chronic inflammatory disease including asthma. Our previous studies showed corresponding changes of cytokines and leukotriene B4 (LTB4) between brain and lung tissues in antigen-challenged asthmatic rats. Here, we investigated how the increased LTB4 level in brain interacts with HPA axis in regulating antigen-induced asthmatic response in sensitized rats. METHODS: Ovalbumin-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v) 30 min before challenge. Lung resistance (RL) and dynamic lung compliance (Cdyn) were measured before and after antigen challenge. Inflammatory response in lung tissue was assessed 24 h after challenge. Expression of CRH mRNA and protein in hypothalamus were evaluated by RT-PCR and Western Blot, and plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using the ELISA kits. RESULTS: Antigen challenge decreased pulmonary function and induced airway inflammation, evoked HPA axis response in sensitized rats. Administration of LTB4 via i.c.v markedly attenuated airway contraction and inflammation. Meanwhile, LTB4 via i.c.v markedly increased CORT and ACTH level in plasma before antigen challenge, and followed by further increases in CORT and ACTH levels in plasma after antigen challenge in sensitized rats. Expression of CRH mRNA and protein in hypothalamus were also significantly increased by LTB4 via i.c.v in sensitized rats after antigen challenge. These effect were completely blocked by pre-treatment with BLT1 receptor antagonist U75302 (10 ng), but not by BLT2 antagonist LY255283. CONCLUSIONS: LTB4 administered via i.c.v down-regulates the airway contraction response and inflammation through activation of the HPA axis via its BLT1 receptor. This study expands our concept of the regulatory role of intracranial inflammatory mediators in inflammatory diseases including asthma. The favourable effects of LTB4 on the HPA axis may help to explain the phenomenon of self-relief after an asthmatic attack.

Leukotriene B4, administered via intracerebroventricular injection, attenuates the antigen-induced asthmatic response in sensitized guinea pigs.

BACKGROUND: Despite intensive studies focused on the pathophysiology of asthmatic inflammation, little is known about how cross-talk between neuroendocrine and immune systems regulates the inflammatory response during an asthmatic attack. We recently showed corresponding changes of cytokines and leukotriene B4 (LTB4) in brain and lung tissues of antigen-challenged asthmatic rats. Here, we investigated how LTB4 interacts with the neuroendocrine-immune system in regulating antigen-induced asthmatic responses in sensitized guinea pigs. METHODS: Ovalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v.) 30 min before challenge. Airway contraction response was evaluated using Penh values before and after antigen challenge. The inflammatory response in lung tissue was evaluated 24 h after challenge. The LTB4 content of lung and brain homogenate preparations was detected by reversed phase high-performance liquid chromatography (RP-HPLC). Plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using ELISA kits. RESULTS: Antigen challenge impaired pulmonary function and increased inflammatory cell infiltration in lung tissue. These responses could be significantly suppressed by LTB4, 30 ng i.c.v., in ovalbumin-sensitized guinea pigs. LTB4 content of lung and brain homogenates from antigen-challenged guinea pigs was significantly increased. In addition, administration of LTB4 via i.c.v. markedly increased CORT and ACTH level in plasma before antigen challenge, and there were further increases in CORT and ACTH levels in plasma after antigen challenge. U75302, 100 ng i.c.v., completely blocked the effects of LTB4. In addition, U75302, 100 ng via i.c.v. injection, markedly decreased LTB4 content in lung homogenates, but not in brain homogenates. CONCLUSIONS: Increased LTB4 levels in brain during asthmatic attacks down-regulates airway contraction response and inflammation through the BLT1 receptor. Stimulation of the hypothalamic-pituitary-adrenal axis by LTB4 may result in an increase in systemic glucocorticoids which, in turn, would feed back to suppress the asthmatic response.

Cytochrome P450 epoxygenases, soluble epoxide hydrolase, and the regulation of cardiovascular inflammation.

The cytochrome P450 (CYP) epoxygenase enzymes CYP2J and CYP2C catalyze the epoxidation of arachidonic acid to epoxyeicosatrienoic acids (EETs), which are rapidly hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). It is well-established that CYP epoxygenase-derived EETs possess potent vasodilatory effects; however, the cellular effects of EETs and their regulation of various inflammatory processes have become increasingly appreciated in recent years, suggesting that the role of this pathway in the cardiovascular system extends beyond the maintenance of vascular tone. In particular, CYP epoxygenase-derived EETs inhibit endothelial activation and leukocyte adhesion via attenuation of nuclear factor-kappaB activation, inhibit hemostasis, protect against myocardial ischemia-reperfusion injury, and promote endothelial cell survival via modulation of multiple cell signaling pathways. Thus, the CYP epoxygenase pathway is an emerging target for pharmacological manipulation to enhance the cardiovascular protective effects of EETs. This review will focus on the role of the CYP epoxygenase pathway in the regulation of cardiovascular inflammation and (1) describe the functional impact of CYP epoxygenase-derived EET biosynthesis and sEH-mediated EET hydrolysis on key inflammatory process in the cardiovascular system, (2) discuss the potential relevance of this pathway to pathogenesis and treatment of cardiovascular disease, and (3) identify areas for future research.

Oral administration of allergen extracts from Dermatophagoides farinae desensitizes specific allergen-induced inflammation and airway hyperresponsiveness in rats.

Clinically sublingual immunotherapy (SLIT) by using allergen extracts effectively alleviates the symptoms of allergic rhinitis and asthma. Supposed that oral administration of high-dose of allergen extracts imitates SLIT and may prevent IgE-related responses in allergic diseases, we investigated the effects of oral administration of allergen extracts from Dermatophagoides farinae (Derf) on allergen-induced inflammation and airway hyperresponsiveness (AHR) in a model of asthmatic rat. After administration to the specific Derf-sensitized rats with Derfdrop solution containing Derf1 and Derf2 extracts derived from Derf, the effects of Derfdrop on AHR, inflammatory cell accumulation, cytokine production in the bronchoalveolar lavage fluid and lung tissue, as well as serum IgE and IgG levels were investigated. Results indicated that Derfdrop not only dose-dependently prevented the AHR in response to methacholine, but also significantly reduced the serum total and allergen-specific IgE levels, all the maximal effects were achieved at dose of 5 mg/kg/d, and were as comparable as those of dexamethasone at dose of 1.0 mg/kg/d. Furthermore, oral administration of Derfdrop not only dose-dependently elevated allergen-specific serum IgG levels and reduced total and allergen-specific IgE levels, but also normalized the imbalance between the Th1 cytokine, IFN-gamma and Th2 cytokine, IL-4. Finally, oral administration of Derfdrop significantly reduced Goblet cell hyperplasia and eosinophilia in the Derf-sensitized allergic rat model. These data suggest that Derfdrop effectively improves specific allergen-induced inflammation and AHR in Derf-sensitized and -challenged rats and provide with the rationale for clinical SLIT by using Derfdrop in a specific allergen-induced asthma.

Indole derivatives as potent inhibitors of 5-lipoxygenase: design, synthesis, biological evaluation, and molecular modeling.

A series of novel indole derivatives was designed, synthesized and evaluated by cell-based assays for their inhibitory activities against 5-LOX in rat peritoneal leukocytes. Most of them (30 out of 35) showed an inhibitory potency higher than the initial screening hit 1a (IC(50)=74 microM). Selected compounds for concentration-response studies showed prominent inhibitory activities with IC(50) values ranging from 0.74 microM to 3.17 microM. Four compounds (1m, 1s, 4a, and 6a) exhibited the most potent inhibitory activity compared to that of the reference drug (Zileuton), with IC(50) values less than 1 microM. Molecular modeling studies for compounds 1a, 3a, 4a, and 6a were also presented. The excellent in vitro activities of this class of compounds may possess potential for the treatment of LT-related diseases.

Anti-asthmatic effects of Perilla seed oil in the guinea pig in vitro and in vivo.

The aim of this study was to investigate the anti-asthmatic effects of Perilla seed oil in vitro and in vivo in sensitized guinea pigs. Aerosolized antigen caused an immediate bronchoconstriction. Perilla seed oil per os inhibited the increase in lung resistance and the decrease in dynamic lung compliance in a dose-dependent manner with an ED50 (95 % confidence interval, CI) of 1.10 (0.98 - 1.24) g/kg and 1.07 (0.94 - 1.22) g/kg, respectively. Infiltration of leukocytes, mononuclear cells, eosinophils and neutrophils induced by inhaling antigen was also inhibited by Perilla seed oil in a dose-dependent manner with an ED50 (95 % CI) of 1.00 (0.86 - 1.15), 1.24 (1.10 - 1.38), 0.63 (0.51 - 0.77) and 0.61 (0.38 - 0.98) g/kg, respectively. Perilla seed oil (5 - 500 microg/mL) inhibited the slow reaction substance of anaphylaxis (SRS-A) release induced by antigen challenge in lung tissue of sensitized guinea pigs. It also inhibited calcium ionophore (A(23187))-induced leukotriene (LT) D4 release from the lung tissue of non-sensitized guinea pigs in a concentration-dependent manner with an IC50 (95 % CI) of 50 (36 - 69) microg/mL. These results indicate that Perilla seed oil may improve lung function in asthma by controlling eicosanoid production and suppressing LT generation.

Effects of ciclamilast, a new PDE 4 PDE4 inhibitor, on airway hyperresponsiveness, PDE4D expression and airway inflammation in a murine model of asthma.

PDE4 (phosphodiesterase-4) plays a critical role in pathogenesis of allergic asthma and chronic obstructive pulmonary disease (COPD). PDE4 inhibitors are presently under clinical development for the treatment of asthma and/or COPD. Ciclamilast, a new PDE4 inhibitor, is a piclamilast (RP 73401) structural analogue, but has a more potent inhibitory effect on PDE4 and inflammation in the airway tissues and less side effects than that of piclamilast. In this study, we elucidate primarily on the roles of compound on PDE4 enzyme in physiological and pathological processes in a mouse model of asthma. The sensitized/challenged mice were reexposed to ovalbumin and airway response to inhaled methacholine was monitored. Orally administration of ciclamilast, in a dose-dependent manner, significantly inhibited changes in lung resistance and lung dynamic compliance, as well as upregulation of cAMP-PDE activity, increase of PDE4D mRNA expression, but not PDE4B from lung tissue in the murine model. In addition, the compound dose-dependently reduced mRNA expression of eotaxin, tumor necrosis factor (TNF)-alpha and interleukin (IL)-4, but slightly increased mRNA expression of interferon (IFN)-gamma from lung tissue. Further, levels of eotaxin, TNF-alpha and IL-4, and eosinophil and neutrophil accumulation in bronchoalveolar lavage fluid were also significantly reduced. Pathological examination, goblet cell hyperplasia and inflammatory cells infiltration in lung tissue were suppressed by treatment with ciclamilast. A significant correlation was observed between the increases in PDE4D mRNA expression and airway hyperresponsiveness. These studies confirm that inhibitory effect of ciclamilast on airway hyperresponsiveness includes its inhibiting PDE4D mRNA expression, down-modulating PDE4 activity, anti-inflammation and anti-mucus hypersecretion, and ciclamilast may have therapeutic potential for the treatment of asthma.

Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma.

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.

Effects of cryptoporus polysaccharide on rat allergic rhinitis associated with inhibiting eotaxin mRNA expression.

Aqueous extract from the fruiting body of Cryptoporus volvatus has been reported to present anti-tumor, anti-allergy, anti-inflammation and immunomodulatory activities. However, the effect mechanisms of anti-allergy and anti-inflammation are poorly understood. The aim of study is to evaluate whether Cryptoporus polysaccharides (CP) extracted from fruiting body of Cryptoporus volvatus decrease the development of nasal symptoms, airway hyperresponsiveness (AHR) to methacholine (MCh) and the infiltration of eosinophils in nasal mucosa in rat model of allergic rhinitis, and investigate a possible action mechanism of CP by detecting the expression of eotaxin mRNA in nasal mucosa and lung tissues. Rats were immunized with ovalbumin and consecutive topical antigen instillation was performed. Repeated intranasal ovalbumin challenge caused rhinitis symptom, AHR to MCh, eosinophil infiltration and histological alterations into the nasal mucosa and increase of eotaxin mRNA expression in nasal mucosa and lung tissue were examined. Pretreatment with CP 3, 9 and 27 mg kg(-1) (ig) decreased the numbers of sneezing 27.4%, 38.4% and 44.3% and nasal rubbing 27.5%, 34.9% and 47.7% comparison with model group, respectively. CP caused a dose-related inhibition of MCh-induced AHR. CP 27 mg kg(-1) decreased the expression of eotaxin mRNA in the nasal mucosa by 35%. These results suggest CP can relieve the symptom, eosinophil infiltration and injury of tissue in nasal mucosa and lung tissue and AHR of allergic rhinitis in rats. Its action mechanism may be associated with the decrease of eotaxin mRNA expression.

The modulatory effects of the volatile oil of ginger on the cellular immune response in vitro and in vivo in mice.

The aim of this study was to investigate the immunomodulatory effects of the volatile oil of ginger (Zingiber officinale Roscoe) in vitro and in vivo in mice. In vitro, the volatile oil of ginger (0.001-10 ng/mL) significantly inhibited T lymphocyte proliferation (P < 0.01), decreased the number of the total T lymphocytes and T helper cells (P < 0.01) in a concentration-dependent manner, but increased the percentage of T suppressor cells to the total T lymphocytes in the mice. In addition, the volatile oil of ginger (0.001-10 ng/mL) inhibited IL-1alpha secretion by the mice peritoneal macrophages in a concentration-dependent manner. In vivo, oral administration of the volatile oil of ginger in the doses of 0.125, 0.25 and 0.5 g/kg body weight dose-dependently weakened the delayed type of hypersensitivity response to 2,4-dinitro-1-fluorobenzene in the sensitized mice (P < 0.05). These results suggest that the volatile oil of ginger influences both cell-mediated immune response and nonspecific proliferation of T lymphocyte, and may exert beneficial effects in a number of clinical conditions, such as chronic inflammation and autoimmune diseases.