Calcium responses mediated by type 2 IP3-receptors are required for osmotic volume regulation of retinal glial cells in mice

Prevention of osmotic swelling of retinal glial (Müller) cells is required to avoid detrimental decreases in the extracellular space volume during intense neuronal activity. Here, we show that glial cells in slices of the wildtype mouse retina maintain the volume of their somata constant up to ∼4 min of perfusion with a hypoosmolar solution. However, calcium chelation with BAPTA/AM induced a rapid swelling of glial cell bodies. In glial cells of retinas from inositol-1,4,5-trisphosphate-receptor type 2-deficient (IP₃R2^−/−) mice, hypotonic conditions caused swelling of the cell bodies without delay. Exogenous ATP (acting at P2Y₁ receptors) prevented the swelling of glial cells in retinal slices from wildtype but not from IP₃R2^−/− mice. Müller cells from IP₃R2^−/− mice displayed a strongly reduced amplitude of the ATP-evoked calcium responses as compared to cells from wildtype mice. It is concluded that endogenous calcium signaling mediated by IP₃R2 is required for the osmotic volume regulation of retinal glial cells.     

Temporal and Spatial Distribution of the Cannabinoid Receptors (CB1, CB2) and Fatty Acid Amide Hydroxylase in the Rat Ovary

Although the effects of Δ⁹‐tetrahydrocannabinol (THC) on ovarian physiology have been known for many decades, its mechanism of action in the rat ovary remains poorly understood. The effects of THC and endocannabinoids on many cell types appear to be mediated through the G‐protein‐coupled CB₁ and CB₂ receptors. Evidence also suggests that the concentration of the endocannabinoid anandamide is regulated by cellular fatty acid amide hydrolase (FAAH). Therefore, we examined the rat ovary for the presence of CB₁ and CB₂ receptors and FAAH. The CB₁ receptor was present in the ovarian surface epithelium (OSE), the granulosa cells of antral follicles, and the luteal cells of functional corpus luteum (CL). The granulosa cells of small preantral follicles, however, did not express the CB₁ receptor. Western analysis also demonstrated the presence of a CB₁ receptor. In both preantral and antral follicles, the CB₂ receptor was detected only in the oocytes. In the functional CL, the CB₂ receptor was detected in the luteal cells. FAAH was codistributed with CB₂ receptor in both oocytes and luteal cells. FAAH was also present in the OSE, subepithelial cords of the tunica albuginea (TA) below the OSE, and in cells adjacent to developing preantral follicles. Western analysis also demonstrated the presence of FAAH in oocytes of both preantral and antral follicles. Our observations provide potential explanation for the effects of THC on steroidogenesis in the rat ovary observed by earlier investigators and a role for FAAH in the regulation of ovarian anandamide. Anat Rec 293:1425–1432, 2010. © 2010 Wiley‐Liss, Inc.     

Inactivation of the CB2 receptor accelerated the neuropathological deterioration in TDP‐43 transgenic mice,a model of amyotrophic lateral sclerosis

The activation of the cannabinoid receptor type‐2 (CB₂) afforded neuroprotection in amyotrophic lateral sclerosis (ALS) models. The objective of this study was to further investigate the relevance of the CB₂ receptor through investigating the consequences of its inactivation. TDP‐43(A315T) transgenic mice were crossed with CB₂ receptor knock‐out mice to generate double mutants. Temporal and qualitative aspects of the pathological phenotype of the double mutants were compared to TDP‐43 transgenic mice expressing the CB₂ receptor. The double mutants exhibited significantly accelerated neurological decline, such that deteriorated rotarod performance was visible at 7 weeks, whereas rotarod performance was normal up to 11 weeks in transgenic mice with intact expression of the CB₂ receptor. A morphological analysis of spinal cords confirmed an earlier death (visible at 65 days) of motor neurons labelled with Nissl staining and ChAT immunofluorescence in double mutants compared to TDP‐43 transgenic mice expressing the CB₂ receptor. Evidence of glial reactivity, measured using GFAP and Iba‐1 immunostaining, was seen in double mutants at 65 days, but not in TDP‐43 transgenic mice expressing the CB₂ receptor. However, at 90 days, both genotypes exhibited similar changes for all these markers, although surviving motor neurons of transgenic mice presented some morphological abnormalities in absence of the CB₂ receptor that were not as evident in the presence of this receptor. This faster deterioration seen in double mutants led to premature mortality compared with TDP‐43 transgenic mice expressing the CB₂ receptor. We also investigated the consequences of a pharmacological inactivation of the CB₂ receptor using the selective antagonist AM630 in TDP‐43 transgenic mice, but results showed only subtle trends towards a greater deterioration. In summary, our results confirmed the potential of the CB₂ receptor agonists as a neuroprotective therapy in ALS and strongly support the need to progress towards an evaluation of this potential in patients.     

The emerging role of the endocannabinoid system in cardiovascular disease

Endocannabinoids are endogenous bioactive lipid mediators present both in the brain and various peripheral tissues, which exert their biological effects via interaction with specific G-protein-coupled cannabinoid receptors, the CB₁ and CB₂. Pathological overactivation of the endocannabinoid system (ECS) in various forms of shock and heart failure may contribute to the underlying pathology and cardiodepressive state by the activation of the cardiovascular CB₁ receptors. Furthermore, tonic activation of CB₁ receptors by endocannabinoids has also been implicated in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes, such as plasma lipid alterations, abdominal obesity, hepatic steatosis, inflammation, and insulin and leptin resistance. In contrast, activation of CB₂ receptors in immune cells exerts various immunomodulatory effects, and the CB₂ receptors in endothelial and inflammatory cells appear to limit the endothelial inflammatory response, chemotaxis, and inflammatory cell adhesion and activation in atherosclerosis and reperfusion injury. Here, we will overview the cardiovascular actions of endocannabinoids and the growing body of evidence implicating the dysregulation of the ECS in a variety of cardiovascular diseases. We will also discuss the therapeutic potential of the modulation of the ECS by selective agonists/antagonists in various cardiovascular disorders associated with inflammation and tissue injury, ranging from myocardial infarction and heart failure to atherosclerosis and cardiometabolic disorders.     

Nrf2 is closely related to allergic airway inflammatory responses induced by low-dose diesel exhaust particles in mice

We have recently reported that disruption of nuclear erythroid 2 P45-related factor 2 (Nrf2) enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles (DEP) in mice. C57BL/6 Nrf2 knockout (Nrf2^−/−) mice and wild-type (Nrf2^+/+) mice were further exposed to low-dose DEP for 7 h/day, 5 days/week, for a maximum of 8 weeks. After exposure to DEP for 5 weeks, allergic airway inflammation was generated in the mice by intraperitoneal sensitization with OVA followed by intranasal challenge. Nrf2^−/− mice exposed to relatively low-dose DEP showed significantly increased percentage changes relative to the OVA alone group in terms of airway hyperresponsiveness (AHR) and inflammatory cells, levels of IL-5 and thymus and activation regulated chemokine (TARC) in bronchoalveolar lavage (BAL) fluid than did Nrf2^+/+ mice. Lung tissues of Nrf2^−/− mice after DEP exposure showed inflammatory cell infiltrates, and increased PAS staining-positive mucus cell hyperplasia. In contrast, the percentage changes relative to the OVA group in the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in whole blood was higher in Nrf2^+/+ mice than in Nrf2^−/− mice. By using Nrf2^−/− mice, it was shown for the first time that relatively low-dose DEP exposure induces oxidant stress, and that host anti-oxidant responses play a key role in the development of DEP-induced exacerbation of allergic airway inflammation.     

Persistence of reduced aggression in vasopressin 1b receptor knockout mice on a more “wild” background

It has been previously reported that vasopressin 1b receptor knockout (Avpr1b^−/−) mice have reduced levels of aggressive behavior compared to wildtype littermates. However, as the background of the mice was always a mixture of 129/SvJ and C57BL/6, we wanted to determine if the phenotype persisted when our laboratory line was crossed with a wild-derived sub-species of house mice. To this end, we crossed our Avpr1b^−/− mice with Mus musculus castaneus, one of the few sub-species that will breed with laboratory strains. Subsequent F₂ offspring were tested in a resident-intruder behavioral test to assess aggressive behavior. We found that even on this more “wild” background, Avpr1b^−/− mice continued to demonstrate longer attack latencies and fewer attacks in a resident-intruder test than wildtype littermates. These findings are consistent with previous reports of reduced aggressive behavior in Avpr1b^−/− mice and show that the deficit does persist on a different background strain. Further, these findings confirm the importance of the Avpr1b to normal displays of social forms of aggressive behavior.     

TLR2- and 4-independent immunomodulatory effect of high molecular weight components from Ascaris suum

Components of high molecular-weight (PI) obtained from Ascaris suum extract down-regulate the Th1/Th2-related immune responses induced by ovalbumin (OVA)-immunization in mice. Furthermore, the PI down-modulates the ability of dendritic cells (DCs) to activate T lymphocytes by an IL-10-mediated mechanism. Here, we evaluated the role of toll like receptors 2 and 4 (TLR2 and 4) in the modulatory effect of PI on OVA-specific immune response and the PI interference on DC full activation. An inhibition of OVA-specific cellular and humoral responses were observed in wild type (WT) or in deficient in TLR2 (TLR2^−/−) or 4 (TLR4^−/−) mice immunized with OVA plus PI when compared with OVA-immunized mice. Low expression of class II MHC, CD40, CD80 and CD86 molecules was observed in lymph node (LN) cells from WT, TLR2^−/− or TLR4^−/− mice immunized with OVA plus PI compared with OVA-primed cells. We also verified that PI was able to modulate the activation of DCs derived from bone marrow of WT, TLR2^−/− or TLR4^−/− mice induced in vitro by agonists of TLRs, as observed by a decreased expression of class II MHC and costimulatory molecules and by low secretion of pro-inflammatory cytokines. Its effect was accompanied by IL-10 synthesis. In this sense, the modulatory effect of PI on specific-immune response and DC activation is independent of TLR2 or TLR4.     

Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy

Background

Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy.

Objective

We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy.

Methods and results

Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1^−/− and SphK2^−/− mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4⁺ effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals.

Conclusion

Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.     

Enlarged extracellular space of aquaporin-4–deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers

Aquaporin-4 (AQP4) water channels expressed on glia have been implicated in maintaining the volume of extracellular space (ECS). A previous diffusion study employing small cation tetramethylammonium and a real-time iontophoretic (RTI) method demonstrated an increase of about 25% in the ECS volume fraction (α) in the neocortex of AQP4^−/− mice compared to AQP4^+/+ mice but no change in the hindrance imposed to diffusing molecules (tortuosity λ). In contrast, other diffusion studies employing large molecules (dextran polymers) and a fluorescence recovery after photobleaching (FRAP) method measured a decrease of about 10%–20% in λ in the neocortex of AQP4^−/− mice. These conflicting findings on λ would imply that large molecules diffuse more readily in the enlarged ECS of AQP4^−/− mice than in wild type but small molecules do not. To test this hypothesis, we used integrative optical imaging (IOI) to measure tortuosity with a small Alexa Fluor 488 (molecular weight [MW] 547, λ_AF) and two large dextran polymers (MW 3000, λ_dex3 and MW 75,000, λ_dex75) in the in vitro neocortex of AQP4^+/+ and AQP4^−/− mice. We found that λ_AF=1.59, λ_dex3=1.76 and λ_dex75=2.30 obtained in AQP4^−/− mice were not significantly different from λ_AF=1.61, λ_dex3=1.76, and λ_dex75=2.33 in AQP4^+/+ mice. These IOI results demonstrate that λ measured with small and large molecules each remain unchanged in the enlarged ECS of AQP4^−/− mice compared to values in AQP4^+/+ mice. Further analysis suggests that the FRAP method yields diffusion parameters not directly comparable with those obtained by IOI or RTI methods. Our findings have implications for the role of glial AQP4 in maintaining the ECS structure.     

Mapping of the binding sites for the OX1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX₁ receptors. Altogether ten chimaeras between OX₁ and OX₂ orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability (K_B) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX₂. As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX₁ and OX₂ receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.     

CD8α⁺ dendritic cells improve collagen-induced arthritis in CC chemokine receptor (CCR)-2 deficient mice

Objective

Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3⁺ regulatory T cells (T_reg), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2^−/−) mice had impaired DCs migration and reduced CD8α⁺ DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2^−/− mice, we tested the hypothesis that CD8α⁺ DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α⁺ DCs in Ccr2^−/− and SKG mice.

Methods

To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8α⁺ DCs.

Results

Flt3L-mediated expansion of endogenous CD8α⁺ DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α⁺ DCs ameliorated arthritis in Ccr2^−/− mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype.

Conclusion

CD8α⁺ DCs were tolerogenic to the development of arthritis. CD8α⁺ DCs deficiency heightened the sensitivity to arthritis in Ccr2^−/− mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2^−/− mice was T cell-independent.     

Functional relevance of NLRP3 inflammasome-mediated interleukin (IL)-1β during acute allergic airway inflammation

Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However, certain symptomatic individuals remain at risk for serious morbidity and mortality, which prompts the identification of novel therapeutic targets and treatment strategies. Thus, using an adjuvant-free T helper type 2 (Th2) murine model, we have deciphered the role of interleukin (IL)-1 signalling during allergic airway inflammation (AAI). Because functional IL-1β depends on inflammasome activation we first studied asthmatic manifestations in specific inflammasome-deficient [NACHT, LRR and PYD domains-containing protein 3 (NLRP3^−/−) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC^−/−)] and IL-1 receptor type 1^−/− (IL-1R1^−/−) mice on the BALB/c background. To verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung pathology, airway hyperresponsiveness and ovalbumin (OVA)-specific immune responses. In the absence of NLRP3 inflammasome-mediated IL-1β release all symptoms of AAI were reduced, except OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling reduced asthmatic development, we administered the IL-1R antagonist anakinra (Kineret®) during critical immunological time-points: sensitization or challenge. Amelioration of asthmatic symptoms was only observed when anakinra was administered during OVA challenge. Our findings indicate that blocking IL-1 signalling could be a potential complementary therapy for allergic airway inflammation.     

A novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors CB2R over CB1R

The type 2 cannabinoid receptors (CB₂R) have gained much attention recently due to their important regulatory role in a host of pathophysiological processes. However, the exact biological function of CB₂R and how this function might change depending on disease progression remains unclear and could be better studied with highly sensitive and selective imaging tools for identifying the receptors. Here we report the first near infrared fluorescence imaging probe (NIR760-XLP6) that binds preferentially to CB₂R over the type 1 cannabinoid receptors (CB₁R). The selectivity of the probe was demonstrated by fluorescence microscopy using DBT-CB₂ and DBT-CB₁ cells. Furthermore, in mouse tumor models, NIR760-XLP6 showed significantly higher uptake in DBT-CB₂ than that in DBT-CB₁ tumors. These findings indicate that NIR760-XLP6 is a promising imaging tool for the study of CB₂R regulation.     

Immunochemical characterization of mouse brain-associated theta (Thy-1) antigen.

The Thy-1 antigens or rat brain and thymus have been isolated and chemically characterized, but those of mice have not been identified. Moreover, it is uncertain whether the antigens are glycolipids or glycoproteins. This study with highly purified preparations of gangliosides GM₁, ¹GD_1a, GD_1b and GT_1b from bovine brain and several ganglioside fractions from mouse brain showed that Thy-1 activity does not reside in gangliosides, but rather in the chloroform-methanol-insoluble residue of brain remaining after extraction of gangliosides. The antigen could be solubilized from this residue with a non-ionic detergent. The antigenic activity of the solubilized preparation was heat-labile but resistant to periodate. The chemical properties of the Thy-1 antigen of mouse brain are discussed.     

Evidences of cannabinoids-induced modulation of paroxysmal events in an experimental model of partial epilepsy in the rat

The anticonvulsant effect of cannabinoids (CB) has been shown to be mediated by the activation of the CB₁ receptor. This study evaluates the anticonvulsant activity of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]-1-naphthalenylmethanone (WIN55,212-2, CB agonist) alone or preceded by the administration of N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, selective CB₁ antagonist) in an experimental in vivo model of complex partial seizures (maximal dentate gyrus activation – MDA) in the rat. WIN55,212-2 (21 mg kg⁻¹) exerted an anticonvulsant effect, significantly reduced by the pre-treatment with AM251 (1 mg kg⁻¹, 30 min interval). Surprisingly, AM251, administered alone at the same dose, failed to induce any modification in MDA responses. Our data suggest the involvement of the CB system in the inhibitory control of hyperexcitability phenomena in a model of acute partial epilepsy. Although the MDA model per se does not induce a basal activation of CB₁ receptors, as suggested by the lack of efficacy of AM251 when administered alone, the partial suppression of WIN55,212-2-induced effects in rats pre-treated with AM251 allows to hypothesise that the WIN55,212-2-induced antiepileptic effect is strictly linked to an increased CB₁ receptor activation or to the involvement of further receptor subtypes.     

The Effects of Lactobacillus rhamnosus on the Prevention of Asthma in a Murine Model

Purpose

Lactobacilli are probiotic bacteria that are effective in the management of allergic diseases or gastroenteritis. It is hypothesized that such probiotics have immunoregulatory properties and promote mucosal tolerance. Our goal was to investigate whether Lactobacillus casei rhamnosus Lcr35 could inhibit airway inflammation in an ovalbumin (OVA)-induced murine model of asthma.

Methods

BALB/c mice aged 6 weeks were used in the present study. Lactobacillus casei rhamnosus Lcr35 was administered daily, starting 1 week prior to the first OVA sensitization (group 1) and 2 days before the first 1% OVA airway challenge (group 2). Mice that received only saline at both sensitization and airway challenge time points were used as negative controls (group 3), and those that had OVA-induced asthma were used as positive controls (group 4). Airway responsiveness to methacholine was assessed, and bronchoalveolar lavage (BAL) was performed. At the endpoint of the study, total IgE as well as OVA-specific IgE, IgG₁ and IgG_2a in serum was measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated.

Results

Airway hyperresponsiveness, total cell counts and the proportion of eosinophils in BAL fluid were significantly decreased in group 1 compared with group 4 (P<0.05). Total serum IgE levels were also significantly decreased in group 1 compared with group 4. Serum levels of OVA-specific IgE, IgG₁ and IgG_2a were not significantly influenced by treatment with Lcr35. There was significantly less peribronchial and perivascular infiltration of inflammatory cells in group 1 compared with group 4; however, there were no significant differences in methacholine challenge, BAL, serology or histology between groups 2 and 4.

Conclusions

Oral treatment with Lcr35 prior to sensitization can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. These results suggest that Lcr35 may have potential for preventing asthma.     

DNA methyltransferase 1 is essential for initiation of the colon cancers

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1^−/−). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR⁺) and CD44-positive and CD24-positive (CD44⁺CD24⁺) cell rates were lower in DNMT1^−/− cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1^−/− cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1^−/− cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.     

Over‐expression of the LTC4 synthase gene in mice reproduces human aspirin‐induced asthma

Background The pathogenesis of aspirin‐induced asthma (AIA) is presumed to involve the aspirin/non‐steroidal anti‐inflammatory drug (NSAID)‐induced abnormal metabolism of arachidonic acid, resulting in an increase in 5‐lipoxygenase (5‐LO) metabolites, particularly leukotriene C₄ (LTC₄). However, the role of LTC₄ in the development of AIA has yet to be conclusively demonstrated. Objective The aim of this study was to evaluate the contribution of the lipid product LTC₄ secreted by the 5‐LO pathway to the pathogenesis of AIA. Methods To evaluate antigen‐induced airway inflammation, the concentrations of T‐helper type 2 cytokine in bronchoalveolar lavage fluid (BALF) obtained from LTC₄ synthase‐transgenic (Tg) and wild‐type (WT) mice after challenge with ovalbumin were measured. Subsequently, the ex vivo and in vivo effects of the NSAID sulpyrine were investigated in these Tg and WT mice by measuring the secretion of LTC₄ from sulpyrine‐treated BAL cells and the levels of LTC₄ in BALF following challenge with sulpyrine. Finally, the sulpyrine‐induced airway response by the administration of pranlukast, an antagonist of the cysteinyl (cs)‐LT1 receptor, was analysed. Results The concentrations of IL‐4, ‐5, and ‐13 in BALF from Tg mice were significantly higher than those in WT mice. In addition, sulpyrine augmented the secretion of LTC₄ in BALF and by BAL cells in Tg mice, but not in WT mice. Additionally, the increased airway resistance induced by sulpyrine could be reduced by treatment with pranlukast. Furthermore, the secretion of LTC₄ from mast cells, eosinophils, and macrophages was increased in the allergen‐stimulated LTC₄ synthase gene Tg mice, even in the absence of sulpyrine, as well as in BAL cells after sulpyrine. Conclusion and clinical relevance The over‐expression of the LTC₄ synthase in a mouse asthma model also replicates the key features of AIA. And our study supports that cys‐LTs play a major role in the pathogenesis of AIA in patients with chronic asthma. Cite this as: H. Hirata, M. Arima, Y. Fukushima, K. Honda, K. Sugiyama, T. Tokuhisa and T. Fukuda, Clinical & Experimental Allergy, 2011 (41) 1133–1142.