AM404, paracetamol metabolite,prevents prostaglandin synthesis in activated microglia by inhibiting COX activity

Background

N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated.

Method

In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA.

Results

Our results show that AM404 inhibited LPS-mediated prostaglandin E₂ (PGE₂) production in OHSC, and LPS-stimulated PGE₂ release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE₂, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD₂ and the formation of reactive oxygen species (8-iso-PGF₂ alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis.

Conclusions

This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia.

     

Colitis generates remote antinociception in rats: the role of the l-arginine/NO/cGMP/PKG/KATP pathway and involvement of cannabinoid and opioid systems

Objective and design

The aim of this study was to investigate the possible involvement of the NO/cGMP/PKG/K _ATP ⁺ pathway, cannabinoids and opioids in remote antinociception associated with 2,4,6-trinitrobenzene sulph onic acid (TNBS)-induced colitis.

Methods

TNBS-induced colitis was induced by intracolonic administration of 20 mg of TNBS in 50 % ethanol. After induction, carrageenan (500 μg/paw) or prostaglandin (PG) E₂ (100 ng/paw) was injected in the rat’s plantar surface and hypersensitivity was evaluated by the electronic von Frey test. Rats were pre-treated with l-Noarg one hour before carrageenan injection. l-Arginine was given 10 min before l-Noarg injections. ODQ, KT 5823, glibenclamide (Glib), naloxone and AM 251 or AM 630 were administered 30 min prior to carrageenan or PGE₂ treatments.

Results

Colitis induction by TNBS reduced PGE₂ or carrageenan-induced hypersensitivity. Antinociception produced by TNBS-induced colitis was reversed significantly (P < 0.05) by l-Noarg, ODQ, KT 5823, glibenclamide, naloxone, AM251 and AM630 treatments.

Conclusions

TNBS-induced colitis causes antinociception in the rat paw. This disorder appears to be mediated by activation of the NO/cGMP/PKG/K_ATP pathway, endocannabinoids and endogenous opioids. This information may contribute to a better understanding of peripheral neurological dysfunctions occurring in Crohn’s disease.     

Foxp3<Superscript>+</Superscript> Regulatory T Cells,Th17 Effector Cells,and Cytokine Environment in Inflammatory Bowel Disease

Background  

Inflammatory bowel disease (IBD) is thought to result from an aberrant immune response. Inflammation in IBD may be caused by the loss of homeostasis between CD4⁺ CD25^high Foxp3⁺ regulatory cells (T _reg) and proinflammatory Th17 cells. The aim of this study was to investigate T _reg and Th17 cells in the peripheral blood and intestinal mucosa of IBD patients and to assess the mucosal cytokine environment.     

Fractalkine receptor (CX<Subscript>3</Subscript>CR1) deficiency sensitizes mice to the behavioral changes induced by lipopolysaccharide

Background  

Interactions between fractalkine (CX₃CL1) and fractalkine receptor (CX₃CR1) regulate microglial activation in the CNS. Recent findings indicate that age-associated impairments in CX₃CL1 and CX₃CR1 are directly associated with exaggerated microglial activation and an impaired recovery from sickness behavior after peripheral injection of lipopolysaccharide (LPS). Therefore, the purpose of this study was to determine the extent to which an acute LPS injection causes amplified and prolonged microglial activation and behavioral deficits in CX₃CR1-deficient mice (CX₃CR1^-/-).     

Extracellular ATP and the P2X<Subscript>7</Subscript>receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

Background  

During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X₇ receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1^G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X₇ receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1^G93A astrocytes.     

Enteroaggregative Escherichia coli induced increase in intracellular calcium concentration modulates cytoskeletal F-actin rearrangement and bacterial entry in INT-407 cells

Background

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen, associated with cases of acute and persistent diarrhoea worldwide. The pathogenesis of EAEC is yet to be understood. In intestinal epithelium, an increase in [Ca²⁺]_i has been attributed due to the action of different enteric pathogens. EAEC was shown to increase [Ca²⁺]_i in HEp-2 cells.The present study was undertaken to investigate the effect of EAEC induced increase in [Ca²⁺]_i oncultured human intestinal epithelial cells.

Methods

INT-407 cells were infected with EAEC (T8 strain) in the absence and presence of dantrolene (inhibitor of release of Ca²⁺ from intracellular stores)/verapamil (L-type Ca²⁺ channel blocker)/BAPTA-AM (Ca²⁺ chelator)/U73122 (PLC inhibitor)/Cytochalasin-D (inhibitor of actin polymerization). [Ca²⁺]_i was estimated using Fura-2/AM. Cytoskeletal rearrangement was assessed by F-actin staining using TRITC-phalloidin. The invasiveness of EAEC-T8 to INT-407 cells was checked by electron microscopy and invasion assay.

Results

A significant increase in [Ca²⁺]_i was observed in EAEC-T8 infected INT-407 cells, which was reduced in presence of dantrolene/verapamil/U73122. EAEC-T8 could induce cytoskeletal F-actin polymerization in INT-407 cells and was found to be invasive in nature. The cytoskeletal rearrangement as well as invasion of EAEC-T8 was attenuated in presence of U73122/dantrolene/BAPTA-AM/verapamil/cytochalasin D.

Conclusions

EAEC induced increase in [Ca²⁺]_i seems to play a major role in host cytoskeletal F-actin rearrangements leading to invasion of the organism.

General significance

Our study undoubtedly will lead to an improved understanding of EAEC-pathogenesis.     

An improved indirect competitive Elisa for aflatoxin m1 in milk powders using novel monoclonal antibodies

Among three newly prepared monoclonal anti‐aflatoxin M₁ antibodies, named AM.1, AM.2 and AM.3, both AM.1 and AM.3 possessed high specificity and sensitivity towards aflatoxin M₁, while AM.2 exhibited lower specificity. Employing AM.3 and horseradish peroxidase‐labelled second antibody, an improved ELISA was developed with a detection limit of 1.0 pg ml^‐1 of aflatoxin M₁ in solution. The contents of aflatoxin M₁ in powdered milks suspended in water were assayed by the indirect ELISA. The detection limit was 5 pg g^‐1dry weight, and no clean‐up procedures were required. The reliability of the present ELISA with monoclonal antibody AM.3 was confirmed with reference powdered milks for aflatoxin M₁. A limited ELISA survey showed the presence of aflatoxin M₁ in commercial milk powders sampled in France, the US and Thailand at levels of 30–418 pg g^‐1, and it was confirmed by an improved HPLC analysis.     

Sialic acid residues play a pivotal role in α1-acid glycoprotein (AGP)-induced generation of reactive oxygen species in chemotactic peptide pre-activated neutrophil granulocytes

Objective  

We have recently shown that terminal sialic acid residues are essential for α₁-acid glycoprotein (AGP)-induced Ca²⁺ mobilization in neutrophils. The aim of the present study was to establish the importance of sialic acid residues on AGP in modulating human neutrophil functions, with emphasis on the generation of reactive oxygen species (ROS).     

Gp91<Superscript>phox</Superscript> (NOX2) in classically activated microglia exacerbates traumatic brain injury

Background  

We hypothesized that gp91^phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂ ^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phox knockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phox generation.     

Cellular localization of kinin B1 receptor in the spinal cord of streptozotocin-diabetic rats with a fluorescent [Nα-Bodipy]-des-Arg9-bradykinin

Background  

The kinin B₁ receptor (B₁R) is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B₁R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nα-Bodipy]-des-Arg⁹-BK (BdABK) and selective antibodies.     

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats

Background

The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet.

Objective

The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs.

Methods

Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by ³H thymidine (³H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically.

Results

Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in ³H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G₀/G₁ phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [³H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium.

Conclusion

ORC1 gene silencing causes rat VSMCs to enter a reversible G₀ quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.     

Superoxide generation by alveolar macrophages from aged rats: improvement by in vitro treatment with IFN-γ

Alveolar macrophages (AM) from aged rats show an impaired oxidative response, but it is unclear whether or not this is due to the inability of these cells to be activated. To elucidate this, we investigated the capacity of AM from young (16-week-old) and aged (100-week-old) rats to become primed with recombinant rat interferon-γ (IFN-γ) for increased phorbol myristate acetate (PMA)-elicited O₂⁻ production, utilizing an MCLA-dependent chemiluminescent assay. We also compared concanavalin A- or Bacillus Calmette Guerin (BCG)-induced IFN-γ production by the spleen cells of young and aged animals. The data indicated that AM freshly harvested from non-sensitized aged rats produced less O₂⁻ than those from young animals. A similar result was obtained in BCG-sensitized rats. However, AM from aged rats were primed with in vitro treatment with IFN-γ for increased rate of O₂⁻ production to an equivalent level of that by AM from young animals. In addition, the ability of spleen cells to produce IFN-γ was well maintained in aged rats. These results suggest that AM function is suppressed in the lungs of aged animals. Our observation that the decreased AM function in aged rats can be reversed is important because it suggests that appropriate treatment may reduce the incidence and mortality of respiratory infections in the elderly.     

Test-retest variability of high resolution positron emission tomography (PET) imaging of cortical serotonin (5HT2A) receptors in older, healthy adults

Background  

Position emission tomography (PET) imaging using [¹⁸F]-setoperone to quantify cortical 5-HT_2A receptors has the potential to inform pharmacological treatments for geriatric depression and dementia. Prior reports indicate a significant normal aging effect on serotonin 5HT_2A receptor (5HT_2AR) binding potential. The purpose of this study was to assess the test-retest variability of [¹⁸F]-setoperone PET with a high resolution scanner (HRRT) for measuring 5HT_2AR availability in subjects greater than 60 years old. Methods: Six healthy subjects (age range = 65–78 years) completed two [¹⁸F]-setoperone PET scans on two separate occasions 5–16 weeks apart.     

Improved Growth and Colchicine Concentration in Gloriosa Superba on Mycorrhizal Inoculation Supplemented with Phosphorus-Fertilizer

Background

Gloriosa superba produces an array of alkaloids including colchicine, a compound of interest in the treatment of various diseases. The tuber of Gloriosa superba is a rich source of colchicine which has shown anti-gout, anti-inflammatory, and anti-tumor activity. However, this promising compound remains expensive and Gloriosa superba is such a good source in global scale. Increase in yield of naturally occurring colchicine is an important area of investigation.

Materials and Methods

The effects of inoculation by four arbuscular mycorrhizal (AM), fungi, Glomus mossae, Glomus fasciculatum, Gigaspora margarita and Gigaspora gilmorei either alone or supplemented with P-fertilizer, on colchicine concentration in Gloriosa superba were studied. The concentration of colchicine was determined by high-performance thin layer chromatography.

Results

The four fungi significantly increased concentration of colchicine in the herb. Although there was significant increase in concentration of colchicine in non-mycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in colchicine concentration may not be entirely attributed to enhanced P-nutrition and improved growth. Among the four AM fungi Glomus mossae was found to be best. The total colchicine content of plant (mg / plant) was significantly high in plants inoculated with Glomus mossae and 25 mg kg⁻¹phosphorus fertilizer (348.9 mg /plant) while the control contain least colchicine (177.87 mg / plant).

Conclusion

The study suggests a potential role of AM fungi in improving the concentration of colchicine in Gloriosa superba tuber.     

The variation of AkT/TSC1-TSC1/mTOR signal pathway in hepatocytes after partial hepatectomy in rats

Objective

The aim of this study was to investigate the role and regulatory mechanisms of Akt/TSC1-TSC2/mTOR signal pathway on the hepatocyte growth and proliferation after partial hepatectomy in rats.

Methods

We used the animal model of 70% hepatectomy, separated and cultivated hepatocytes. According to the different time points after partial hepatectomy, it could be grouped into 0 h, 2 h, 6 h, 24 h and 72 h. According to the different kinds of specific inhibitor in the nutritive medium after the separation of hepatocytes, it could be grouped into Triciribine (TR), Rapamycin (RA) and Control (CO). We investigated ³H-Leucine incorporation into protein, the cross section areas of hepatocytes, and detected cell cycle through FCM. The expressions of phosphorylated protein TSC2 and mTOR were observed.

Results

(1) The content of phosphorylated protein TSC2 in group CO began to increase at 2 h and got to the peak at 6 h but declined at 24 h. The content of phosphorylated protein TSC2 in group RA had the same variation with that of phosphorylated protein TSC2 in group CO. (2) At the time point of 0 h, 2 h, 6 h and 24 h after operation, the incorporation efficiency of ³H-Leucine in groups RA and TR was different from that in group CO in statistics (P < 0.01). (3) It could be seen that the cross section areas of hepatocytes in groups RA and TR were different from that in group CO in statistics at 2 h and 6 h after operation (P < 0.05). (4) Comparing with the other two inhibitor groups (TR and RA), the number of cells during the period of G₀/G₁ in group CO became fewer, while the number of cells during the period of S and G₂/M grew obviously (referring to Fig. 8). After operation, each time point was different from the inhibitor groups obviously (P < 0.05 or P < 0.01). The peak declined greatly at 24 h and 72 h after operation.

Conclusions

These data strongly suggest the effects of Akt/TSC1-TSC2/mTOR signal pathway on hepatocyte growth, protein synthesis and cell cycle, and prove its contribution to liver regeneration.     

Phénotypes de l’alpha-1-antitrypsine dans la population togolaise : mise en évidence d’une fréquence élevée de l’allèle rare Pi dans une ethnie isolat

Objectives

The study, which is a ethnobiologic characterization, investigated α₁-antitrypsin gene polymorphism in the togolese ethnic groups. We aimed to determine the existence of rare or deficient alleles predisposing to pulmonary or hepatic genetic diseases.

Patients and methods

We focused our study on healthy subjects of two samples by comparing 205 Adélé from relative isolated ethnic group alive in mountain region and 255 subjects from pluriethnic population living on Atlantic coastal region. Data analysis was performed by α₁-antitrypsin level quantification and serum isoelectric focusing.

Results

The two alleles Pi^M et Pi^F frequencies are respectively 0.834 and 0.166 in Adélé; 0.989 and 0.011 in the subjects from pluriethnic population. Phenotypes MM and FM distribution in the two groups is significantly different (p < 0.001). However, α₁-antitrypsin polymorphism does not significantly influence proteinic and lipidic profiles of the subjects in the two samples.

Conclusion

The Pi^F allele of α₁ antitrypsin is rare allele in the world global populations. Its very high frequency in Adélé explained by preferential endogamic marriage in this ethnic group. Compared to the subjects from pluriethnic population, more than 30 Adélé subjects present a higher risk to develop pulmonary diseases according to isoform F properties.     

Factors affecting SOCE activation in mammalian skeletal muscle fibers

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca²⁺ entry, SOCE. Sarcoplasmic reticulum (SR) Ca²⁺ stores were depleted by repetitive exposures to high K⁺ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca²⁺. SR Ca²⁺ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca²⁺ (5 mM) increased basal [Ca²⁺]_i under two modalities. In mode 1 [Ca²⁺]_i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca²⁺]_i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca²⁺ entry. In nine FCCP-poisoned fibers, the initial rate of Ca²⁺ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca²⁺, we show that during depletion [Ca²⁺]_mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca²⁺]_i, associated with SOCE activation, diminishes upon external Na⁺ withdrawal. Na⁺ entry through the SOCE pathway and activation of the reversal of Na⁺/Ca²⁺ exchanger could explain this SOCE modulation by Na⁺.     

Kinetics of oxygen uptake by cells potentially used in a tissue engineered trachea

Synthetic polymer scaffold seeded with autologous cells have a clinical translational potential. A rational design oriented to clinical applications must ensure an efficient mass transfer of nutrients as a function of specific metabolic rates, especially for precariously vascularized tissues grown in vitro or integrated in vivo. In this work, luminescence lifetime-based sensors were used to provide accurate, extensive and non-invasive measurements of the oxygen uptake rate for human mesenchymal stem cells (hMSCs), tracheal epithelial cells (hTEpiCs) and human chondrocytes (hCCs) within a range of 2–40% O₂ partial pressure. Estimated Michaelis–Menten parameters were: V_max = 0.099 pmol/cell⋅h and K_M = 2.12 × 10⁻⁷ mol/cm³ for hMSCs, V_max = 1.23 pmol/cell⋅h and K_M = 2.14 × 10⁻⁷ mol/cm³ for hTEpiCs, V_max = 0.515 pmol/cell⋅h and K_M = 1.65 × 10⁻⁷ mol/cm³ for hCCs. Kinetics data served as an input to a preliminary computational simulation of cell culture on a poly-ethylene terephthalate (PET) tracheal scaffold obtaining an efficient mass transfer at cell density of 10⁶ cell/cm³. Oxygen concentration affected the glucose uptake and lactate production rates of cells that adapted their metabolism according to energy demand in hypoxic and normoxic conditions.     

Release of platelet-activating factor (PAF-acether) and arachidonic acid metabolites from alveolar macrophages

Human, monkey and rat alveolar macrophages (AM) release PAF-acether in a dose-dependent fashion in the presence of 1 to 5 g/ml ionophore A 23187 (2.5 pmol of PAF-acether from 2.5×10⁵ cells) but not in the presence of zymosan. Arachidonic acid (AA) metabolites released from AM from these species were studied. Thromboxane A₂ TxA₂)—detected by its action on rabbit arteries—was released from human, monkey and rat AM upon addition of 0.5 mM AA. This release was inhibited by aspirin and indomethacin. Lipoxygenase and cyclooxygenase AA metabolites from rat AM were identified using high efficiency glass capillary column gas chromatography coupled to mass spectrometry. The cyclooxygenase metabolites PGF₂, E₂ and D₂ and TxB₂ were identified. The lipoxygenase-dependent AA metabolites were explored using aspirin-pretreated AM. Only 12 HETE was found.These data indicate that AM secrete several substances with bronchoconstrictive activity: PGF₂, D₂, TxA₂ and PAF-acether. Therefore an active role of AM in human and experimental bronchoconstriction must be considered.