B-cell-specific-peroxisome proliferator-activated receptor γ deficiency augments contact hypersensitivity with impaired regulatory B cells.

Nuclear receptor peroxisome proliferator-activated receptor γ (PPAR-γ) activation can prevent immunoinflammatory disorders and diabetes. B cells play protective roles during inflammation as well. However, the roles of endogenous PPAR-γ in the regulatory properties of B cells to relieve inflammation remain unknown. Here, we developed B-cell-specific PPAR-γ knockout (B-PPAR-γ-/- ) mice and found that the conditional deletion of PPAR-γ in B cells resulted in exaggerated contact hypersensitivity (CHS). Meanwhile, interferon-γ (IFN-γ) of CD4+ CD8+ T cells was up-regulated in B-PPAR-γ-/- mice in CHS. This showed that the regulatory function of B cells in B-PPAR-γ-/- mice declined in vivo. Whereas splenic CD5+ CD1dhi regulatory B-cell numbers and peripheral regulatory T-cell numbers were not changed in naive B-PPAR-γ-/- mice. Loss of PPAR-γ in B cells also did not affect either CD86 or FasL expression in splenic CD5+ CD1dhi regulatory B cells after activation. Notably, interleukin-10 (IL-10) production in CD5+ CD1dhi regulatory B cells reduced in B-PPAR-γ-deficient mice. In addition, functional IL-10-producing CD5+ CD1dhi regulatory B cells decreased in B-PPAR-γ-/- mice in the CHS model. These findings were in accordance with augmented CHS. The current work indicated the involvement of endogenous PPAR-γ in the regulatory function of B cells by disturbing the expansion of IL-10-positive regulatory B cells.

Heat Shock Protein 90 Facilitates Latent HIV Reactivation through Maintaining the Function of Positive Transcriptional Elongation Factor b (p-TEFb) under Proteasome Inhibition.

The persistence of HIV in resting memory CD4+ T cells at a latent state is considered as the major barrier on the path to achieve a cure for HIV. Proteasome inhibitors (PIs) were previously reported as latency reversing agents (LRAs) but the mechanism underlying this function is yet unclear. Here we demonstrate that PIs reactivate latent HIV ex vivo without global T cell activation, and may facilitate host innate immune responses. Mechanistically, latent HIV reactivation induced by PIs is mediated by heat shock factor 1 (HSF1) via the recruitment of the heat shock protein (HSP) 90-positive transcriptional elongation factor b (p-TEFb) complex. Specifically, HSP90 downstream HSF1 gives positive feedback to the reactivation process through binding to cyclin-dependent kinase 9 (CDK9) and preventing it from undergoing degradation by the proteasome. Overall, these findings suggest proteasome inhibitors as potential latency reversing agents. In addition, HSF1/HSP90 involved in HIV transcription elongation, may serve as therapeutic targets in HIV eradication.

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

AIM: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. METHODS: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. RESULTS: Aconine (0.125, 0.25 µmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, ß3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. CONCLUSION: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Effect of different preoperative fasting time on safety and postoperative complications of painless gastrointestinal endoscopy for polyps in patients.

OBJECTIVE: To determine the effect of different preoperative fasting time on safety and postoperative complications of painless gastrointestinal endoscopy for polyps in patients. METHODS: Enrolled patients were assigned to an observation group and a control group by the random number table method (each n=68). Before operation, each patient in the observation group was fasted from solids for 6 h and from liquids for 2 h, while each one in the control group was fasted from solids for 8-12 h and from liquids for 4 h according to the conventional method. The levels of blood glucose, insulin, potassium and sodium in patients before and after operation were determined, and their hunger and thirst were recorded before anesthesia. Additionally, the incidences and degrees of vomiting and nausea among the patients after anesthesia and operation were recorded. RESULTS: Before operation, the observation group showed higher levels of blood glucose, insulin, serum potassium and serum sodium than the control group (all P<0.001), while after operation, the observation group showed lower levels of blood glucose and insulin and higher levels of serum potassium and serum sodium than the control group (all P<0.001). In addition, the degrees and incidences of hunger and thirst in patients of the observation group were significantly lower than those in the control group before operation (P<0.01), and the degrees and incidences of nausea and vomiting in the observation group were also notably lower than those in the control group before and after operation (both P<0.05). CONCLUSION: For patients undergoing painless gastrointestinal endoscopy for polyps, shortening their fasting time from solids and liquids before operation can stabilize their blood glucose, insulin and electrolyte levels before and after operation, relieve their thirst and hunger before operation, and reduce the incidences of postoperative nausea and vomiting.

[Determination of optical parameters in thin films by transmittance spectra].

The high tin-doped SiO2 films prepared by CVD will bring some crystals, but sol gel can control the accurate chemical component of the films. So it can produce high tin-doped materials. In the present paper, the tin-doped SiO2 films were prepared by a sol gel dip-coating process on glass substrates at concentrations of 66 mol% and 75 mol%. Then dipping was performed several times to enhance the thickness of the films. The transmittance spectrum was obtained by spectrophotometer. Compared with the films prepared in a thin film on a thick substrate previously, the model here has been modified to describe the transmittance of a thick film (substrate) sandwiched between two thin films (coating method). The authors used the curves method to calculate the film's parameters. The result showed that uhe refractive index of different samples was increased with the wave-length. And the film thickness was about 900 nm.

TLR4 supports the expansion of FasL+CD5+CD1dhi regulatory B cells, which decreases in contact hypersensitivity.

Certain B cells termed as "regulatory B cells" (Bregs) can suppress the ongoing immune responses and a splenic CD5+CD1dhi Breg subset identified earlier was shown to exert its regulatory functions through secretion of IL-10. Though FasL expression is an alternative mechanism of immune suppression used by B cells, little is known about the FasL expressing CD5+CD1dhi Bregs. In this study, we isolated splenocytes or splenic CD19+ B cells and compared the efficiency of toll-like receptor(TLR)4 ligand (lipopolysaccharide) with TLR9 ligand (CpG), anti-CD40 and TLR9 ligand (CpG) plus anti-CD40 on the FasL expression of splenic CD5+CD1dhi Bregs by flow cytometry. FasL expression in CD5+CD1dhi B cells was rapidly increased after TLR4 ligation. Intriguingly, anti-CD40 and CpG plus anti-CD40 combinations failed to stimulate FasL expression in CD5+CD1dhi B cells although the IL-10 production was up-regulated in this subset. In addition, LPS and other B10-cell inducers increased the expression of surface molecules like CD86 and CD25, which are correlated to the regulatory functions of B cells. Furthermore, NF-κB and NF-AT inhibitors decreased the TLR4-activated FasL expression in CD5+CD1dhi B cells. Then we sorted splenic CD5+CD1dhi Bregs using flow cytometry and found that TLR4-activated CD5+CD1dhi Bregs suppressed the proliferation of CFSE-labeled CD4+ T cells in vitro, which was partly blocked by anti-FasL antibody. In oxazolone-sensitized mice having contact hypersensitivity, FasL expression in splenic CD5+CD1dhi B cells was decreased compared to the control group after TLR4 ligation. Our findings suggest that the regulatory function of CD5+CD1dhi B cells could be partly mediated by Fas-FasL pathway and this FasL expressing CD5+CD1dhi Bregs might participate in the regulation of inflammatory diseases.

Artesunate suppresses RANKL-induced osteoclastogenesis through inhibition of PLCγ1-Ca2+-NFATc1 signaling pathway and prevents ovariectomy-induced bone loss.

Bone lytic diseases including osteoporosis, rheumatoid arthritis, and bone metastatic tumors affect hundreds of millions people worldwide. Targeting over-activated osteoclasts as an anti-resorptive treatment becomes an important strategy to treat osteolytic diseases. Artesunate is a compound derived from artemisinin (qinghaosu) and has been used to treat malaria and rheumatoid arthritis clinically in China, but its role in osteolysis is unknown. Here, we found that artesunate could suppress RANKL-induced osteoclastogenesis and bone resorption from 1.56 to 12.5µM. Artesunate obviously reduced RANKL-induced NF-κB-luc activity at 50µM, but had no effects on RANKL-induced NF-κB activation (NF-κB luciferase activity, IκB-α degradation and nuclear NF-κB p65 protein level) from 3.125 to 12.5µM in pre-osteoclastic RAW264.7 cells. Interestingly, artesunate could significantly inhibit RANKL-induced NFATc1 activation measured by NFAT luciferase activity, NFATc1 mRNA and nuclear NFATc1 protein levels from 3.125 to 12.5µM. Further study revealed that artesunate inhibited RANKL up-regulated PLCγ1 activation, intracellular calcium, and calcineurin (PP2B-Aα) protein expression from 3.125 to 12.5µM. In addition, the NFATc1 targeted osteoclast-specific genes expression including cathepsin K, MMP-9, and TRAP was reduced by artesunate. Finally, we showed that artesunate was able to reverse the bone loss in an ovariectomized mouse model in vivo accompanied with reduced RANKL, RANKL/OPG, and TRAP-5b levels. This study indicates that artesunate inhibits RANKL-induced osteoclastogenesis and bone loss by inhibiting PLCγ1-Ca2+-calcineurin-NFATc1 pathway. Collectively, our data suggest that artesunate is a potential treatment option against RANKL-mediated osteolytic bone disease.

Potentiating effect of tramadol on methamphetamine-induced behavioral sensitization in mice.

RATIONALE: Polydrug abuse is a common phenomenon in human drug addicts. Previous studies have shown that both tramadol (TRAM) and methamphetamine (METH) share the ability to modulate brain monoaminergic (dopamine, 5-hydroxytryptamine, and noradrenaline) systems that may be involved in behavioral sensitization to METH. Therefore, we hypothesized that there would be an interaction between TRAM and METH on behavioral sensitization induced by METH. OBJECTIVES: To investigate whether TRAM affects METH-induced behavioral sensitization. METHODS: Male Kunming mice were subjected to two regimens of drugs: (1) Mice were injected with TRAM (1-16 mg/kg, i.p.) alone or a combination of TRAM and METH (1 mg/kg, i.p.) once daily for 7 days. After 7 drug-free days (on day 15), animals were challenged with the corresponding TRAM dose or METH (1 mg/kg, i.p.). On days 1, 7, and 15, locomotion was monitored in the open field test after the last injection. (2) Mice received METH (1 mg/kg, i.p.) once daily for 7 days, followed by 7 drug-free days. On day 15, a challenge of TRAM (1-16 mg/kg, i.p.) or TRAM plus METH (1 mg/kg, i.p.) was given and then locomotor activity was quantified. RESULTS: TRAM or METH challenge did not induce hyperlocomotion in mice chronically treated with TRAM, and TRAM challenge was insufficient to induce subsequent hyperlocomotion in METH-sensitized mice. However, TRAM significantly increased METH-induced hyperlocomotion. TRAM plus METH-sensitized mice showed a significantly greater hyperlocomotor response to METH challenge than METH-sensitized mice. Furthermore, TRAM (16 mg/kg, i.p.) plus METH (1 mg/kg, i.p.) challenge enhanced the sensitized locomotor response compared to METH-alone (1 mg/kg, i.p.) challenge in METH-sensitized mice. CONCLUSIONS: TRAM fails to produce behavioral sensitization, and there is no apparent cross-sensitization between TRAM and METH. However, TRAM can increase METH-induced hyperlocomotion and potentiate the development and expression of behavioral sensitization to METH.

[Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

Celecoxib antagonizes the cytotoxicity of oxaliplatin in human esophageal cancer cells by impairing the drug influx.

It has been demonstrated that COX-2-selective inhibitor celecoxib shows synergy with oxaliplatin for suppressing tumor growth. However, the benefit of adding celecoxib to oxaliplatin-based regimen in human esophageal cancer is largely unknown. In the present study, we demonstrated that celecoxib antagonized oxaliplatin-induced cytotoxicity and apoptosis independent of COX-2 inhibition in human esophageal cancer cells. Celecoxib decreased cellular oxaliplatin accumulation and Pt-DNA adduction formation due to reduced drug influx. Celecoxib alone or combined with oxaliplatin substantially reduced the expression of organic cation transporter 2 (OCT2). To this end, OCT2 knockdown was sufficient to reduce oxaliplatin uptake, connecting OCT2 expression to oxaliplatin accumulation. Moreover, oxaliplatin combined with celecoxib also showed no beneficial effect when compared with monotherapy in esophageal cancer cell-xenografted nude mice. To conclude, our data provide evidence that the addition of celecoxib to oxaliplatin-containing regimens for patients with OCT2-expressing cancers should be cautious.

Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors.

Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.

Association of the iPLA2ß gene with bipolar disorder and assessment of its interaction with TRPM2 gene polymorphisms.

Altered intracellular calcium homeostasis and oxidative stress are involved in the pathophysiology of bipolar disorder (BD)-I. To explore the genes contributing to these abnormalities, we examined the association with BD of the iPLA2ß (PLA2G6), a signaling enzyme that mobilizes the arachidonic acid signaling cascade and activates oxidative stress, and assessed whether it interacts genetically with type 2 transient receptor potential channel gene (TRPM2), an oxidative stress-responsive calcium channel implicated both functionally and genetically in BD-I. Two tag single nucleotide polymorphisms rs4375 and rs3788533 in iPLA2ß were genotyped in 446 White case-control individuals and 296 BD families using a 5'-nuclease TaqMan assay. The results were analyzed using χ-test and transmission disequilibrium tests, and Haploview. In a secondary analysis, we tested gene-gene interactions between TRPM2 and iPLA2ß on BD vulnerability by logistic regression using a case-only design in PLINK. iPLA2ß-rs3788533 showed a borderline association with BD-I in patients with a history of psychosis in both case-control and family designs. Association with BD as a whole was observed in the family study (significant over transmissions of rs3788533-allele C, P=0.015, PBonferroni=0.03, TDTPHASE). A borderline interaction was found between rs749909 within TRPM2 and rs4375 within iPLA2ß (Puncorrected=0.009), on the basis of the case-only design analyzed with PLINK. A significant association of iPLA2ß variants with BD-I and a trend gene-gene interaction between iPLA2ß and TRPM2 provides additional support for the notion that genetic variation in these two functionally implicated candidates contributes toward the risk and pathophysiology of this illness.