Suppressive effect of β,β-dimethylacryloyl alkannin on activated dendritic cells in psoriasis by the TLR7/8 pathway

β,β-dimethylacryloyl alkannin (DMA) is a key component of Lithospermum and possesses good efficacy for treating psoriasis. DMA inhibits activated dendritic cells (DCs), but the mechanism is unknown. Therefore, this study aimed to explore the modulation of the TLR7/8 pathway by DMA in psoriasis-activated DCs. Models of psoriasis-like skin lesions were established using BALB/c mice; 8 mice were treated with DMA (2.5 mg/kg). Bone marrow cells were isolated and induced into DCs using R848, a TLR7/8 agonist. Splenic CD11c + cells were detected by flow cytometry. Skin CD11c + cells were detected by immunofluorescence. TLR7, TLR8, MYD88, and IRAKM proteins were detected by Western blot. The effects of DMA on surface molecules of DCs were observed by flow cytometry. mRNA expression of inflammatory factors was detected by qRT-PCR. Secreted cytokines were detected by cytometric bead array. Compared with the model group, psoriasis-like skin lesions were alleviated by DMA, the splenic CD11c + cells were significantly decreased (P < 0.01), and CD11c + cell numbers in skin lesions were decreased (P < 0.01). Expression levels of TLR7, MYD88, and IRAKM were significantly decreased (P < 0.05). R848-stimulated DCs showed increased expression of I-A/I-E, CD80, and CD86 (P < 0.01), increased IL-23 and IL-1β mRNA and secretion (P < 0.05), and increased TLR7, TLR8, MYD88, and IRAKM expression (P < 0.01); DMA inhibited all of these effects of the TLR7/8 pathway activation by R848 (P < 0.05). In conclusion, DMA could inhibit psoriasis-activated DCs via the TLR7/8 pathway.     

MicroRNA-106b regulates pro-allergic properties of dendritic cells and Th2 polarisation by targeting early growth response-2 in vitro

Recent evidence has suggested that miRNA is implicated in the immune response of allergic and inflammatory diseases. However, little is known about its role in the mechanism that underlies the establishment of pro-allergic DCs in allergic rhinitis is not fully understood. This study assessed whether and how microRNA (miR)-106b regulates the pro-allergic properties of DCs upon allergen stimulation in vitro. Bone marrow-derived dendritic cells (BMDCs) were generated and stimulated with ovalbumin (OVA) to identify the miRNA expression profile. After transfection with miR-106b mimics and inhibitors OVA-activated BMDCs were further evaluated for surface marker expression using flow cytometry, cytokine production using ELISA and subsequent effects on Th2 cell polarisation using flow cytometry. Moreover, the upstream controllers and potential target proteins of miR-106b were examined in a western blot analysis. Results showed that MiR-106b expression was significantly inhibited in activated BMDCs upon OVA stimulation (p < 0.05). Surface marker expression (e.g., MHC class II, CD80 and CD86) was significantly upregulated after the transfection of an miR-106b inhibitor (p < 0.05), and the proportion of GATA-3⁺ T cells was significantly increased among CD4⁺ T cells that were cocultured with miR-106b inhibitor-pretreated BMDCs (p < 0.05). Conversely, IL-12 production from OVA-activated BMDCs and the proportion of T-bet⁺ T cells increased significantly in a coculture of CD4⁺ T cells and miR-106b mimics-transfected BMDCs (p < 0.05). The early growth response (Egr)-2 was identified via luciferase reporter assays as a target gene of miR-106b, and significant Egr-2 upregulation was observed in OVA-activated BMDCs following transfection with a miR-106b inhibitor (p < 0.05). In conclusion, our results suggest that miR-106b negatively regulates the pro-allergic properties of BMDCs and subsequent Th2 polarisation upon OVA stimulation and might represent a promising therapeutic target for allergic inflammation.     

Aflatoxins B1, B2 and G1 modulate cytokine secretion and cell surface marker expression in J774A.1 murine macrophages

Aflatoxins are fungal products which occur in food and feed. They are potent hepatocarcinogens, and are known to cause immunosuppression. We investigated the effect of aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂) and aflatoxin G₁ (AFG₁) exposure, alone and in combination, on the secretion of key pro- and anti-inflammatory cytokines from the murine macrophage cell line, J774A.1. Exposure of macrophages to low doses of aflatoxin (0.01 or 0.1 ng/mL) resulted in a statistically significant change in the secretion of a number of cytokines following stimulation with lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls. Specifically, treatment with AFB₁ or AFB₂ alone significantly decreased (P < 0.01) the secretion of the anti-inflammatory cytokine interleukin (IL) 10 (IL-10), while the secretion of the pro-inflammatory cytokine IL-6 was significantly increased (P < 0.01). In addition, aflatoxin exposure affected expression levels of key cell surface markers involved in the inflammatory response. Toll-like receptor 2 (TLR2) and Cluster of Differentiation 14 (CD14) expression levels decreased significantly (P < 0.01), but Toll-like receptor 4 (TLR4) expression was unaffected. This data provides further insight into the mechanisms by which aflatoxins modulate the host immune response to exert their immunosuppressive activity.     

The effect of safranal,a constituent of Crocus sativus (saffron), on tracheal responsiveness,serum levels of cytokines,total NO and nitrite in sensitized guinea pigs

BackgroundThe effect of safranal (one of the constituents of Crocus sativus) on ovalbumin (OVA) sensitized guinea pigs was examined.MethodsOne group of sensitized guinea pigs were given drinking water alone (group S), three groups drinking water containing three concentrations of safranal and one group contain dexamethasone (S + D). Tracheal responses (TR) of the animals to methacholine as effective concentration causing 50% of maximum response (EC₅₀ M), TR to 0.1% OVA, relative to contraction induced by 100 μM methacholine, IL-4, IFN-γ, total NO and nitrite levels in serum were measured.ResultsThe TR to both methacholine and OVA, the level of total NO, nitrite and IL-4 significantly increased but IFN-γ and IFN-γ/IL-4 ratio was decreased in group S compared controls (p < 0.05 to p < 0.001). The TR to both methacholine and OVA in treated animals with dexamethasone and all concentrations of safranal were significantly decreased compared to S group (p < 0.01 to p < 0.001). The level of serum IL-4 in treated guinea pigs was significantly decreased but IFN-γ and IFN-γ/IL-4 ratio was increased compared to S group (p < 0.01 to p < 0.001). The levels of total NO and nitrite were significantly decreased in treated groups compared to sensitized group (p < 0.05 to p < 0.001).ConclusionThese results showed a preventive effect for safranal on tracheal responses and serum cytokine, total NO and nitrite levels as well as increased Th1/Th2 balance in sensitized guinea pigs.     

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3–5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 × 10⁵ cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 ± 0.21 vs. 3.23 ± 0.89 mg/dl, p < 0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-α and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile.     

Adenosine deaminase activity and immunoglobulin levels as potential systemic biomonitors of occupational hazards and health status in municipal solid waste management workers

Immune status of waste management workers (WMW) with underlying systemic inflammation was assessed to identify useful immune-related biomarkers of occupational health and safety. Clinical history of WMW revealed high prevalence of respiratory symptoms alongside gastrointestinal and musculoskeletal complaints relative to control. Systemic inflammation, characterized by significant (p < 0.001) elevation of erythrocyte sedimentation rate and C-reactive protein, was associated with marked increase in concentration and prevalence of IgA (p < 0.05), IgG (p < 0.01) and adenosine deaminase activity (ADA) (p < 0.01) in WMW. Haematological changes include significant (p < 0.01) increase in lymphocytes, monocytes and total leukocytes. Eosinophils also increased significantly (p < 0.001) while haemoglobin, packed cell volume and neutrophil decreased significantly (p < 0.05). Receiver operating characteristic curve and multivariate analyses revealed ADA (p < 0.002) and IgG (p < 0.05) as important immune markers respectively for assessing sub-clinical effects of occupational exposure. Our data suggest ADA and IgG as useful immune health and safety indicators in WMW.     

Dietary fatty acid content influences the expression of genes involved in the lipid turnover and inflammation in mouse colon and spleen

BackgroundDietary interventions can improve gastrointestinal (GI) symptoms. We determined the effects of fatty acids (FAs) supplementation with medium- and long-chain saturated FAs on mouse GI motility and correlated them with the expression of genes for free FA receptors (FFAR)1-4, FA binding protein 4 (FABP4) and inflammation.MethodsForty-eight BalbC were assigned to: standard diet (STD), diet rich in medium-chain saturated FAs (COCO) and long-chain saturated FAs (HF) (7% by weight). Body weight (BW) and food intake (FI) were monitored for 8-weeks. GI motility was determined by fecal pellet output (FPO) and colon bead expulsion tests. FABP4 inhibitor, BMS309403 (1 mg/kg, ip) was injected to half of each group 2 days/week. mRNA expression of FABP4, (FFAR)1-4, and pro-inflammatory cytokines were measured in colonic and splenic tissues using real-time PCR.ResultsCOCO and HF decreased FI. COCO accelerated overall GI transit (p < 0.05). COCO increased the mRNA expression of FFAR2 (p < 0.001) and TNFα (p < 0.01); HF increased the expression of FABP4 and FFAR4 (p < 0.05), and FFAR2 (p < 0.001) in the colon, and decreased FFAR1 and FFAR4 (p < 0.001), TNFα (p < 0.01) and IL-1β (p < 0.05) in splenic tissues. BMS309403 decreased the FI and delayed colonic transit in STD+BMS and COCO+BMS vs. STD (p < 0.05). HF+BMS increased colonic expression of FFAR3 (p < 0.01), TNFα (p < 0.01), IL-6 (p < 0.01), and reduced FFAR4 (p < 0.05); COCO + BMS decreased TNFα (p < 0.01).ConclusionDiversification in the dietary lipid content affected GI motility in mice and the expression of FFARs and pro-inflammatory cytokines in vivo.     

Physiological interactions between the hypothalamic-pituitary-gonadal axis and spleen in rams actively immunized against GnRH

Hypothalamic-pituitary-gonadal (HPG) axis is strongly implicated in the regulation of immune system. The objective was to determine the effects of immunocastration on splenic reproduction- and immunity-related gene expressions, and serum cytokine profiles in rams. Forty rams were randomly allocated into three groups: control (n = 14); surgically castrated (n = 13); or immunized (n = 13) against 100 μg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at 6 months of age (with a booster 2 months later). Blood samples (for hormone and immune cytokine profiles) were collected at 1-month intervals until rams were slaughtered (10 months). Compared to intact controls, anti-GnRH immunization reduced (P < 0.05) serum concentrations of LH, FSH, and testosterone. Reduced testosterone abrogated its inhibitor feedback effect on the synthesis of GnRH in spleen, as evidenced by increased (P < 0.05) protein content and mRNA expressions of GnRH, and simultaneously decreased (P < 0.05) mRNA expressions of androgen receptor in spleen. In parallel with the increased GnRH production in spleen, the mRNA expressions of interleukin (IL)-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α) as well as lymphocyte marker CD4, CD8 and CD19 molecules were increased (P < 0.05) in spleen. Consistently, serum concentrations of IL-2, IL-4, IL-6, TNF-α were increased (P < 0.05) in rams following immunization. Similarly, deprivation of testosterone by surgical castration also increased (P < 0.05) GnRH and thus immune cytokine expressions in spleen. Collectively, our data suggested that immunocastration increased GnRH production in spleen by abrogating the inhibitory feedback effects from testosterone, consequently improving the immune markers of spleen and serum immune cytokines in rams.     

Exposure of mice to benzo(a)pyrene impairs endometrial receptivity and reduces the number of implantation sites during early pregnancy

Benzo(a)pyrene (BaP) is a ubiquitous environmental pollutant. Studies have demonstrated it to be an endocrine-disrupting chemical that can cause adverse effects on the female reproductive system. However, the effect of BaP on early pregnancy has not been reported. We investigated the effect of BaP on endometrial receptivity and embryo implantation. Pregnant mice were dosed with BaP at 0.2, 2 and 20 mg/kg/day from day 1 (D1) to day 5 (D5) of gestation. Exposure to BaP impaired the morphology of the endometrium and decreased the number of implantation sites (p_0.2 = 0.006, p₂ = 0.167, p₂₀ = 0.003). Levels of estrodiol (p < 0.001, for three treatment group compare with control group) and progesterone-4 in plasma were elevated in BaP-treatment groups (p_0.2 < 0.001, p₂ < 0.001, p₂₀ = 0.032). Expression of estrogen receptor-α was up-regulated (p_0.2 = 0.002, p₂ = 0.131, p₂₀ = 0.024) whereas expression of the progesterone receptor was down-regulated (p_0.2 < 0.001, p₂ = 0.064, p₂₀ = 0.021). Levels of receptivity-related genes HoxA10 (p_0.2 < 0.001, p₂ = 0.135, p₂₀ < 0.001) and E-cadherin (p_0.2 = 0.002, p₂ = 0.624, p₂₀ = 0.137) were changed by BaP. These results revealed that BaP can disrupt the balance of estrogen and progesterone, influence expression of their receptors and downstream related genes, lead to changes in endometrium receptivity, and reduce of the number of implantation sites.     

Solid lipid nanoparticles as anti-inflammatory drug delivery system in a human inflammatory bowel disease whole-blood model

Standard treatment for inflammatory bowel diseases (IBD) necessitates frequent intake of anti-inflammatory and/or immunosuppressive drugs, leading to significant adverse events.To evaluate the role solid lipid nanoparticles (SLN) play as drug delivery system in enhancing anti-inflammatory activity for drugs such as dexamethasone and butyrate in a human inflammatory bowel diseases whole-blood model. ELISA assay and the peripheral blood mononuclear cell (PBMC) cytokine mRNA expression levels were evaluated by quantitative SYBR Green real-time RT-PCR to determine the IL-1β, TNF-α, IFN-γ and IL-10 secretion in inflammatory bowel diseases patients’ PBMC culture supernatants. There was a significant decrease in IL-1β (p < 0.01) and TNF-α (p < 0.001) secretion, whilst IL-10 (p < 0.05) secretion significantly increased after cholesteryl butyrate administration, compared to that of butyrate alone at the highest concentration tested (100 μM), at 24 h exposure. There was a significant decrease in IL-1β (p < 0.01), TNF-α (p < 0.001) and IL-10 (p < 0.001) secretion after dexamethasone loaded SLN administration, compared to dexamethasone alone at the highest concentration tested (250 nM) at 24 h exposure. No IFN-γ was detected under any conditions and no cytotoxic effects observed even at the highest concentration tested.The incorporation of butyrate and dexamethasone into SLN has a significant positive anti-inflammatory effect in the human inflammatory bowel disease whole-blood model.     

Interleukin-23 mediates the pathogenesis of LPS/GalN-induced liver injury in mice

BackgroundInterleukin-23 (IL-23) is required for T helper 17 (Th17) cell responses and IL-17 production in hepatitis B virus infection. A previous study showed that the IL-23/IL-17 axis aggravates immune injury in patients with chronic hepatitis B virus infection. However, the role of IL-23 in acute liver injury remains unclear.ObjectiveThe purpose of this study was to determine the role of the inflammatory cytokine IL-23 in lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute liver injury in mice.MethodsSerum IL-23 from patients with chronic hepatitis B virus (CHB), acute-on-chronic liver failure (ACLF) and healthy individuals who served as healthy controls (HCs) was measured by ELISA. An IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody was administered intravenously at the time of challenge with LPS (10 μg/kg) and GalN (400 mg/kg) in C57BL/6 mice. Hepatic pathology and the expression of Th17-related cytokines, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and the stabilization factor Csf3 were assessed in liver tissue.ResultsSerum IL-23 was significantly upregulated in ACLF patients compared with CHB patients and HCs (P < 0.05 for both). Serum IL-23 was significantly upregulated in the non-survival group compared with the survival group of ACLF patients, which was consistent with LPS/GalN-induced acute hepatic injury in mice (P < 0.05 for both). Moreover, after treatment, serum IL-23 was downregulated in the survival group of ACLF patients (P < 0.001). Compared with LPS/GalN mice, mice treated with either an IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody showed less severe liver tissue histopathology and significant reductions in the expression of Th17-related inflammatory cytokine, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and stabilization factors Csf3 within the liver tissue compared with LPS/GalN mice (P < 0.05 for all).ConclusionHigh serum IL-23 was associated with mortality in ACLF patients and LPS/GalN-induced acute liver injury in mice. IL-23 neutralizing antibodies attenuated liver injury by reducing the expression of Th17-related inflammatory cytokines, neutrophil chemoattractants and stabilization factors within the liver tissue, which indicated that IL-23 likely functions upstream of Th17-related cytokine and chemokine expression to recruit inflammatory cells into the liver.     

Donor pre-treatment with tacrolimus reduces transplant vasculopathy

We tested whether transplant arteriosclerosis can be reduced by pre-treatment of the donor with immunosuppressive agents, using a rat allogeneic aorta transplantation model.Donor rats received no pre-treatment, or tacrolimus, methylprednisolone, rapamycin, or mycofenolate mofetil (MMF) 16 and 2 h before explantation of the grafts. Eight weeks after transplantation, aorta allografts were harvested. Percent intima area/intima + media area (I/I + M), inflammatory cells and in situ MMP-2 and -9 activity were determined. In pre-transplantation biopsies, MMP-2 and -9 ratio, and mRNA levels for genes of interest were determined.In pre-transplantation biopsies we found no differences in MMP-2/9 ratio, and Bcl-2, Bax, TGF-β, HO-1, p21, and HIF-1α mRNA expression between the groups.Aorta allografts, pre-treated with tacrolimus, showed significantly lower I/I + M ratio compared to untreated controls (p < 0.01). Pre-treatment with methylprednisolone, rapamycin or MMF did not significantly reduce I/I + M ratio. In situ MMP-2/MMP-9 activity was significantly reduced in grafts treated with tacrolimus and rapamycin compared to controls (p < 0.05). Immunohistochemistry revealed a high number of CD4+ cells and high CD4/CD8 ratio in grafts pre-treated with tacrolimus.Donor pre-treatment with tacrolimus significantly reduces transplant arteriosclerosis and is associated with reduced in situ MMP-2/MMP-9 activity and increased number of CD4+ cells.     

EEG spectral phenotypes: Heritability and association with marijuana and alcohol dependence in an American Indian community study

Native Americans have some of the highest rates of marijuana and alcohol use and abuse, yet neurobiological measures associated with dependence on these substances in this population remain unknown. The present investigation evaluated the heritability of spectral characteristics of the electroencephalogram (EEG) and their correlation with marijuana and alcohol dependence in an American Indian community. Participants (n = 626) were evaluated for marijuana (MJ) and alcohol (ALC) dependence, as well as other psychiatric disorders. EEGs were collected from six cortical sites and spectral power determined in five frequency bands (delta 1.0–4.0 Hz, theta 4.0–7.5 Hz, alpha 7.5–12.0 Hz, low beta 12.0–20.0 Hz and high beta/gamma 20–50 Hz). The estimated heritability (h²) of the EEG phenotypes was calculated using SOLAR, and ranged from 0.16 to 0.67. Stepwise linear regression was used to detect correlations between MJ and ALC dependence and the spectral characteristics of the EEG using a model that took into account: age, gender, Native American Heritage (NAH) and a lifetime diagnosis of antisocial personality and/or conduct disorder (ASPD/CD). Increases in spectral power in the delta frequency range, were significantly correlated with gender (p < 0.001) and marijuana dependence (p < 0.003). Gender, age, NAH and ASPD/CD were all significantly (p < 0.001) correlated with theta, alpha and beta band power, whereas alcohol dependence (p < 0.01), gender (p < 0.001), and ASPD/CD (p < 0.001) were all correlated with high beta/gamma band power. These data suggest that the traits of EEG delta and high beta/gamma activity are correlated with MJ dependence and alcohol dependence, respectively, in this community sample of Native Americans.     

Assessment of toxicological effects of blood microsampling in the vehicle dosed adult rat

Historically, satellite groups are often used for rodent toxicokinetic profiling because of the haematological consequences of blood sampling. If microsampling is shown to be toxicologically benign, its adoption in rat studies would enable comparison of exposure and toxicity in individual animals (as happens in non-rodent studies) as well as obviating need for satellite groups.MethodsGroups of 10 male (200–300 g) and female (150–250 g) rats aged 10 weeks were vehicle dosed and either left unsampled, conventional blood volume sampled (6 × 200 μL) or microsampled (6 × 32 μL) on Days 1 and 14. At termination on Day 15, clinical pathology plus liver and spleen weights and histopathology were obtained.ResultsAll clinical pathology parameters were within background range. However, compared to unsampled controls, conventional volume sampled rats showed a statistically significant (p < 0.001) decrease in haemaglobin, haematocrit and red blood cell count, an increase in reticulocytes (at least p < 0.01), increased AST and GLDH and, in males only, an increase in monocytes and neutrophils. In contrast, microsampled animals showed no changes except for a slight, toxicologically insignificant decrease in haemoglobin concentration (15.0 g/dL compared to the unsampled group mean of 14.4 g/dL) in females (p < 0.05) and a small increase in monocytes (p < 0.05) in males.ConclusionMicrosampling of adult rats is possible without adverse toxicological consequences.     

Vitamin D3 enhances bactericidal activity of macrophage against Pseudomonas aeruginosa

BackgroundThe bioactive form of vitamin D3, i.e.1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) vitamin D has been shown to modulate monocytes/macrophages physiology and its response against bacterial infections. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterial pathogen that can most frequently be fatal in immunocompromised infected people.MethodsWe investigated the ex vivo effect of 1,25(OH)₂D₃ on monocyte-derived macrophages function against P. aeruginosa infection.ResultsRelative vitamin D receptor (VDR) mRNA expression was significantly increased in infected and 1,25(OH)₂D₃-treated macrophages compared to controls (p < 0.01). Treatment with 1,25(OH)₂D₃ markedly resulted in up-regulation of nitric oxide (NO) and IL-1β production and down-regulation of IL-10 levels (respectively, p = 0.029, p = 0.048 and p = 0.008). Additionally, 1,25(OH)₂D₃ significantly increased M1/M2 macrophage ratio (p < 0.05) and slightly reduced intracellular bacterial development. Furthermore, the arginase activity, P. aeruginosa phagocytosis and killing were significantly increased in cells that were both infected and 1,25(OH)₂D₃-treated compared to the infected, but not 1,25(OH)₂D₃-treated macrophages (respectively, p < 0.001, p < 0.01 and p < 0.001).ConclusionsWe show in this study that bioactive from of vitamin D [1,25-dihydroxyvitamin D3 (1,25D3)] can enhance M1 macrophage polarization and their bactericidal protective activity against P. aeruginosa. Future works would involve improving the treatment response through dose-dependent effect studies, both in ex vivo and in vivo models.     

Effects of N-(morpholinomethyl)- p-isopropoxyphenylsuccinimide on the protective action of different classical antiepileptic drugs against maximal electroshock-induced tonic seizures in mice

BackgroundThe aim of this study was to determine the effects of N-(morpholinomethyl)-p-isopropoxy-phenylsuccinimide (MMIPPS) on the protective action of four classical antiepileptic drugs (AEDs: carbamazepine [CBZ], phenobarbital [PB], phenytoin [PHT] and valproate [VPA]) against maximal electroshock (MES)-induced seizures in mice.MethodsTonic hind limb extension (seizure activity) was evoked in adult male albino Swiss mice by a current (sine-wave, 25 mA, 500 V, 50 Hz, 0.2 s stimulus duration) delivered via auricular electrodes. Total brain concentrations of AEDs were measured to determine the characteristics of interaction between MMIPPS and classical AEDs in the mouse MES model.ResultsMMIPPS administered intraperitoneally (ip) at 100 mg/kg significantly elevated the threshold for electroconvulsions in mice (p < 0.01). MMIPPS at doses of 25 and 50 mg/kg had no impact on the threshold for electroconvulsions in mice. Moreover, MMIPPS (50 mg/kg) significantly enhanced the anticonvulsant activity of PB and VPA(p < 0.05), but not that of CBZ or PHT, in the MES test in mice. Pharmacokinetic studies revealed that MMIPPS (50 mg/kg) did not alter total brain concentrations of PB, but significantly elevated total brain concentrations of VPA in mice (p < 0.05).ConclusionsThe enhanced anticonvulsant action of PB byMMIPPS in themouseMESmodel and lack of any pharmacokinetic interaction between drugs make the combination of MMIPPS with PB of pivotal importance for further experimental and clinical studies. Pharmacokinetic increase in total brain VPAconcentration seems to be responsible for the enhanced anticonvulsant action of VPAby MMIPPS in the mouse MES model. The combinations of MMIPPS with CBZ and PHT are neutral from a preclinical viewpoint.     

GV1001 immunotherapy ameliorates joint inflammation in a murine model of rheumatoid arthritis by modifying collagen-specific T-cell responses and downregulating antigen-presenting cells

This study investigated whether GV1001 may be useful for treating rheumatoid arthritis (RA). Two collagen-induced arthritis (CIA) experiments showed that therapeutic, but not preventive, GV1001 treatment reduced the severity of joint inflammation in CIA. The third CIA experiment indicated that, compared to vehicle treatment, therapeutic GV1001 treatment was associated with a significantly smaller area under the curve for the overall clinical joint score over the 98 day observation period (p < 0.05). GV1001 treatment was also associated with lower Day 98 serum IL-6 levels (p < 0.01) and histological joint scores (p < 0.05). Moreover, splenocytes harvested from the GV1001-treated mice exhibited lower basal and collagen-stimulated production of IFN-γ and IL-6 on Days 49 and 98 than the splenocytes from vehicle-treated mice. The fourth and fifth experiments indicated that earlier treatment resulted in a better response.In addition, human (THP-1) and murine (RAW 264.7) macrophages and fibroblast-like synoviocytes (FLS) from RA patients were used for in vitro analyses. GV1001 treatment of lipopolysaccharide-stimulated macrophages derived from THP-1 and RAW 264.7 monocytes significantly reduced TNF-α and IL-6 secretion (THP-1: all p < 0.05; RAW 264.7: all p < 0.01). However, GV1001 treatment did not affect IL-6 expression in TNFα-stimulated RA FLS.GV1001 reduced the clinical joint scores, serum IL-6 levels, and histological joint scores of mice with CIA. In addition, GV1001 lowered the collagen-stimulated IFN-γ and IL-6 production of murine T-cells and reduced the TNF-α and IL-6 production of macrophages in vitro. Thus, GV1001 may ameliorate joint inflammation by modifying T-cell reactions to the triggering autoantigen and by reducing macrophage cytokine production.     

TLR4-HMGB1 signaling pathway affects the inflammatory reaction of autoimmune myositis by regulating MHC-I

ObjectivesTo analyze the effects of TLR4 on the expression of the HMGB1, MHC-I and downstream cytokines IL-6 and TNF-α, and to investigate the biological role of the TLR4-HMGB1 signaling pathway in the development of the autoimmune myositis.MethodsWe built mice models with experimental autoimmune myositis (EAM) and used the inverted screen experiment to measure their muscle endurance; we also examined inflammatory infiltration of muscle tissues after HE staining; and we assessed the expression of MHC-I using immunohistochemistry. In addition, peripheral blood mononuclear cells (PBMC) were extracted and flow cytometry was utilized to detect the effect of IFN-γ on the expression of MHC-I. Furthermore, PBMCs were treated with IFN-γ, anti-TLR4, anti-HMGB1 and anti-MHC-I. Real-time PCR and western blotting were employed to examine the expressions of TLR4, HMGB1 and MHC-I in different groups. The ELISA method was also utilized to detect the expression of the downstream cytokines TNF-α and IL-6.ResultsThe expressions of TLR4, HMGB1 and MHC-I in muscle tissues from mice with EAM were significantly higher than those in the control group (all P < 0.05). After IFN-γ treatment, the expressions of TLR4, HMGB1, MHC-I, TNF-α and IL-6 in PBMCs significantly increased (all P < 0.05). The treatment of anti-TLR4, anti-HMGB1 and anti-MHC-I could significantly downregulate the expression of MHC-I (all P < 0.05). In addition, anti-TLR4 and anti-HMGB1 significantly reduced the expression of TNF-α and IL-6 (all P < 0.05).ConclusionsThe TLR4-HMGB1 signaling pathway affects the process of autoimmune myositis inflammation by regulating the expression of MHC-I and other pro-inflammatory cytokines.     

Association of CD40 − 1C/T polymorphism with cerebral infarction susceptibility and its effect on sCD40L in Chinese population

This study aims to determine whether functional polymorphism of CD40 is associated with the cerebral infarction (CI) susceptibility, and to investigate the effect of CD40 gene polymorphism on CD40 mRNA expression in PBMCs and plasma sCD40L concentration. A case–control study was performed in 402 CI patients and 693 controls. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The expressions of CD40 mRNA and plasma sCD40L concentration were determined. The distribution of TT genotype and the frequency of T allele in CI patients were significantly higher than those in the controls (P < 0.05). The frequency of T allele was also significantly higher in the male subjects and the elder subjects (P < 0.05) when stratified analysis was carried out. The PBMCs from CI patients showed significantly higher CD40 mRNA expression than controls (P < 0.01), the CD40 mRNA expression from TT genotype was higher than other genotypes (P < 0.05). TT genotype subjects also showed the highest plasma sCD40L concentration in the male CI patients (P < 0.01). CD40 − 1C/T polymorphism may interfere CI susceptibility, and the T allele may be associated with increased risk of CI. The CD40 − 1C/T polymorphism is also a regulator of CD40 expression and plasma sCD40L concentration.     

Daphnoretin modulates differentiation and maturation of human dendritic cells through down-regulation of c-Jun N-terminal kinase

Daphnoretin, an active constituent of Wikstroemia indica C.A. Meys, has been shown possessing anti-cancer activity. In this study, we examined the effect of daphnoretin on differentiation and maturation of human myeloid dendritic cells (DCs). After treatment with daphnoretin (0, 1.1, 3.3, 10 and 30 μM) to initiate monocytes, the recovery rate of DCs was reduced in a dose-dependent manner. The mature DCs differentiated in the presence of daphnoretin had fewer and shorter dendrites. Daphnoretin modulated DCs differentiation and maturation in terms of lower expression of CD1a, CD40, CD83, DC-SIGN, and HLA-DR. Daphnoretin inhibited the allostimulatory activity of DCs on proliferation of naive CD4⁺ CD45⁺ RA⁺ T cell. On the mitogen-activated protein kinase, daphnoretin down-regulated the lipopolysaccharide-augmented expression of phosphorylated c-Jun N-terminal kinase (pJNK), but not p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of JNK by anisomycin reversed the effect of daphnoretin on daphnoretin-inhibited pJNK expression and dendrite formation of DCs. In disease model related to maturation of DCs, daphnoretin suppressed the acute rejection of skin allografts in mice. Our results suggest that daphnoretin modulated differentiation and maturation of DCs toward a state of atypical maturation with impaired allostimulatory function and this effect may go through down-regulation of phosphorylated JNK.