Mitogenic Response Research Articles

Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partialagonist carrying triple mutations (FGF1ΔHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1ΔHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 invitro and invivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

MicroRNAs-143 and -145 induce epithelial to mesenchymal transition and modulate the expression of junction proteins.

Transforming growth factor (TGF)-β is one of the major inducers of epithelial to mesenchymal transition (EMT), a crucial program that has a critical role in promoting carcinoma's metastasis formation. MicroRNAs-143 and -145, which are both TGF-β direct transcriptional targets, are essential for the differentiation of vascular smooth muscle cells (VSMC) during embryogenesis, a TGF-β-dependent process reminiscent of EMT. Their role in adult tissues is however less well defined and even ambiguous, as their expression was correlated both positively and negatively with tumor progression. Here we show that high expression of both miRs-143 and -145 in mouse mammary tumor cells expressing constitutively active STAT3 (S3C) is involved in mediating their disrupted cell-cell junctions. Additionally, miR-143 appears to have a unique role in tumorigenesis by enhancing cell migration in vitro and extravasation in vivo while impairing anchorage-independent growth, which may explain the contradictory reports about its role in tumors. Accordingly, we demonstrate that overexpression of either miRNA in the non-transformed mammary epithelial NMuMG cells leads to upregulation of EMT markers and of several endogenous TGF-β targets, downmodulation of a number of junction proteins and increased motility, correlating with enhanced basal and TGF-β-induced SMAD-mediated transcription. Moreover, pervasive transcriptome perturbation consistent with the described phenotype was observed. In particular, the expression of several transcription factors involved in the mitogenic responses, of MAPK family members and, importantly, of several tight junction proteins and the SMAD co-repressor TGIF was significantly reduced. Our results provide important mechanistic insight into the non-redundant role of miRs-143 and -145 in EMT-related processes in both transformed and non-transformed cells, and suggest that their expression must be finely coordinated to warrant optimal migration/invasion while not interfering with cell growth.

Serotonin Signaling Through the 5-HT1B Receptor and NADPH Oxidase 1 in Pulmonary Arterial Hypertension.

Serotonin can induce human pulmonary artery smooth muscle cell (hPASMC) proliferation through reactive oxygen species (ROS), influencing the development of pulmonary arterial hypertension (PAH). We hypothesize that in PASMCs, serotonin induces oxidative stress through NADPH-oxidase-derived ROS generation and reduced Nrf-2 (nuclear factor [erythroid-derived 2]-like 2) antioxidant systems, promoting vascular injury. HPASMCs from controls and PAH patients, and PASMCs from Nox1-/- mice, were stimulated with serotonin in the absence/presence of inhibitors of Src kinase, the 5-HT1B receptor, and NADPH oxidase 1 (Nox1). Markers of fibrosis were also determined. The pathophysiological significance of our findings was examined in vivo in serotonin transporter overexpressing female mice, a model of pulmonary hypertension. We confirmed thatserotonin increased superoxide and hydrogen peroxide production in these cells. For the first time, we show that serotonin increased oxidized protein tyrosine phosphatases and hyperoxidized peroxiredoxin and decreased Nrf-2 and catalase activity in hPASMCs. ROS generation was exaggerated and dependent on cellular Src-related kinase, 5-HT1B receptor, and the serotonin transporter in human pulmonary artery smooth muscle cells from PAH subjects. Proliferation and extracellular matrix remodeling were exaggerated in human pulmonary artery smooth muscle cells from PAH subjects and dependent on 5-HT1B receptor signaling and Nox1, confirmed in PASMCs from Nox1-/- mice. In serotonin transporter overexpressing mice, SB216641, a 5-HT1B receptor antagonist, prevented development of pulmonary hypertension in a ROS-dependent manner. Serotonin can induce cellular Src-related kinase-regulated Nox1-induced ROS and Nrf-2 dysregulation, contributing to increased post-translational oxidative modification of proteins and activation of redox-sensitive signaling pathways in hPASMCs, associated with mitogenic responses. 5-HT1B receptors contribute to experimental pulmonary hypertension by inducing lung ROS production. Our results suggest that 5-HT1B receptor-dependent cellular Src-related kinase-Nox1-pathways contribute to vascular remodeling in PAH.

Role of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Androgen-Mediated Cell Viability in Prostate Cancer Cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10μM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p<0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46±3%; 1μM, 23±3%*; 10μM, 28±3%*) and by an increase in apoptosis (vehicle, 2.1±0%; 1μM, 16±4%*; 10μM, 31±3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1nM) was abrogated by 30*-42%* by 1-10μM HET0016. Incubation with 20-HETE (5-1000nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10μM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.

Hematopoietic stem cell transplantation for RelB deficiency

Hematopoietic stem cell transplantation for RelB deficiency

Modeling Combined Immunosuppressive and Anti-inflammatory Effects of Dexamethasone and Naproxen in Rats Predicts the Steroid-Sparing Potential of Naproxen.

Dexamethasone (DEX), a widely prescribed corticosteroid, has long been the cornerstone of the treatment of inflammation and immunologic dysfunctions in rheumatoid arthritis. Corticosteroids are frequently used in combination with other antirheumatic agents such as nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs to mitigate disease symptoms and minimize unwanted effects. We explored the steroid dose-sparing potential of the NSAID naproxen (NPX) with in vitro and in vivo studies. The single and joint suppressive effects of DEX and NPX on the in vitro mitogen-induced proliferation of T lymphocytes in blood and their anti-inflammatory actions on paw edema were investigated in female and male Lewis rats with collagen-induced arthritis (CIA). As expected, DEX was far more potent than NPX in these systems. Mathematical models incorporating an interaction term ψ were applied to quantitatively assess the nature and intensity of pharmacodynamic interactions between DEX and NPX. Modest synergistic effects of the two drugs were found in suppressing the mitogenic response of T lymphocytes. A pharmacokinetic/pharmacodynamic/disease progression model integrating dual drug inhibition quantitatively described the pharmacokinetics, time-course of single and joint anti-inflammatory effects (paw edema), and sex differences in CIA rats, and indicated additive effects of DEX and NPX. Further model simulations demonstrated the promising steroid-sparing potential of NPX in CIA rats, with the beneficial effects of the combination therapy more likely in males than females.

In vitro T lymphocyte proliferation by carboxyfluorescein diacetate succinimidyl ester method is helpful in diagnosing and managing primary immunodeficiencies.

Functional studies besides routine laboratory tests for the definitive diagnosis of T lymphocyte disorders with isolated T or combined T/B-cell immunodeficiencies are important. We hereby summarized our experience with a carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay for the assessment of mitogenic T-cell proliferation responses in primary immunodeficiency (PID) patients who have not been diagnosed yet or genetically analyzed, but classified as probably having T-cell defects. Unclassified patients (n=46) and controls (n=25) were evaluated for T-cell disorders with CFSE-based assay. CD3+ blast cells after PHA-L stimulation were significantly lower in patients (31.1±28.8) than controls (67.9±8.79; P<.001). Nine patients with low and four patients with normal CD3 values had severely decreased blastic transformation. The proliferation response decreased mostly in combined immunodeficiency group. Sixteen of them had impaired proliferation responses. Appropriate molecular genetical analyses were planned after thorough evaluation of each patient. In vitro lymphocyte cell proliferation analysis by CFSE method is a reliable and practical choice for the assessment of mitogenic T lymphocyte responses in yet unclassified PID patients for targeting further genetical analyses.

Release kinetics and mitogenic capacity of collagen barrier membranes supplemented with secretome of activated platelets - the in vitro response of fibroblasts of the periodontal ligament and the gingiva

BackgroundPlatelet preparations can stimulate the healing process and have mitogenic properties. We hypothesized that collagen barrier membranes (CBM), clinically used in guided bone regeneration and guided tissue regeneration, can serve as carriers for platelet secretome.MethodsSecretome was generated from washed platelets and unwashed platelets (washed/unwashed PSEC) and lyophilized onto CBM. Overall appearance of CBM was evaluated by scanning electron microscopy. The impact of PSEC on cell attachment was measured based on fluorescence microscopy with DiI-labeled cells. To assess the release kinetics, supernatants of CBM were collected and medium was replaced at hour 1–48. The mitogenic effect was evaluated with periodontal fibroblasts. Furthermore, the release of total protein, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF) β1 was measured.ResultsCBM overall appearance and cell attachment was not modulated by PSEC. Supernatants taken after one hour induced a mitogenic response in fibroblasts and showed the highest levels of total protein, TGFβ1 and PDGF-BB. These effects decreased rapidly in subsequent supernatants. While supernatants of CBM loaded with unwashed PSEC induced a stronger mitogenic response than supernatants of CBM loaded with washed PSEC this difference between the PSEC preparations was not observed when cells were seeded on 48–hours-washed CBM.ConclusionsCBM release platelet-derived factors in continuously declining release kinetics.

Difference in release kinetics of unwashed and washed platelet-released supernatants from bone substitute materials: the impact of platelet preparation modalities.

In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and β-tricalcium phosphate. Scanning electron microscopy images were taken. Supernatants of bone substitutes were collected at hours 1, 3, 6, 24, and 48 and medium was replaced. We evaluated the protein content with the bicinchoninic acid assay and the effect on proliferation using bioassays with human periodontal fibroblasts. Release of growth factors from the loaded bone substitutes was measured based on the platelet-derived growth factor isoform (PDGF-BB) and thrombin immunoassays. Furthermore, we assessed DNA and RNA content of washed PRS and unwashed PRS. Unwashed PRS showed higher total protein concentrations than washed PRS, while the concentration of PDGF-BB, thrombin, DNA, RNA and their mitogenic effect was not significantly different. The bone substitute materials adsorbed protein over time but no significant changes in overall appearance was found. Supernatants collected from unwashed PRS-loaded bone substitute after 1 h induced a potent mitogenic response in periodontal fibroblasts. This pro-mitogenic capacity of the supernatants decreased over the observation period. Supernatants of washed PRS-loaded bone substitutes did not induce a substantial mitogenic effect. Levels of PDGF-BB, thrombin and protein were higher in supernatants of unwashed PRS-loaded bone substitutes than of washed PRS-loaded bone substitutes. Bone substitutes loaded with unwashed PRS, but not bone substitutes loaded with washed PRS show continuously declining release kinetics. These data suggest that plasma components in platelet preparations can modify the release kinetics profile.

Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation.

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.

Aldose Reductase Inhibitor Protects against Hyperglycemic Stress by Activating Nrf2-Dependent Antioxidant Proteins.

We have shown earlier that pretreatment of cultured cells with aldose reductase (AR) inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG-) induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM)-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1) and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK-α1 in Thp1 cells. Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins.

In vitro effect of copper treatments on mitogenic response and superoxide dismutase expression of lead exposed leukocytes from periparturient dairy cows

The aim of this study was to evaluate whether copper (Cu) treatment in lead (Pb)-exposed lymphocytes can modulate their proliferation and superoxide dismutase (SOD) expression. Blood samples were collected from advanced pregnant crossbred dairy cows at –30, –15, 0, 15, and 30 days of calving and evaluated for lymphocytes proliferation and mRNA expression of SOD. A fixed number of lymphocytes (2×106) were cultured for 72 h with 10–4, 10–5, and 10–6 molar (M) levels of Pb. Pb-exposed lymphocytes were treated with 30, 35 and 40 micro molar (µM) Cu. Mitogenic response of lymphocytes and mRNA expression of SOD reduced as the days of parturition advanced and were noted to be lowest at the day of calving. Reduced mitogenic response of lymphocytes and mRNA expression of SOD was reported in Pb-treated groups as compared to control. Addition of Cu in the Pb- exposed lymphocytes culture improved lymphocytes proliferation and relative mRNA SOD expression. Lymphocytes proliferation and relative mRNA SOD expression was reported highest in 40 µM Cu-treated groups. These results indicated that Cu can ameliorate the adverse effects of Pb on lymphocytes proliferation and SOD expression in periparturient dairy cows.

Effect of zinc treatments on cadmium exposed periparturient bovine lymphocytes in vitro on their proliferation and superoxide dismutase (SOD) expression

This study was conducted to evaluate effect of cadmium (Cd) on lymphocyte proliferation and mRNA expression of Cu/Zn superoxide dismutase (SOD) and to determine whether zinc (Zn) treatment in Cd-exposed lymphocytes can modulate lymphocyte proliferation and SOD expression. Blood samples were collected from crossbred transition dairy cow at –30, –15, 0, 15 and 30 days of calving and evaluated for lymphocytes proliferation and SOD expression. Isolated lymphocytes were cultured with 10–3, 10–4, 10–5 and 10–6 molar (M) levels of Cd for 72 h. Adverse effect of transitional stress and Cd on lymphocyte proliferation and mRNA SOD expression was counteracted by 50, 55 and 60 micromolar (µM) Zn. Mitogenic response of lymphocyte and mRNA expression of SOD reduced as the days of parturition advanced. Lymphocyte proliferation and mRNA SOD expression showed negative correlation with Cd levels. Treatment of Zn in the Cd-exposed lymphocyte culture improved lymphocyte proliferation and relative abundance of SOD mRNA expression. In summary, Zn can ameliorate adverse effect of transitional stress and Cd on lymphocyte proliferation and SOD expression in dairy cows.

Adipose stromal cells differentiation toward smooth muscle cell phenotype diminishes their vasculogenic activity due to induction of activin A secretion.

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell-contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC-lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ1 ) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre-differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre-differentiated ASCs were used. Pre-differentiated ASCs had an inferior mitogenic response to EC-produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co-culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFβ1 induced secretion of activin A. Augmenting co-culture incubation media with anti-activin A IgG restored the ability of pre-differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFβ1 or activin A-induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co-injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.

Immunotoxic effects of gold and silver nanoparticles: Inhibition of mitogen-induced proliferative responses and viability of human and murine lymphocytes in vitro

Understanding the effects of nanoparticles (NP) on immune cell functions is essential in designing safe and effective NP-based in vivo drug delivery systems. The immunomodulatory potential of gold nanoparticles (GNP) and silver nanoparticles (SNP) was investigated in vitro using murine splenic and human peripheral blood lymphocytes (PBL) in terms of effects on viability and mitogen-induced proliferation. Hydrodynamic size and number of NP were characterized using NP tracking analysis (NTA); modal diameters of GNP and SNP were 28 (±1.5) and 66 (± 2.7) nm, respectively, with a unimodal distribution. Lymphocytes were incubated with GNP or SNP in the presence/absence of B- or T-cell mitogens and proliferative responses then determined using [3H]-thymidine incorporation. Concanavalin A (T-cell-specific) and lipopolysaccharide- (B-cell-specific) stimulated responses of murine splenic lymphocytes, as well as phytohemagglutinin (T-cell-specific) and pokeweed mitogen- (B-and T-cell specific) induced responses of human lymphocytes, were significantly inhibited by GNP (25–200 μg/ml) and SNP (12.5–50 μg/ml). However, [3H]-thymidine incorporation by unstimulated lymphocytes was unaffected in the presence of GNP or SNP. Viability of lymphocytes was determined using trypan blue dye exclusion and was significantly inhibited only at 200 μg GNP/ml and 25 or 50 μg SNP/ml. As mitogen responses are most useful to provide supportive mechanistic information on primary immunotoxicologic functional observations, and so far more comprehensive data (in vivo and in vitro) is still needed, the results nevertheless suggest to us that GNP and SNP might potentially be able to modulate immune responses by impacting on lymphocyte activation.

Ameliorating proliferation and superoxide dismutase mRNA expression of cadmium exposed leukocytes through copper treatment in transitional dairy cows

The study was conducted to determine the effect of cadmium on lymphocyte proliferation and mRNA superoxide dismutase (SOD) expression and to evaluate whether copper treatment in Cd exposed lymphocytes can modulate their proliferation and SOD mRNA expression. Blood samples were collected from crossbred transitional dairy cows at –30, –15, 0, 15, and 30 days of calving and evaluated for lymphocytes proliferation and SOD expression. A fixed number of lymphocyte were cultured for 72 h with 10-3, 10-4, 10-5 and 10-6 M levels of Cd. Effect of different Cd levels on proliferation and mRNA SOD expression was counteracted by 30, 35 and 40 μM Cu. Mitogenic response of lymphocyte and mRNA expression of SOD reduced as the days of parturition advanced and was noted lowest at the day of calving. Lymphocyte proliferation and mRNA SOD expression showed negative correlation with Cd levels. Addition of Cu in the Cd exposed lymphocyte culture improved lymphocyte proliferation and relative abundance of SOD mRNA expression and increase was reported highest in 40 μM Cu treated lymphocytes. These results indicated that Cu can ameliorate adverse effect of Cd on lymphocyte proliferation and SOD expression in transitional dairy cows.

Dendritic cell-based vaccines in treating recurrent herpes labialis: Results of pilot clinical study

ABSTRACTRecurrent herpes simplex labialis caused predominantly with herpes simplexvirus 1(HSV-1) is a major problem, for which various treatments have minimal impact. Given the important role of the immune system in controlling virus infection, an activation of virus-specific immune responses, in particular,using dendritic cell (DCs) vaccines, seems to be a promising approach for the treatment of patients with frequent recurrences of herpes labialis. The current paper presents the results of a pilot study of the safety and efficacy of DC vaccines in 14 patients with recurrent HSV-1 infections. DCs were generated in presence of GM-CSF and IFN-alpha and were loaded with HSV-1 recombinant viral glycoprotein D (HSV1gD). DCs cells were injected subcutaneously as 2 courses of vaccination during 9 months. Immunotherapy with DCs did not induce any serious side effects and resulted in more than 2-fold reduction in the recurrence rate and significant enhancement of the inter-recurrent time during the 9 months of treatment and subsequent 6-month follow-up period. An obvious clinical improvement was accompanied with an induction of an antigen-specific response to HCV1gD and a normalization of reduced mitogenic responsiveness of mono-nuclear cells. According to long-term survey data (on average 48 months after the beginning of therapy), 87% of respondents reported the decreased incidence of recurrent infection. At this time, most patients (85.7%) responded to HCV1gD stimulation. The data obtained suggests that dendritic cell vaccines may be a promising new approach for the treatment of recurrent labial herpes.

Mitogenic response of human carcinoma cells to the liver-derived hormone FGF21

Mitogenic response of human carcinoma cells to the liver-derived hormone FGF21

Expression of Slit3/Robo signal pathway in mouse aortic smooth muscle cell and its impact on proliferation and migration

To observe the expression of neural axon guidance molecules Slit3 and Robo receptors in mouse aortic smooth muscle cell(MASMC) and investigate the effect of exogenous Slit3 protein on migration and proliferation of MASMC. The primary cultured MASMC were identified by immunofluorescent assay. The expression of Slit3/Robo signal pathway was detected by RT-PCR and immunocytochemical staining. MASMC were divided into 6 groups: the negative control group (DMEM medium containing bovine serum albumin 86 μg/L), Slit3 0 μg/L group (DMEM medium without Slit3), Slit3 24 μg/L group (DMEM medium containing Slit3 24 μg/L), Slit3 40 μg/L group (DMEM medium containing Slit3 40 μg/L), Slit3 80 μg/L group (DMEM medium containing Slit3 80 μg/L) and the positive control group (DMEM medium containing platelet derived growth factor 10 μg/L). The effects of exogenous Slit3 on MASMC proliferation and migration were detected by CCK-8 and scratched cells and transwell chambers respectively. (1) The mRNA and protein expressions of Slit2, Slit3, Robo1 and Robo4 were detected in MASMC. mRNA level of Slit2 was lower than Slit3 (P<0.05) and there were no significant difference between mRNA level of Robo1 and Robo4. (2) The mitogenic responses of MASMC were significantly enhanced in Slit3 24 μg/L group, Slit3 40 μg/L group and Slit3 80 μg/L group compared with negative control group (1.13±0.04, 1.19±0.02, 1.18±0.08 and 0.64±0.10 respectively, all P<0.05). The mitogenic activity of MASMC was the strongest in Slit3 40 μg/L group (compared with positive control group 1.27±0.05, P>0.05). (3)The autonomous migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group (cell scratch width were (0.40±0.03)cm, (0.32±0.03)cm, (0.30±0.02)cm and (0.49±0.01)cm respectively, all P<0.05). The autonomous migration activity of MASMC was the strongest in Slit3 80 μg/L group (compared with positive control group (0.22±0.01)cm, P>0.05). The transmembrane migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group (the number of cell migration were 46.67±2.23, 65.33±3.43, 81.67±4.22 and 39.33±2.03 respectively, all P<0.05). The transmembrane migration activity of MASMC was the strongest in Slit3 80 μg/L group (compared with positive control group 84.00±2.02, P>0.05). Slit2, Slit3, Robo1 and Robo4 were expressed in MASMC, and exogenous Slit3 could promote proliferation and migration of MASMC in vitro.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.