Biotransformation of ginsenosides Rb<Subscript>1</Subscript>, Rg<Subscript>3</Subscript> and Rh<Subscript>2</Subscript> in rat gastrointestinal tracts

Background  

Ginsenosides such as Rb₁, Rg₃ and Rh₂ are major bioactive components of Panax ginseng. This in vivo study investigates the metabolic pathways of ginsenosides Rb₁, Rg₃ and Rh₂ orally administered to rats.     

Fingolimod targeting protein phosphatase 2A differently affects IL‐33 induced IL‐2 and IFN‐γ production in CD8+ lymphocytes

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720‐P and then internalizes sphingosine‐1‐phosphate receptors, preventing lymphocyte sequestration. IL‐33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720‐P, and S1P on IL‐33 induced formation of IL‐2 and IFN‐ γ , by using IL‐33 receptor overexpressing EL4 cells, primary CD8 ⁺ T cells, and splenocytes. EL4‐ST2 cells released IL‐2 after IL‐33 stimulation that was inhibited dose‐dependently by FTY720‐P but not FTY720. In this system, S1P increased IL‐2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL‐2 release. In primary CD8 ⁺ T cells and splenocytes IL‐33/IL‐12 stimulation induced IFN‐ γ , which was prevented by FTY720 but not FTY720‐P, independently from intracellular phosphorylation. The inhibition of IFN‐ γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN‐ γ formation and the inhibition of IFN‐ γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.     

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells,limits metastatic development in vivo,and modulates the immune system

OBJECTIVE:

Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines.

METHODS:

We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed.

RESULTS:

FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma.

CONCLUSION:

Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.     

Effects of different dose of FTY720 on lymphocyte cell cycle arrest in cardiac transplantation model of rats

Purposes: To probe into immunosuppressive effect and cell cycle arrest effect of different dose of FTY720.

Method: we study immunosuppressive effect and cell cycle arrest effect of different dose of FTY720 and time and dose dependence of FTY720 with control and random method in vivo, respectively.

Results: (i) survival time of graft in rat cardiac transplantation model is prolonged in group treated with FTY720. It is longer in group treated with FTY720 10?mg/kg than that in group treated with FTY720 2?mg/kg. (ii) The mean sum of lymphocyte in periphery blood decrease obviously 2?h after administration, reaches its peak 4?h after administration, and then maintain at low level stably. The mean sum of lymphocyte shows a linear relationship with dose of FTY720 and decrease with increasing of dose of FTY720. (iii) The percent of cell in G1-G0 increased in group treated with FTY720, not only in thymus but also in lymph node; this is more prevalent in the group treated with FTY720 10?mg/kg than that in group treated with FTY720 2?mg/kg. Similarly, the percent of cell in S and G2-M decreased in group treated with FTY720, not only in thymus but also in lymph node, and this decrease is more associated with group treated with FTY720 10?mg/kg than the one treated with FTY720 2?mg/kg.

Conclusion: FTY720 is an effective immunosuppressant and immunosuppressive effect of FTY720 show a tendency of time and dose dependent. Effect of cell cycle arrest of FTY720 is related to dose of this new immunosuppressant.     

Anti-oxidative effects of the biennial flower of <Emphasis Type="Italic">Panax notoginseng</Emphasis> against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cytotoxicity in cultured PC12 cells

Background  

Radix notoginseng is used in Chinese medicine to improve blood circulation and clotting; however, the pharmacological activities of other parts of Panax notoginseng have yet to be explored. The present study reports the anti-oxidative effects of various parts of Panax notoginseng.     

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES:

FTY720 modulates CD4⁺T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment.

METHODS:

F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis.

RESULTS:

The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4⁺ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft.

CONCLUSIONS:

Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.     

An agonist sensitive,quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E<Subscript>2</Subscript> or E<Subscript>1</Subscript> activity through subtype EP<Subscript>1</Subscript> receptor: Suitable for high throughput screening

Background  

Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E₁ (PGE₁) and E₂ (PGE₂) can be identified in the water-soluble non-purified fraction. PGE₁ is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE₂ is an inflammatory molecule.     

Multiple interactions between the alpha<Subscript>2C</Subscript>- and beta<Subscript>1</Subscript>-adrenergic receptors influence heart failure survival

Background  

Persistent stimulation of cardiac β₁-adrenergic receptors by endogenous norepinephrine promotes heart failure progression. Polymorphisms of this gene are known to alter receptor function or expression, as are polymorphisms of the α_2C-adrenergic receptor, which regulates norepinephrine release from cardiac presynaptic nerves. The purpose of this study was to investigate possible synergistic effects of polymorphisms of these two intronless genes (ADRB1 and ADRA2C, respectively) on the risk of death/transplant in heart failure patients.     

Rapid T<Subscript>1</Subscript> quantification based on 3D phase sensitive inversion recovery

Background  

In Contrast Enhanced Magnetic Resonance Imaging fibrotic myocardium can be distinguished from healthy tissue using the difference in the longitudinal T ₁ relaxation after administration of Gadolinium, the so-called Late Gd Enhancement. The purpose of this work was to measure the myocardial absolute T ₁ post-Gd from a single breath-hold 3D Phase Sensitivity Inversion Recovery sequence (PSIR). Equations were derived to take the acquisition and saturation effects on the magnetization into account.     

FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats, III. Increase in frequency of CD62L-positive T cells in Peyer's patches by FTY720-induced lymphocyte homing.

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.     

<Emphasis Type="Italic">E. coli</Emphasis> lipopolysaccharide attenuates adenosine A<Subscript>1</Subscript> receptor-mediated increase in plasma exudation from the hamster cheek pouch

Objective and design  

To determine whether exposure to E. coli lipopolysaccharide (LPS) modulates adenosine A₁ receptor-induced increase in plasma exudation from the intact hamster cheek pouch microcirculation.     

Human CD81 directly enhances T<Subscript>h</Subscript>1 and T<Subscript>h</Subscript>2 cell activation,but preferentially induces proliferation of T<Subscript>h</Subscript>2 cells upon long-term stimulation

Background  

CD81, a cell-surface protein of the tetraspanin superfamily, has been shown to costimulate T cell activation in murine T cells, and is involved in development of Th2 immune responses in mice.     

Effects of 1,25-(OH)<Subscript>2</Subscript>D<Subscript>3</Subscript> on the expressions of vitamin D receptor,STAT5 and cytoskeletal rearrangement in human monocytes incubated with sera from type 2 diabetes patients and diabetic nephropathy patients with uremia

Objective  

To explore the effects of 1,25-(OH)₂D₃ and lipopolysaccharide (LPS) plus human recombinant interleukin-15 (IL-15) on expression of vitamin D receptor (VDR) and STAT5, and cytoskeletal rearrangement in human monocytes incubated with sera from type 2 diabetes (T2DM) patients and diabetic nephropathy (DN) patients with uremia.     

Key role for spinal dorsal horn microglial kinin B<Subscript>1</Subscript>receptor in early diabetic pain neuropathy

Background  

The pro-nociceptive kinin B₁ receptor (B₁R) is upregulated on sensory C-fibres, astrocytes and microglia in the spinal cord of streptozotocin (STZ)-diabetic rat. This study aims at defining the role of microglial kinin B₁R in diabetic pain neuropathy.     

Transient T cell accumulation in lymph nodes and sustained lymphopenia in mice treated with FTY720

FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) is an orally available immunomodulatory agent that induces severe peripheral blood lymphopenia. Most studies of these lymphopenic effects have been limited to short-term exposure to FTY720. FTY720 alters the ability of lymphocytes to respond to sphingosine-1-phosphate (S1P) through S1P receptors, particularly S1P1. FTY720 affects different leukocyte populations and their trafficking through major lymphoid organs. We show the dynamics of CD4 T, CD8 T, and B lymphocyte recirculation in all major lymphoid compartments during 21-day FTY720 treatment of normal C57BL/6 mice. Following a transient increase in peripheral lymph nodes and Peyer's patches, lymphocyte recirculation reaches a new steady state. Other lymphoid organs show transient changes in lymphocyte composition with various patterns. At 21 days of FTY720 treatment, total body lymphocyte content is reduced by 20% and blood lymphocytes by 80%. Modeling suggests that the new steady state is due to a combination of reduced naive lymphocyte release from the thymus and a transient reduction of lymphocyte egress from lymph nodes. Our data indicate that the commonly held belief that FTY720 blocks lymphocyte egress from lymph nodes cannot fully explain the lymphocyte dynamics observed with prolonged treatment.     

Local delivery of FTY720 in PCL membrane improves SCI functional recovery by reducing reactive astrogliosis

FTY720 has recently been approved as an oral drug for treating relapsing forms of multiple sclerosis, and exerts its therapeutic effect by acting as an immunological inhibitor targeting the sphingosine-1-phosphate (S1P) receptor subtype (S1P1) of T cells. Recently studies demonstrated positive efficacy of this drug on spinal cord injury (SCI) in animal models after systemic administration, albeit with significant adverse side effects. We hereby hypothesize that localized delivery of FTY720 can promote SCI recovery by reducing pathological astrogliosis. The mechanistic functions of FTY720 were investigated in vitro and in vivo utilizing immunofluorescence, histology, MRI and behavioral analysis. The in vitro study showed that FTY720 can reduce astrocyte migration and proliferation activated by S1P. FTY720 can prolong internalization of S1P1 and exert antagonistic effects on S1P1. In vivo study of SCI animal models demonstrated that local delivery of FTY720 with polycaprolactone (PCL) membrane significantly decreased S1P1 expression and glial scarring compared with the control group. Furthermore, FTY720-treated groups exhibited less cavitation volume and neuron loss, which significantly improved recovery of motor function. These findings demonstrated that localized delivery of FTY720 can promote SCI recovery by targeting the S1P1 receptor of astrocytes, provide a new therapeutic strategy for SCI treatment.     

H5N1 Influenza Vaccine Formulated with AS03<Subscript>A</Subscript> Induces Strong Cross-Reactive and Polyfunctional CD4 T-Cell Responses

Objective  

Adjuvantation of an H5N1 split-virion influenza vaccine with AS03_A substantially reduces the antigen dose required to produce a putatively protective humoral response and promotes cross-clade neutralizing responses. We determined the effect of adjuvantation on antibody persistence and B- and T-cell-mediated immune responses.     

Chemical genetic analysis of FTY720‐ and Ca2+‐sensitive mutants reveals a functional connection between FTY720 and membrane trafficking

FTY720, a sphingosine‐1‐phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca²⁺ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720‐sensitive mutants in terms of their relevance to the Ca²⁺ homeostasis and identified the 16 F TY720‐ and C a²⁺‐s ensitive mutants (fcs mutants). Most of the FTY720‐sensitive mutants showed elevated Ca²⁺ levels and exhibited Ca²⁺ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca²⁺ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca²⁺ increase, inhibited the secretion of acid phosphatase in the wild‐type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase‐associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720‐sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca²⁺ homeostasis.     

Sphingosine‐1‐phosphate receptor 1 signalling in T cells: trafficking and beyond

Sphingosine-1-phosphate (S1P) is a lipid second messenger that signals via five G protein-coupled receptors (S1P_1–5). S1P receptor (S1PR) signalling is associated with a wide variety of physiological processes including lymphocyte biology, their recirculation and determination of T-cell phenotypes. The effect of FTY720 (Fingolimod, Gilenya™) to regulate lymphocyte egress and to ameliorate paralysis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis led to the use of FTY720 as a first-line oral agent for treatment of relapsing–remitting multiple sclerosis. However, a significant body of research suggests that S1P signalling may participate in diverse immune regulatory functions other than lymphocyte trafficking. This review article discusses the current knowledge of S1P signalling in the fate and function of T regulatory, T helper type 17 and memory T cells in health and disease.     

Role of sphingosine 1-phosphate receptor type 1 in lymphocyte egress from secondary lymphoid tissues and thymus

Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays a central role in the immune system. Homing of lymphocytes from blood into secondary lymphoid tissues beyond high endothelial venules is highly dependent on the interaction between the chemokines CCL19, CCL21, CXCL12, and CXCL13, and their receptors CCR7, CXCR4 and CXCR5. However, the molecular mechanism（s） of lymphocyte egress from secondary lymphoid tissues to lymph remained unclear. We have found a new class of immunomodulator, FTY720 by chemical modification of vegetative wasp-derived natural product, ISP-I （myriocin）. FTY720 has been shown to be highly effective in experimental allograft and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating activity in these models. The reduction of circulating lymphocytes by FTY720 is caused by sequestration of lymphocytes into secondary lymphoid tissues and thymus. FTY720 is rapidly converted to （S）-enantiomer of FTY720-phosphate [（S）-FTY720-P] by sphingosine kinase 2 in vivo. （S）-FTY720-P acting as a potent agonist of S1P receptor type 1 （S1P1）, induces long-term down-regulation of S1P1 on lymphocytes, and thereby inhibits the migration of lymphocytes toward S1P. Thus, it is presumed that FTY720-induced lymphocyte sequestration is due to the inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymus by its active metabolite （S）-FTY720-P. Throughout the analysis of the mechanism of action of FTY720, it is clarified that S1P/S1P1 interaction plays an important role for lymphocyte egress from secondary lymphoid tissues and thymus.