G1-4 A,an arabinogalactan polysaccharide from Tinospora cordifolia increases dendritic cell immunogenicity in a murine lymphoma model

The immunogenicity of dendritic cells (DC) is known to increase with their maturation state and both are induced by microbial products like LPS. In this study, we have investigated the effect of G1-4A, a polysaccharide isolated from Indian medicinal plant, Tinospora cordifolia on phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDC) and its ability to be used as an adjuvant in immunotherapy. G1-4A, enhanced surface expression of CD40, CD80, CD86, MHCII by BMDC in vitro and splenic DC in vivo. T cell allostimulatory activity and secretion of IL-12 and TNFα by BMDC were also increased. Treatment with G1-4A resulted in decreased phagocytosis and increased antigen processing that are characteristic of mature DC. G1-4A treated DC cross presented exogenous antigens on a MHC I background which resulted in the activation of cytotoxic T cells. These cells thus activated could cause lysis of target tumor cells in vitro. Administration of tumor lysate pulsed G1-4A treated DC resulted in decreased tumor burden in preventive as well as therapeutic tumor challenge experiments in a murine lymphoma model. These results thus confirm that G1-4A could be a promising nontoxic maturation agent to be potentially used in DC based immunotherapy of tumor.     

Activation of murine macrophages by G1-4A,a polysaccharide from Tinospora cordifolia,in TLR4/MyD88 dependent manner

Macrophages are centrally placed in the innate immune system and their activation is crucial to the generation of appropriate immune response in the event of any pathogenic invasion, tumorigenesis or other human diseases. Many plant derived polysaccharides are known to activate macrophages. In the present study, effects of G1-4A, a polysaccharide derived from Tinospora cordifolia, on the activation of macrophages were investigated. Our data demonstrated the up regulation of expression of TNF-α, IL-β, IL-6, IL-12, IL-10 and IFN-γ in RAW 264.7 cell line and peritoneal macrophages after G-14A treatment. Nitric oxide levels were also enhanced along with up-regulation of NOS2 expression in murine macrophages post G1-4A treatment. Further, G1-4A treatment up-regulated the surface expression of MHC-II and CD-86 in macrophages. Using siRNA against TLR4, MyD88 and anti-TLR4 blocking antibodies, we established that G1-4A activated macrophages by classical pathway in TLR4-MyD88 dependent manner. Additionally, G1-4A treatment activated p38, ERK and JNK MAPKs in macrophages. Using pharmaceutical inhibitors of above MAPKs we concluded that G1-4A activates the macrophages by activation of p38, ERK and JNK MAPKs in RAW264.7 macrophages. Thus our data suggests the activation of macrophages by classical pathway after treatment of G1-4A.     

Baicalin inhibits macrophage activation by lipopolysaccharide and protects mice from endotoxin shock

Baicalin (BA) exhibits anti-inflammatory effect in vivo and in vitro and is used to treat inflammatory diseases. Here, we report that BA inhibits the activation of macrophage and protects mice from macrophage-mediated endotoxin shock. The experiments in vitro showed BA suppressed the increased generation of nitric oxide (NO) and expression of inducible nitric oxide synthase (iNOS) induced by LPS or Interferon-gamma (IFN-gamma) without directly affecting iNOS activity in RAW264.7 cells and peritoneal macrophages. Similarly, BA inhibited the production of reactive oxidative species (ROS), whereas augmented the level of intracellular superoxide dismutase (SOD). Moreover, BA inhibited the production of inflammatory mediators including tumor necrosis factor (TNF)-alpha, endothelin (ET)-1 and thromboxane A2 (TXA2) induced by lipopolysaccharide (LPS) in RAW264.7 cells. In animal model, BA protected mice from endotoxin shock induced by d-galactosamine (D-GalN)/LPS possibly through inhibiting the production of cytokine and NO. Collectively, BA inhibited the production of inflammatory mediators by macrophage and may be a potential target for treatment of macrophage-mediated diseases.     

乙酸钠对D-GalN/LPS所致内毒素休克的保护作用

目的研究乙酸钠(NaAc)对小鼠急性肝损伤的保护作用及药理机制。方法分别用不同剂量的NaAc 2.50、2.30、2.00、1.75 g/kg对受试动物预处理30 min,然后腹腔注射D-氨基半乳糖/脂多糖(D-GalN/LPS)诱导小鼠急性肝衰竭,观察肝脏组织结构的变化,测定血清ALT活性及一氧化氮(NO)的含量。结果 NaAc预处理可显著降低动物死亡率,明显抑制血清ALT活性,降低NO的产生,减轻肝组织损伤。结论 NaAc对D-GalN/LPS诱导的急性肝损伤具有显著的保护作用。     

Pirfenidone suppresses tumor necrosis factor-alpha,enhances interleukin-10 and protects mice from endotoxic shock

A new experimental drug, pirfenidone (5-methyl-1-phenyl-1H-pyridine-one; S-7701), has been reported to have beneficial effects for the treatment of certain fibrotic diseases. We investigated the anti-inflammatory properties in murine endotoxic shock to determine the pharmacological characteristics. The present study describes the prophylactic effect, cytokine regulatory profiles and therapeutic effect of pirfenidone in murine endotoxic shock, which was induced in mice using an intraperitoneal (i.p.) injection of lipopolysaccharide and D-galactosamine. First, we examined the prophylactic effect and cytokine regulatory profiles. A single oral administration of pirfenidone prior to lipopolysaccharide/D-galactosamine challenge inhibited the production of circulating tumor necrosis factor-alpha (TNF-alpha), interleukin-12 and interferon-gamma, markedly enhanced that of interleukin-10, and offered protection from subsequent lethal symptoms in a dose-dependent manner. Second, we examined the therapeutic effect. A single oral administration of pirfenidone 1, 2, 3, 4 and 5 h post lipopolysaccharide/D-galactosamine challenge provided protection against lethal shock in a time-dependent manner. At the histopathological level, apoptotic positive cells were found to be suppressed in the liver. The transforming growth factor (TGF)-beta1 level was markedly elevated in the liver of lipopolysaccharide/D-galactosamine-challenged mice, suppressed in pirfenidone-treated mice. These findings may offer an alternative for both protective and therapeutical treatment of several human acute or chronic inflammatory diseases by pirfenidone.     

A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice

To assess the chemoprotective properties of a polysaccharide from Strongylocentrotus nudus eggs (SEP), myelosuppressed and immunosuppressed mouse models were generated by administration of cyclophosphamide (Cy) and then treated with SEP. SEP (16 mg/kg/d) remarkably increased spleen and thymus indices, activated the proliferation of leukocytes and erythrocytes and platelets from peripheral blood, and exhibited co-mitogenic activity on ConA- or LPS-stimulated splenocytes in a dose-dependent manner. An increased percentage of CD34⁺ cells in bone marrow of Cy-treated mice was also observed. Furthermore, SEP elevated CD4⁺ T lymphocyte counts as well as the CD4/CD8 ratio dose-dependently, and it increased interleukin-2 (IL-2), IgA, IgM, and IgG levels in the sera of Cy-treated mice. Pre-incubation with TLR2 and TLR4 blocking antibodies inhibited splenocyte proliferation and its IL-2 secretion. Finally, SEP significantly induced Akt phosphorylation in splenocytes from Cy-treated mice, suggesting that chemoprotection by SEP was mediated through the PI3K/Akt signaling pathway. These findings indicate that SEP plays an important role in the protection against myelosuppression and immunosuppression in Cy-treated mice and could be a potential immunomodulatory agent.     

Endothelial glutathione-S-transferase A4-4 protects against oxidative stress and modulates iNOS expression through NF-kappaB translocation

Our recent work in endothelial cells and human atherosclerotic plaque showed that overexpression of glutathione-S-tranferases (GSTs) in endothelium protects against oxidative damage from aldehydes such as 4-HNE. Nuclear factor (NF)-kappaB plays a crucial role during inflammation and immune responses by regulating the expression of inducible genes such as inducible nitric oxide synthase (iNOS). 4-HNE induces apoptosis and affects NF-kappaB mediated gene expression, but conflicting results on 4-HNE's effect on NF-kappaB have been reported. We compared the effect of 4-HNE on iNOS and the NF-kappaB pathway in control mouse pancreatic islet endothelial (MS1) cells and those transfected with mGSTA4, a alpha-class GST with highest activity toward 4-HNE. When treated with 4-HNE, mGSTA4-transfected cells showed significant upregulation of iNOS and nitric oxide (NO) through (NF)-kappaB (p65) translocation in comparison with wild-type or vector-transfected cells. Immunohistochemical studies of early human plaques showed lower 4-HNE content and upregulation of iNOS, which we take to indicate that GSTA4-4 induction acts as an enzymatic defense against high levels of 4-HNE, since 4-HNE accumulated in more advanced plaques, when detoxification and exocytotic mechanisms are likely to be overwhelmed. These studies suggest that GSTA4-4 may play an important defensive role against atherogenesis through detoxification of 4-HNE and upregulation of iNOS.     

Folic acid protects motor neurons against the increased homocysteine, inflammation and apoptosis in SOD1 G93A transgenic mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by selective degeneration of motor neurons. Mutations in copper/zinc superoxide dismutase (SOD1) account for 20% cases of familial ALS (fALS), but the underlying pathogenetic mechanisms are largely unknown. Using SOD1^G93A mice model of ALS, we demonstrated that mutation in SOD1 caused a significant increase in the level of plasma homocysteine (Hcy). To investigate whether Hcy-lowering therapy is beneficial to this disease, we applied folic acid (FA) and vitamin B12 which are important factors involved in the Hcy metabolism to assess the neuroprotective effect of FA and B12 in the SOD1^G93A mice. Our results showed FA or FA + B12 treatment significantly delayed the disease onset and prolonged the lifespan, accompanied by the significant reduction of motor neuron loss. Furthermore, we found that FA or FA + B12 treatment significantly attenuated the plasma Hcy level, suppressed the activation of microglia and astrocytes, and inhibited the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-) in spinal cord. Moreover, FA or FA + B12 treatment decreased the levels of cleaved caspase-3 and poly(ADP-ribose)polymerase (PARP) but up-regulated the level of anti-apoptotic protein Bcl-2. However, B12 treatment alone did not show any significant benefit to this disease. These results provide evidence to demonstrate that elevated Hcy is involved in the pathogenesis of fALS and FA therapy may have therapeutic potential for the treatment of the disease.     

Water-soluble polysaccharide from Eleutherococcus senticosus stems attenuates fulminant hepatic failure induced by D-galactosamine and lipopolysaccharide in mice

The aim of this study was to investigate whether Eleutherococcus senticosus stems could attenuate D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice. E. senticosus, known as Siberian ginseng, is a popular folk medicine used as a tonic in Asia. Preparations of E. senticosus used in this study were as follows; (i) 70% ethanol extract (ii) water extract (iii) ethanol-soluble part of the water extract (iv) polysaccharide obtained as an 80% ethanol insoluble of the water extract. Preparations were given by intraperitoneal (300 mg/kg and 50 mg/kg) or oral (300 mg/kg) injection at 12 hr and 1 hr before a D-galactosamine/lipopolysaccharide injection. The intraperitoneal injection of water extract and polysaccharide significantly lowered serum levels of tumour necrosis factor-alpha, aspartate transaminase and alanine transaminase, improved the histologic changes in liver, inhibited hepatocyte apoptosis confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA fragmentation assay, and suppressed the lethality induced by D-galactosamine/lipopolysaccharide. The oral administration of water extract and polysaccharide also reduced serum aspartate transaminase, alanine transaminase and tumour necrosis factor-alpha levels. In contrast 70% ethanol extract and ethanol-soluble part of the water extract had no protective effect when treated intraperitoneally or orally. These results indicate E. senticosus stems attenuate fulminant hepatic failure induced by D-galactosamine/lipopolysaccharide in mice and the protective effect is due to water-soluble polysaccharides in E. senticosus stems.     

Inhibition activities of polysaccharide (RG4-1) from Gentiana rigescens against RSV

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in infants and young children. With the emergence of drug-resistant strains of RSV, new antiviral agents are needed urgently. Gentiana rigescens is a kind of Chinese herb, belonging to Gentianaceae, which has long been used as a folk medicine for curing inflammation, bacterial infection, viral infection, and so on. In this research, polysaccharide designated RG4-1 was isolated from G. rigescens by hot water extraction, ethanol precipitation, and macroreticular adsorbing resin column chromatography, and its antiviral activity, cytotoxicity, and possible antiviral mechanisms were assayed by cytopathogenic effect inhibition assay, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and plaque reduction assay. RG4-1 was a fructose-binding lectin. In host cell cultures, RG4-1 was found to be an effective antiviral component against RSV. It showed good inhibitory effect against RSV when it was added 2?h after virus infection with 50% effective concentration of 12.86?μg/ml. RG4-1 also displayed its direct inactivation, attachment inhibition effect, and penetration inhibition effect against RSV. A time-dependent experiment was set up to confirm that RG4-1 blocked RSV infection at early stages of the infection. But RG4-1 seemed to be ineffective against intracellular virus and viral biosynthesis.     

Inhibition activities of polysaccharide (RG4-1) from Gentiana rigescens against RSV

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in infants and young children. With the emergence of drug-resistant strains of RSV, new antiviral agents are needed urgently. Gentiana rigescens is a kind of Chinese herb, belonging to Gentianaceae, which has long been used as a folk medicine for curing inflammation, bacterial infection, viral infection, and so on. In this research, polysaccharide designated RG4-1 was isolated from G. rigescens by hot water extraction, ethanol precipitation, and macroreticular adsorbing resin column chromatography, and its antiviral activity, cytotoxicity, and possible antiviral mechanisms were assayed by cytopathogenic effect inhibition assay, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and plaque reduction assay. RG4-1 was a fructose-binding lectin. In host cell cultures, RG4-1 was found to be an effective antiviral component against RSV. It showed good inhibitory effect against RSV when it was added 2 h after virus infection with 50% effective concentration of 12.86 μg/ml. RG4-1 also displayed its direct inactivation, attachment inhibition effect, and penetration inhibition effect against RSV. A time-dependent experiment was set up to confirm that RG4-1 blocked RSV infection at early stages of the infection. But RG4-1 seemed to be ineffective against intracellular virus and viral biosynthesis.     

A polysaccharide,extract from Grifola frondosa,induces Th-1 dominant responses in carcinoma-bearing BALB/c mice

A polysaccharide, designated as the D-fraction, extracted from maitake (Grifola frondosa), was reported to have anti-tumor effects by activating macrophages and T cells in C3H/HeN mice in which a Th-1 dominant response was established. In this study using BALB/c mice in which a Th-2 response is genetically dominant, D-fraction reduced the expression of Th-2 cytokine interleukin (IL)-4 but markedly increased the expression of Th-1 cytokine interferon (IFN)-gamma in CD4(+) T cells and also increased IL-12p70 production as well as IFN-gamma production by antigen-presenting cells (APCs), suggesting that D-fraction promotes the differentiation into Th-1 cells of CD4(+) T cells through enhancement of IL-12p70 production by APCs.     

Protective effects of an acidic polysaccharide isolated from fruiting bodies of Ganoderma lucidum against murine hepatic injury induced by Propionibacterium acnes and lipopolysaccharide

Fruiting bodies of Ganoderma lucidum were extracted with chloroform followed by hot water, from which an acidic polysaccharide named GLAa was separated and purified using 10% CCl₃COOH followed by column chromatography on DE-52 cellulose and Toyopearl HW-65. Chemical and spectroscopic analyses showed that GLAa was composed of Glc, Gal, Man and Fuc (1:0.013:0.009:0.024), in which β-d-(1→6)-Glc, β-d-(1→3)-Glc and β-d-(1→4)-Glc linkages comprised 34.9, 33.9, and 28.7% of the total linkages, respectively. The average molecular weight of GLAa was 12.5×10⁵ Da and the optical rotation was [a]^{25° \textC}_\textD = - 9.1^° [\alpha ]^{{25^\circ {\text{C}}}}_{{\text{D}}} = - 9.1^\circ ; c =0.55, H₂O. Uronic (mannuronic) acid accounted for 14.8% of the molecule, protein, 0.14% and nitrogen, 0.75%. The hepatoprotective effect of GLAa was evaluated using a mouse model in which hepatic injury was induced with Propionibacterium acnes and lipopolysaccharide (LPS). GLAa (0.4 and 0.8 g kg⁻¹ day⁻¹, b.w., p.o.), significantly prevented increases in serum aspartate aminotransferase and alanine aminotransferase levels in mice exposed to P. acnes–LPS, indicating that GLAa has hepatoprotective activity in vivo.     

2,6-二异丙基苯酚、人参皂苷Rg-1和氯化锂逆转电休克后嗅球切除抑郁大鼠学习记忆障碍

目的观察2,6-二异丙基苯酚与人参皂苷Rg-1和氯化锂对电休克后抑郁模型大鼠学习记忆、海马内谷氨酸浓度和Tau蛋白磷酸化的影响,比较其药理作用机制,为临床治疗提供依据。方法按2×4析因设计设2个干预因素,即ECT干预(2水平:无处置、施行1疗程电休克)和药物干预(4水平:海马CA1区微量注射2,6-二异丙基苯酚、人参皂苷Rg-1、氯化锂,生理盐水)。电休克结束后行Morris水迷宫测认知能力;取海马通过高效液相色谱法测谷氨酸的浓度;免疫印迹法测Tau-5和p-AT8Ser202表达。结果 2,6-二异丙基苯酚、人参皂苷Rg-1和氯化锂可改善电休克后认知障碍;2,6-二异丙基苯酚和人参皂苷Rg-1可减少谷氨酸浓度;2,6-二异丙基苯酚与人参皂苷Rg-1和氯化锂可降低海马中Tau蛋白磷酸化程度。结论 2,6-二异丙基苯酚与人参皂苷Rg-1和氯化锂均可缓解ECT后的Tau蛋白过度磷酸化进而改善电休克后的学习记忆;前两者与降低海马谷氨酸浓度有关。     

Oncoglabrinol C,a new flavan from Oncocalyx glabratus protects endothelial cells against oxidative stress and apoptosis,and modulates hepatic CYP3A4 activity

Active herbal or natural compounds have high chemical diversity and specificity than synthetic drugs. Recently, we have validated the hypoglycemic salutation of Oncocalyx glabratus in rodent model, and demonstrated the activation of PPARα/γ by its newly ioslated flavan derivative Oncoglabrinol C (5,3′-Dihydroxyflavan 7-4′-O-digallate) in liver cells (HepG2). Here we evaluated the potential of Oncoglabrinol C against Dichlorofluorescin (DCFH) and Methylglyoxal (MGO) induced endothelial cells (HUVEC) oxidative and apoptotic damage, including activation of PXR-mediated hepatic CYP3A4. Our MTT assay showed protection of ~57% and ~63.5% HUVEC cells by 10 and 20 μg/ml doses of Oncoglabrinol C, respectively through attenuating DCFH triggered free-radicals. Also, the two doses effectively protected ~53% and ~65.5% cells, respectively by reversing MGO toxicity. In DCFH and MGO treated cells, Oncoglabrinol C (20 μg/ml) effectively downregulated caspase 3/7 activity by ~33% and ~43.5%, respectively. Moreover, in reporter gene (dual-luciferase) assay, Oncoglabrinol C (20 μg/ml) moderately activated hepatic CYP3A4. Molecular docking of Oncoglabrinol C indicated its strong interactions with cellular caspase 3/7, PPARα/γ and PXR proteins, which supported its anti-apoptotic (antagonistic) as well as pro-hypoglycemic and PXR/CYP activating (agonistic) activities. Taken together, our findings demonstrated the potential of Oncoglabrinol C in reversing the endothelial oxidative and apoptotic damage as well as in the activation of hepatic CYP3A4. This warrants further evaluations of Oncoglabrinol C and related compounds towards developing effective and safe drugs against diabetes associated cardiovascular disorders.     

Geniposide, an iridoid glucoside derived from Gardenia jasminoides, protects against lipopolysaccharide-induced acute lung injury in mice

Geniposide, a main iridoid glucoside component of gardenia fruit, has been shown to possess anti-inflammatory activity. However, its potential use for acute lung injury (ALI) has not yet been studied. The aim of this study was to evaluate the anti-inflammatory properties of geniposide using a mouse ALI model. ALI was induced by intranasal injection of lipopolysaccharide (LPS). Pretreatment of mice with geniposide (20, 40, or 80 mg/kg) resulted in a marked reduction in inflammatory cells and total protein concentration in the bronchoalveolar lavage fluid (BALF) of mice. Levels of inflammatory mediators, including tumour necrosis factor- α (TNF- α), interleukin-6 (IL-6), and interleukin-10 (IL-10), were significantly altered after treatment with geniposide. Histological studies using hematoxylin and eosin (H&E) staining demonstrate that geniposide substantially inhibited LPS-induced alveolar wall changes, alveolar haemorrhage, and neutrophil infiltration in lung tissue, with evidence of reduced myeloperoxidase (MPO) activity. In addition, we investigated potential signal transduction mechanisms that could be implicated in geniposide activity. Our results suggest that geniposide may provide protective effects against LPS-induced ALI by mitigating inflammatory responses and that the compound's mechanism of action may involve blocking nuclear factor-kappaB (NF- κB) and mitogen-activated protein kinases (MAPK) signalling pathway activation.     

The lazaroid, U-74389G, inhibits inducible nitric oxide synthase activity, reverses vascular failure and protects against endotoxin shock

The aim of our study was to investigate the effect of the 21-aminosteroid U-74389G [21- < 4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4,9,(11) triene-3,20-dione(z)-2-butenedionate] on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg/kg of Salmonella Enteritidis lipopolysaccharide (LPS). Rats were treated with U-74389G (7.5, 15 and 30 mg/kg i.v.) or vehicle (1 ml/kg i.v.) 5 min after endotoxin challenge. Lipopolysaccharide administration reduced survival rate (0%, 72 h after endotoxin administration) decreased mean arterial blood pressure, enhanced plasma concentration of bilirubin and alanine aminotransferase and increased plasma nitrite concentrations. Lipopolysaccharide injection also increased the activity of inducible NO synthase in the liver and in the aorta. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (1 nM-10 microM). In addition lipopolysaccharide (50 microg/ml for 4 h) in vitro stimulation significantly increased nitrite production in peritoneal macrophages harvested from normal rats. Treatment with U-74389G (15 and 30 mg/kg i.v., 5 min after endotoxin challenge) significantly protected against lipopolysaccharide-induced lethality (90% survival rate 24 h and 80% 72 h after lipopolysaccharide injection, respectively, following the highest dose of the drug), reduced hypotension, ameliorated liver function, decreased plasma nitrite levels, restored the hyporeactivity of aortic rings to their control values and inhibited the activity of inducible NO synthase in the liver and in the aorta. Finally, U-74389G in vitro (12.5, 25 and 50 microM) significantly inhibited nitrite production in endotoxin stimulated peritoneal macrophages. The data suggest that U-74389G may exert beneficial effects in an experimental model of septic shock by inhibiting the activity of the inducible NO synthase.     

Ganoderma tsugae in vivo modulates Th1/Th2 and macrophage responses in an allergic murine model

We have reported that Ganoderma tsugae supplementation alleviates bronchoalveolar inflammation in an airway sensitization and challenge model with female BALB/c mice. However, the effects of G. tsugae supplementation in vivo on serum antibody levels, splenocyte and peritoneal microphage immune responses have not yet been determined. In this study, serum antibody levels, cytokines and splenocyte chemical mediators and peritoneal macrophage cultures from ovalbumin (OVA)-sensitized and -challenged mice were examined after continuously consuming G. tsugae supplementation diets for 5 weeks. The results showed that OVA sensitization and challenge significantly (P < 0.05) decreased the spontaneous production of IL-2 (Th1) cytokine, but significantly (P < 0.05) increased spontaneous and OVA-stimulated IL-4 (Th2) production in splenocyte cultures from experimental mice. OVA administration significantly decreased both spontaneous and LPS/IFN-γ-stimulated IL-1β and IL-6 levels in peritoneal macrophage cultures from experimental mice. However, dietary supplementation with G. tsugae significantly increased spontaneous IL-2 level, but slightly decreased spontaneous IL-4 level in cultured splenocyte supernatants in the experimental groups. G. tsugae supplementation enhanced pro-inflammatory cytokines IL-1β and IL-6 production in cultured peritoneal macrophages. However, the nitric oxide level from cultured peritoneal macrophages and serum OVA-specific IgE and IgG_2a antibody levels was not significantly affected. These results suggest that OVA sensitization and challenge induced a Th2-skewed splenocyte response and decreased peritoneal macrophage cytokine secretion. G. tsugae supplementation in vivo modulated the Th1/Th2 balance and enhanced macrophage immune responses. However, the supplementation diet could not fully reverse the Th2-skewed responses to level of Th1-skewed responses.     

A novel dual regulator of tumor necrosis factor-alpha and interleukin-10 protects mice from endotoxin-induced shock

A pyrimidylpiperazine derivative, N-[1-(4-?[4-(pyrimidin-2-yl)piperazin-1-yl]methyl?phenyl)cycloprop yl] acetamide (Y-39041), is a dual cytokine regulator of tumor necrosis factor (TNF)-alpha and interleukin-10 production. Lipopolysaccharide-induced TNF-alpha release in BALB/c mice was inhibited by the oral treatment with the compound at 10-100 mg/kg (about 80% suppression) while interleukin-10 release was augmented (about 10-fold increase at 30 mg/kg). In addition, Y-39041 (30 mg/kg, p.o.) completely protected mice from lipopolysaccharide-induced death by the treatment before and after lipopolysaccharide injection. The finding that Y-39041 suppresses TNF-alpha production and stimulates interleukin-10 production at the same time provides new insights for the treatment of septic shock, rheumatoid arthritis and Crohn's diseases.     

Overexpression of glutathione S-transferase A1-1 in ECV 304 cells protects against busulfan mediated G2-arrest and induces tissue factor expression

1. The antineoplastic drug busulfan is frequently used in preconditioning regimens for bone marrow transplantation. Pharmacokinetics vary tremendously between patients due to extensive metabolism in the liver via conjugation to glutathione catalysed by glutathione S-transferase (GST) A1-1. Since elevated busulfan plasma levels have been reported to be a risk factor for developing veno-occlusive disease (VOD), metabolism of busulfan may play a pivotal role in the induction of VOD. 2. Therefore, we developed a cell model to investigate the influence of busulfan metabolism on its biological effects. GSTA1-1 cDNA was transfected into the cell line ECV 304 and protein expression was demonstrated by Western blotting. Enzymatic activity could be detected by formation of tetrahydrothiophene. Additionally, effects of busulfan treatment on cell cycle and expression of tissue factor have been investigated. 3. A busulfan-induced G2-arrest was reduced in GSTA1-1-transfected cells, which consequently displayed a significantly higher activity of cdc2 kinase (24.1+/-1.5 AU mg(-1) protein) after busulfan treatment compared to controls (14.7+/-2.3 AU mg(-1) protein; P<0.01). Elevated basal expression of tissue factor in GSTA1-1-transfected ECV 304 cells could be 4 fold increased by busulfan treatment. 4. These data demonstrate that ECV 304 cells transfected with GSTA1-1 provide a valuable tool to assess busulfan metabolism in vitro. Furthermore, overexpression of GSTA1-1 leads to a partial protection against cell cycle effects of busulfan and affects tissue factor expression.