GFPBW1, a ß-glucan from Grifola frondosa as vaccine adjuvant: APCs activation and maturation.

Adjuvants for vaccines with characteristics of improving adaptive immunity particularly via leverage of antigen presenting cells (APCs) are currently lacking. In a previous work we obtained a new soluble 300 kDa homogeneous ß-glucan named GFPBW1 from the fruit bodies of Granola frondosa. GFPBW1 could activate macrophages by targeting dendritic cell associated C-type lectin 1 (Dectin-1)/Syk/NF-κB signaling to achieve antitumour effects. In this study the adjuvant effects of GFPBW1 were explored with OVA-antigen and B16-OVA tumor model. We showed that GFPBW1 (5, 50, 500 µg/mL) dose-dependently promoted activation and maturation of APCs in vitro by increasing CD80, CD86 and MHC II expression. We immunized female mice with OVA in combination with GFPBW1 (50 or 300 µg) twice with an interval of two weeks. GFPBW1 markedly and dose-dependently increased OVA-specific antibody titers of different subtypes including IgG1, IgG2a, IgG2b and IgG3, suggesting that it could serve as an adjuvant for both Th1 and Th2 type immune responses. Furthermore, GFPBW1 in combination with aluminum significantly increased the titers of OVA-specific IgG2a and IgG2b, but not those of IgG1, suggesting that GFPBW1 could be used as a co-adjuvant of aluminum to compensate for Th1 deficiency. For mice immunized with OVA plus GFPBW1, no obvious pathological injury was observed in either major organs or injection sites, and no abnormalities were noted for any of the hematological parameters. When GFPBW1 served as an adjuvant in the B16-OVA cancer vaccine models, it could accomplish entire tumor suppression with preventive vaccines, and enhance antitumour efficacy with therapeutic vaccines. Differentially expressed genes were found to be enriched in antigen processing process, specifically increased tumor infiltration of DCs, B1 cells and plasma cells in the OVA plus GFPBW1 group, in accordance with its activation and maturation function of APCs. Collectively, this study systematically describes the properties of GFPBW1 as a novel potent and safe adjuvant and highlights its great potential in vaccine development.

Eutypenoids A-C: Novel Pimarane Diterpenoids from the Arctic Fungus Eutypella sp. D-1.

Eutypenoids A-C (1-3), pimarane diterpenoid alkaloid and two ring A rearranged pimarane diterpenoids, were isolated from the culture of Eutypella sp. D-1 obtained from high-latitude soil of the Arctic. Their structures, including absolute configurations, were authenticated on the basis of the mass spectroscopy (MS), nuclear magnetic resonance (NMR), X-ray crystallography, and electronic circular dichroism (ECD) analysis. The immunosuppressive effects of eutypenoids A-C (1-3) were studied using a ConA-induced splenocyte proliferation model, which suggested that 2 exhibited potent immunosuppressive activities.

Prenylated benzoylphloroglucinols and xanthones from the leaves of Garcinia oblongifolia with antienteroviral activity.

An acetone extract of the leaves of Garcinia oblongifolia showed antiviral activity against enterovirus 71 (EV71) using a cytopathic effect inhibition assay. Bioassay-guided fractionation yielded 12 new prenylated benzoylphloroglucinols, oblongifolins J-U (1-12), and five known compounds. The structures of 1-12 were elucidated by spectroscopic analysis including 1D- and 2D-NMR and mass spectrometry methods. The absolute configurations were determined by a combination of a Mosher ester procedure carried out in NMR tubes and ECD calculations. Compared to ribavirin (IC50 253.1 µM), compounds 1, 4, and 13 exhibited significant anti-EV71 activity in vitro, with IC50 values of 31.1, 16.1, and 12.2 µM, respectively. In addition, the selectivity indices of these compounds were 1.5, 2.4, and 3.0 in African green monkey kidney (Vero) cells, respectively.

Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity.

AIM: To investigate the immunomodulating activity of astragalosides, the active compounds from a traditional tonic herb Astragalus membranaceus Bge, and to explore the molecular mechanisms underlying the actions, focusing on CD45 protein tyrosine phosphatase (CD45 PTPase), which plays a critical role in T lymphocyte activation. METHODS: Primary splenocytes and T cells were prepared from mice. CD45 PTPase activity was assessed using a colorimetric assay. Cell proliferation was measured using a [(3)H]-thymidine incorporation assay. Cytokine proteins and mRNAs were examined with ELISA and RT-PCR, respectively. Activation markers, including CD25 and CD69, were analyzed using flow cytometry. Activation of LCK (Tyr505) was detected using Western blot analysis. Mice were injected with the immunosuppressant cyclophosphamide (CTX, 80 mg/kg), and administered astragaloside II (50 mg/kg). RESULTS: Astragaloside I, II, III, and IV concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 µg/mL. Astragaloside II (10 and 30 nmol/L) significantly enhanced the proliferation of primary splenocytes induced by ConA, alloantigen or anti-CD3. Astragaloside II (30 nmol/L) significantly increased IL-2 and IFN-γ secretion, upregulated the mRNA levels of IFN-γ and T-bet in primary splenocytes, and promoted CD25 and CD69 expression on primary CD4(+) T cells upon TCR stimulation. Furthermore, astragaloside II (100 nmol/L) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells, which could be blocked by a specific CD45 PTPase inhibitor. In CTX-induced immunosuppressed mice, oral administration of astragaloside II restored the proliferation of splenic T cells and the production of IFN-γ and IL-2. However, astragaloside II had no apparent effects on B cell proliferation. CONCLUSION: Astragaloside II enhances T cell activation by regulating the activity of CD45 PTPase, which may explain why Astragalus membranaceus Bge is used as a tonic herb in treating immunosuppressive diseases.

Oral administration of artemisinin analog SM934 ameliorates lupus syndromes in MRL/lpr mice by inhibiting Th1 and Th17 cell responses.

OBJECTIVE: SM934, an artemisinin derivative, possesses potent antiproliferative and antiinflammatory properties. The aim of this study was to examine the effects and explore the mechanisms of SM934 to treat autoimmune disease in lupus-prone female MRL/lpr mice. METHODS: In vitro, the effects of SM934 on the activation of polyclonal CD4+ T cells and the differentiation of naive CD4+ T cells were examined. In vivo, the preventative or therapeutic effects of SM934 in MRL/lpr mice were investigated. Ex vivo, the mechanisms of treatment were explored according to the immunologic correlates of disease. RESULTS: In vitro, SM934 inhibited interferon-γ (IFNγ) and interleukin-17 (IL-17) production from polyclonal CD4+ T cells activated by T cell receptor engagement and the differentiation of naive CD4+ T cells into Th1 and Th17 cells, but not Treg cells. In vivo, 12-week-old MRL/lpr mice treated with SM934 for 4 weeks showed significantly ameliorated proteinuria and renal lesion severity; decreased levels of blood urea nitrogen, serum IFNγ, and serum anti-double-stranded DNA antibodies; decreased spleen size; and a lower percentage of CD3+B220+CD4-CD8- T cells; 16-week-old MRL/lpr mice treated with SM934 for 8 weeks avoided severe proteinuria and survived longer. Ex vivo, SM934 treatment elevated the percentage of Treg cells, inhibited the development of Th1 and Th17 cells, and impeded the comprehensive activation of STAT-1, STAT-3, and STAT-5 proteins in splenocytes. CONCLUSION: Taken together, the results of this study demonstrated that the artemisinin analog SM934 had therapeutic effects in lupus-prone female MRL/lpr mice by inhibiting both Th1 cell and Th17 cell responses. Moreover, this study indicated that both IFNγ and IL-17 are required for the elicitation and development of murine lupus.

Suppression of Th17 cell differentiation via sphingosine-1-phosphate receptor 2 by cinnamaldehyde can ameliorate ulcerative colitis.

Ulcerative colitis (UC) is chronic disease characterized by diffuse inflammation of the mucosa of the colon and rectum. Although the etiology is unknown, dysregulation of the intestinal mucosal immune system is closely related to UC. Cinnamaldehyde (CA) is a major active compound from cinnamon, is known as its anti-inflammatory and antibacterial. However, little research focused on its regulatory function on immune cells in UC. Therefore, we set out to explore the modulating effects of CA on immune cells in UC. We found that CA reduced the progression of colitis through controlling the production of proinflammatory cytokines and inhibiting the proportion of Th17 cells. Furthermore, the liquid chromatography-mass spectrometry (LC-MS) method was employed for analyzing and differentiating metabolites, data showed that sphingolipid pathway has a great influence on the effect of CA on UC. Meanwhile, sphingosine-1-phosphate receptor 2 (S1P2) and Rho-GTP protein levels were downregulated in colonic tissues after CA treatment. Moreover, in vitro assays showed that CA inhibited Th17 cell differentiation and downregulated of S1P2 and Rho-GTP signaling. Notably, we found that treatment with S1P2 antagonist (JTE-013) weakened the inhibitory effect of CA on Th17 cells. Furthermore, S1P2 deficiency (S1P2-/-) blocked the effect of CA on Th17 cell differentiation. In addition, CA can also improve inflammation via lncRNA H19 and MIAT. To sum up, this study provides clear evidence that CA can ameliorate ulcerative colitis through suppressing Th17 cells via S1P2 pathway and regulating lncRNA H19 and MIAT, which further supports S1P2 as a potential drug target for immunity-mediated UC.

(5R)-5-hydroxytriptolide inhibits the immune response of human peripheral blood mononuclear cells.

AIM: (5R)-5-hydroxytriptolide (LLDT-8) displayed immunosuppressive activities both in vitro and in autoimmune disease models. Here, we aim to further clarify the effect of LLDT-8 on the immune responses of human peripheral blood mononuclear cells (PBMC). METHOD: Cell proliferation of human PBMC from healthy donors was evaluated by [3H]-thymidine uptake. NK cell cytotoxicity was assayed using K562 cells in a [3H] lysis assay. Cytokine production was determined by enzyme-linked immunosorbent assay. The expression of cell surface molecules was detected with flow cytometry. The mRNA expression and the protein phosphorylation levels were detected by RT-PCR and Western immunoblot assay. RESULTS: LLDT-8 at 25 and 50 nM significantly inhibited the PHA- and recall antigens-induced T cell proliferation, and suppressed mixed lymphocyte reaction. LLDT-8 reduced cytokines production (IFN-gamma, IL-2, TNF-alpha) in PHA- and Sac-activated PBMC. LLDT-8 did not alter the increased expression of MHC class I/II and B7.1, but reduced B7.2 by approximately 30%. No effect of LLDT-8 was observed for the expression of T cell activation markers (CD69, CD154). However, LLDT-8 significantly reduced IFN-gamma-expressing T cell percentages and IFN-gamma mRNA transcription in PHA-activated T cells. It also inhibited the phosphorylation levels of JNK and p38. LLDT-8 did not affect NK cytotoxic activity against K562 cells. CONCLUSION: LLDT-8 was a promising immunosuppressant for human immune-related diseases.

Periplocoside A prevents experimental autoimmune encephalomyelitis by suppressing IL-17 production and inhibits differentiation of Th17 cells.

AIM: The aim of this study was to determine the therapeutic effect of Periplocoside A (PSA), a natural product isolated from the traditional Chinese herbal medicine Periploca sepium Bge, in MOG(35-55) (myelin oligodendrocyte glycoprotein 35-55)-induced experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice immunized with MOG(35-55) were treated with (50 mg/kg or 25 mg/kg) or without PSA following immunization and continuously throughout the study. The degree of CNS inflammation was evaluated by H&E staining. Anti-MOG-specific recall responses were analyzed by [3H]-Thymidine incorporation, ELISA, and RT-PCR. The proportion of IL-17-producing T cells was measured by flow cytometry. RESULTS: Oral administration of PSA significantly reduced the incidence and severity of EAE, which closely paralleled the inhibition of MOG(35-55)-specific IL-17 production. Importantly, PSA inhibited the transcription of IL-17 mRNA and RORgammat. Further studies examining intracellular staining and adoptive transfer EAE validated the direct suppressive effect of PSA on Th17 cells. In vitro studies also showed that PSA significantly inhibited the differentiation of Th17 cells from murine purified CD4+ T cells in a dose-dependent manner. CONCLUSION: PSA ameliorated EAE by suppressing IL-17 production and inhibited the differentiation of Th17 cells in vitro. Our results provide new insight into the potential mechanisms underlying the immunosuppressive and anti-inflammatory effects of PSA.

Periplocoside A, a pregnane glycoside from Periploca sepium Bge, prevents concanavalin A-induced mice hepatitis through inhibiting NKT-derived inflammatory cytokine productions.

Periploca sepium Bge, a traditional Chinese herb medicine, is widely used for treating rheumatoid arthritis in china. Periplocoside A (PSA), a pregnane glycoside, is a new nature product compound isolated from P. sepium Bge. We examined the protective effects of PSA, on concanavaline A (ConA)-induced hepatitis. Pretreatment with PSA dramatically ameliorated ConA-induced liver injury, which was characterized by reducing serum alanine transaminase (ALT), pathogenic cytokines of interleukin (IL)-4 and interferon (IFN)-gamma levels, impeding the liver necrosis, and thus elevating the survival rate. In vitro, PSA inhibited IL-4 and IFN-gamma productions of alpha-galactosylceramide (alpha-GalCer) or anti-CD3-activated Natural killer T (NKT) cells. Enzyme Linked Immunosorbent Assay (ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays revealed PSA suppressed IL-4 transcription and IFN-gamma translation. In conclusion, PSA had significantly preventative effect on ConA-induced hepatitis, which was closely associated with inhibition of NKT-derived inflammatory cytokine productions. These findings suggested that PSA has the therapeutic potential for treatment of human autoimmune-related hepatitis.

Anti-inflammatory effects of Z23 on LPS-induced inflammatory responses in RAW264.7 macrophages.

AIM OF THE STUDY: Fissistigma oldhamii (Hemsl.) Merr, a traditional Chinese herb medicine, is used for treating rheumatoid arthritis in China. In our previous study, an effective compound, 7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl) ethyl] propenamide (Z23), from this herb has showed potent immunosuppressive effects both in vitro and in vivo. However, its anti-inflammatory effect and mechanism is still need to explore. MATERIALS AND METHODS: We examined the in vitro effects of Z23 on the production of nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines by lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. RESULTS: Z23 significantly decreased the production of PGE2, NO, tumour necrosis factor alpha (TNFalpha) and IL6 production. Inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX2) gene expression were also significantly reduced. CONCLUSIONS: These results demonstrated that Z23 exerted an anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process. This study provided evidence to understand the therapeutic effects of Fissistigma oldhamii (Hemsl.) Merr and indicated that Z23 has the potential for treatment of various inflammatory diseases where the overproduction of NO, PGE2 and inflammatory cytokines has been shown to play a role, e.g. rheumatoid arthritis.

COX-2 inhibitors ameliorate experimental autoimmune encephalomyelitis through modulating IFN-gamma and IL-10 production by inhibiting T-bet expression.

The COX-2 inhibitors Rofecoxib (Rof) and Lumiracoxib (Lum) were evaluated in experimental autoimmune encephalomyelitis (EAE), the model of multiple sclerosis (MS). Administration of Rof and Lum significantly reduced the incidence and severity of EAE, which was associated with the inhibition of MOG 35-55 lymphocyte recall response, anti-MOG 35-55 T cell responses, and modulation of cytokines production. In vitro Rof and Lum inhibited primary T cells proliferation and modulated cytokine production. These findings highlight the fact that Rof and Lum likely prevents EAE by modulating Th1/Th2 response, and suggest its utility in the treatment of MS and other autoimmune diseases.

Suppressive effect of a novel water-soluble artemisinin derivative SM905 on T cell activation and proliferation in vitro and in vivo.

Artemisinin and its derivatives exhibit potent immunosuppressive activity. The aim of this study was to investigate the suppressive effects of SM905, a new water-soluble artemisinin derivative, on T lymphocytes both in vitro and in vivo, and explore its potential mode of action. The results showed that SM905 had a high inhibitory activity in Concanavalin A (ConA)-induced splenocyte proliferation and mixed lymphocyte reaction, and a relatively low cytotoxicity in vitro. In ovalbumin-immunized mice, oral administration of SM905 dose-dependently suppressed T cell proliferative response to ovalbumin, and inhibited anti-ovalbumin interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by T cells. Further studies showed that SM905 inhibited TCR (T cell receptor)/CD3 plus CD28-mediated primary T cell proliferation and cytokine production (IL-2 and IFN-gamma), and exerted an inhibitory action on the phosphorylation of mitogen-activated protein (MAP) kinases including extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK), and the activation of Ras. The results of this study provided experimental evidence that the new artemisinin derivative SM905 had immunosuppressive effects both in vitro and in vivo. SM905 suppressed T cell activation, which was associated with the inhibition of MAP kinases and Ras activation. Our results suggested a potential of SM905 to be developed as a new type agent for treating T cell-mediated immune disorder.

7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl)ethyl]propenamide (Z23), an effective compound from the Chinese herb medicine Fissistigma oldhamii (Hemsl.) Merr, suppresses T cell-mediated immunity in vitro and in vivo.

Fissistigma oldhamii (Hemsl.) Merr [F. oldhamii], a traditional Chinese herb medicine, is widely used for treating rheumatoid arthritis (RA) in China. Following bioactivity-guided isolation, a representative immunosuppressive compound with low cytotoxicity, 7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl)ethyl]propenamide (Z23), was been identified in this herb medicine. We investigated the immunosuppressive effects of Z23 on T cells in vitro and in vivo. The results showed that Z23 in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A (ConA) and by the mixed lymphocyte culture reaction (MLR), with half inhibitive concentration (IC(50)) values of 6.22 microM and 0.78 microM, respectively. Z23 also dose-dependently inhibited the proliferation and type 1 cytokine (IFN-gamma and IL-2) production of primary T cells stimulated by anti-CD3/CD28 mAbs, but did not affect IL-12 production by mouse peritoneal macrophages (pMphi) stimulated with LPS plus IFN-gamma in vitro. Administration of Z23 (6.25 mg/kg, 12.5 mg/kg, 25 mg/kg, i.p.) dose-dependently suppressed 2,4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) reactions. Furthermore, administration of Z23 (25 mg/kg, i.p.) significantly reduced the incidence and severity of type II bovine collagen (CII)-induced arthritis (CIA), which was associated with the inhibition of CII-specific T cell proliferation and type 1 cytokine (IFN-gamma and IL-2) production. In this study, we report that a representative immunosuppressive compound from F. oldhamii, Z23, effectively inhibits murine immune responses in vitro and in vivo, and that the immunosuppressive effects of Z23 might be attributed to suppression of T cell activation and function and Th1 type cytokine production.

Suppression of (5R)-5-hydroxytriptolide (LLDT-8) on allograft rejection in full MHC-mismatched mouse cardiac transplantation.

BACKGROUND: (5R)-5-hydroxytriptolide (LLDT-8) is a new compound derived from triptolide, which is the major immunosuppressive fraction of Tripterygium wilfordii Hook. F (TWHF). Studies in vitro and in vivo have demonstrated that LLDT-8 had potent immunosuppressive activities. Here we tested LLDT-8 in major histocompatibility complex (MHC)-mismatched cardiac transplantation and investigated the mechanisms underlying the prevention of transplant rejection. METHODS: LLDT-8 was administered orally to recipients in Balb/c to C57BL/6 murine cardiac transplantation model. Allograft survival after transplantation was recorded in recipients. The T cell immunity and cytokine production were observed. Histological analysis was performed. The chemokine and its receptor were analyzed by reverse transcriptase-polymerase chain reaction on cardiac graft RNA. RESULTS: LLDT-8 administered orally significantly induced the survival prolongation of allogeneic cardiac graft. Histological results showed that LLDT-8 well preserved myocardium and significantly reduced infiltration of the graft with inflammatory cells. LLDT-8 decreased IL-2 production in recipient splenocytes stimulated by concanavalin A (ConA) ex vivo. LLDT-8 significantly inhibited the immunoreactivity of recipient to specific donor alloantigens, but preserved immunity to third-party alloantigens and mitogen. However, the flow cytometry analysis of the proportion of CD4+, CD8+ T cell subgroup in recipient spleens showed LLDT-8 had a normalizing effect on the splenic lymphocytes population. LLDT-8 decreased CC chemokine receptor 5 (CCR5) and their ligands macrophage inflammatory protein 1 alpha (MIP-1alpha) and beta (MIP-1beta) mRNA expressions in allografts. CONCLUSION: The results outline the great potential of LLDT-8 as a therapeutic tool in transplant rejection.

(5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative, prevents experimental autoimmune encephalomyelitis via inhibiting T cell activation.

A novel triptolide derivative (5R)-5-hydroxytriptolide (LLDT-8) has been shown to have potent immunosuppressive activities. Here LLDT-8 was evaluated in experimental autoimmune encephalomyelitis (EAE), the model of multiple sclerosis (MS). LLDT-8 reduced the incidence and severity of EAE, which was associated with the inhibition of the MOG 35-55 lymphocyte recall response, anti-MOG 35-55 T cell responses, interleukin (IL)-2 and interferon (IFN)-gamma production. In vitro, LLDT-8 inhibited primary T cells proliferation, division, IL-2 and IFN-gamma production stimulated with anti-CD3/28. These findings highlight the fact that LLDT-8 prevents EAE by suppressing T cell proliferation and activation, with a potential for treatment of MS.

Preventive effects of (5R)-5-hydroxytriptolide on concanavalin A-induced hepatitis.

(5R)-5-hydroxytriptolide (LLDT-8) exhibits strong immunosuppressive activities in vitro and in vivo. Here, we investigated the effects of LLDT-8 on concanavalin A-induced hepatitis. Liver damage was evaluated by serum alanine transaminase (ALT) level and liver histology. The effects of LLDT-8 were determined by measurement of serum cytokines, lymphocyte proliferation assay, flow cytometry analysis of splenic T cell percentage and apoptosis, reverse-transcription polymerase chain reaction (RT-PCR) analysis for gene transcriptions. In LLDT-8-treated mice, serum ALT level and histological damage were markedly attenuated. The beneficial effect of LLDT-8 was closely associated with (i) reduction of serum tumor necrosis factor-alpha, interferon-gamma (IFN-gamma), interleukin-2, interleukin-12, and interleukin-6 levels; (ii) elimination of activated T cells by increasing proapoptotic genes signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor-1 (IRF-1) expression in spleens; (iii) blockade of mRNA expressions for chemokines (monokine induced by IFN-gamma, Mig; IFN-gamma-inducible protein-10, IP-10; IFN-inducible T cell-alpha chemoattractant, I-TAC), vascular adhesion molecule-1 (VCAM-1), and chemokine receptors (C-C chemokine receptor 1, CCR1; C-C chemokine receptor 5, CCR5; C-X-C chemokine receptor 3, CXCR3) in livers. These results suggested the therapeutic potential of LLDT-8 in IFN-gamma/STAT1/IRF-1 signaling- and inflammatory cytokines-mediated immune disorders.

[Simulation on area threshold of urban building land based on water environmental response in watersheds.] / 基于流域水环境响应的城市建设用地面积阈值模拟.

Urban sprawl has impacted increasingly on water environment quality in watersheds. Based on water environmental response, the simulation and prediction of expanding threshold of urban building land could provide an alternative reference for urban construction planning. Taking three watersheds (i.e., Yundang Lake at complete urbanization phase, Maluan Bay at peri-urbanization phase and Xinglin Bay at early urbanization phase) with 2009-2012 observation data as example, we calculated the upper limit of TN and TP capacity in three watersheds and identified the threshold value of urban building land in watersheds using the regional nutrient management (ReNuMa) model, and also predicted the water environmental effects associated with the changes of urban landscape pattern. Results indicated that the upper limit value of TN was 12900, 42800 and 43120 kg, while that of TP was 340, 420 and 450 kg for Yundang, Maluan and Xinglin watershed, respectively. In reality, the environment capacity of pollutants in Yundang Lake was not yet satura-ted, and annual pollutant loads in Maluan Bay and Xinglin Bay were close to the upper limit. How-ever, an obvious upward trend of annual TN and TP loads was observed in Xinglin Bay. The annual pollutant load was not beyond the annual upper limit in three watersheds under Scenario 1, while performed oppositely under Scenario 3. Under Scenario 2, the annual pollutant load in Yundang Lake was under-saturation, and the TN and TP in Maluan Bay were over their limits. The area thresholds of urban building land were 1320, 5600 and 4750 hm2 in Yundang Lake, Maluan Bay and Xinglin Bay, respectively. This study could benefit the regulation on urban landscape planning.

Prevention of graft-versus-host disease by a novel immunosuppressant, (5R)-5-hydroxytriptolide (LLDT-8), through expansion of regulatory T cells.

(5R)-5-hydroxytriptolide (LLDT-8) is a new compound derived from triptolide, which is the major immunosuppressive fraction of Tripterygium wilfordii Hook. F (TWHF). In this study, we demonstrated that administration of LLDT-8 (1 mg/kg/day, p.o.) effectively prevented weight loss and death induced by allo-BMT (BLAB/c, H-2d to C57BL/6, H-2b), and extended survival in allo-BMT model of aGVHD. Following days 7 to 28 after allo-BMT, the allogeneic graft survived by increasing the number of engrafted cells (H-2d) in spleens of recipient mice with LLDT-8 treatment. To construe the immunosuppressive effects of LLDT-8, the splenocytes (H-2d) of LLDT-8 treated recipients (H-2b) were tested for the proliferative responses to donor antigen (H-2d), host antigen (H-2b) and mitogen (ConA) stimulations, respectively, the results indicated that LLDT-8 induced the T cells' unresponsiveness to donor and host antigens, while still maintaining response to ConA; Compared with the vehicle group of GVHD mice, administration of LLDT-8 significantly inhibited T cells to produce IFN-gamma with or without host antigen or ConA stimulation. Further studies indicated LLDT-8 had a normalizing effect on the ratio of CD4+/CD8+ T cells, and increased CD4+CD25+ T regulatory cells with the Foxp3 expression in splenocytes from LLDT-8 treated mice. The results outline the great potential of LLDT-8 as a therapeutic tool to induce suppression in GVHD.

(5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide analog mediates immunosuppressive effects in vitro and in vivo.

A series of triptolide analogs have been successfully synthesized. In the present study we demonstrated one of them, (5R)-5-hydroxytriptolide (LLDT-8), showed low cytotoxicity and relative high immunosuppressive activities as compared with its parent compound triptolide in vitro. The CC50 values of triptolide and LLDT-8 were 2.1+/-0.3 and 256.6+/-73.8 nM, respectively. LLDT-8 significantly inhibited the proliferation of splenocytes induced by concanavalin A (ConA), lipopolysaccharide (LPS), or mixed lymphocyte reaction (MLR), and the IC50 values were 131.7+/-32.4, 171.5+/-17.3, and 38.8+/-5.1 nM, respectively. LLDT-8 (25, 50, 100 nM) dose-dependently reduced the production of Th1 type cytokines (IFN-gamma, IL-2) and inflammatory cytokines (TNF-alpha, IL-6) in vitro. Administration of LLDT-8 (at the low dose of 0.4 microg/kg, i.p.; 40 microg/kg, p.o.) intensively suppressed 2,4-dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity (DTH) reactions. Treatment with LLDT-8 (40 microg/kg, i.p. and p.o.) also markedly inhibited the sheep red blood cell (SRBC)-induced antibody production in BLAB/c mice. Most importantly, comparing with triptolide, LLDT-8 significantly reduced toxicity, with a 122-fold lower cytotoxicity in vitro and 10-fold lower acute toxicity in vivo. The results suggested that LLDT-8 had immunosuppressive activities in both cellular and humoral immune responses. LLDT-8 might be a potential therapeutic agent for immune-related diseases.

The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3.

CC chemokine receptor (CCR) 5 and CXC chemokine receptor (CXCR)3 are expressed on T helper cell type 1 cells and have been implicated in their migration to sites of inflammation. Our preceding study demonstrated that a nonpeptide synthetic CCR5 antagonist, TAK-779 (N, N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbon-yl]amino]benzyl]-tetrahydro-2H-pyran4-aminium chloride, inhibits the development of experimentally induced arthritis by modulating the migration of CCR5(+)/CXCR3(+) T cells to joints. The present study investigated the functional properties of TAK-779, including the effect of this antagonist on CXCR3 function. For this purpose, transfectants expressing mouse CCR5 (mCCR5) or mCXCR3 and expressing mCCR4 or mCXCR4 as controls were established by introducing each relevant gene into 2B4 T cells and were subjected to the following assays. First, the ligand binding to chemokine receptors was assayed by incubating transfectants with [(125)I]-labeled relevant ligand or with the unlabeled relevant ligand followed by staining with anti-ligand antibody. Second, chemokine-induced lymphocyte function-associated antigen-1 (LFA-1) activation was assayed by measuring the adhesion of cells to microculture plates coated with purified intercellular adhesion molecule-1. Third, chemokine-stimulated chemotaxis was assayed by observing the cell migration through transwells. In these assays, TAK-779 blocked the ligand binding as well as LFA-1 up-regulating and chemotactic function of mCXCR3 and mCCR5 but did not elicit a biologically significant inhibition of those functions of mCCR4 and mCXCR4. These observations indicate the unique target specificity of TAK-779 and explain why this antagonist efficiently blocks the migration of T cells expressing CCR5 and CXCR3 to sites of inflammation.