Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

Quercetin Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis by Inhibiting the Glucose Regulatory Protein 78 Activation.

Background: The inflammatory bowel diseases (IBD) are significantly influenced by apoptosis and endoplasmic reticulum (ER) stress. Aims: To investigate the effects of quercetin on ER stress-mediated apoptosis in a trinitrobenzene sulfonic acid (TNBS) induced experimental IBD model. Study Design: In vivo animal experimental study. Methods: To demonstrate the effect of quercetin in an experimental colitis model, Control, TNBS, and TNBS+quercetin groups were created with 24 Wistar Albino rats. Colitis was induced by intrarectal administration of 25 mg TNBS. In the TNBS+quercetin group, intragastrically 100 mg/kg quercetin was given for 7 days, immediately after colitis induction. In the TNBS-induced experimental IBD model, we evaluated the effects of quercetin on colonic epithelial cell apoptosis, oxidative stress, ER stress, the mitogen-activated protein kinase c-Jun N-terminal kinase, and the nuclear factor kappa B immunoreactivities, the levels of myeloperoxidase and tumor necrosis factor-α, the disease activity index with colonic histopathologic changes. Results: TNBS administration induced an elevated level of disease activity and oxidative stress indices, inflammation markers, and an increase in the immunoreactivities of nuclear factor kappa B and the mitogen-activated protein kinase c-Jun N-terminal kinase in the colon of the colitis group. Glucose regulatory protein 78, caspase-12 immunoreactivities, and epithelial cell apoptosis also were shown in the colon. However, quercetin improved TNBS-induced histopathological alterations, apoptosis, inflammation, oxidative stress, and ER stress. Conclusion: This study suggests that quercetin has a regulatory effect on ER stress-mediated apoptosis, and thus may be beneficial in treating IBD.

A Mixed-ligand Zn(II)-based Coordination Polymer for Selectively Detect 2,4,6-Trinitrophenol (TNP) and Treatment Effect on Periodontal Diseases via Inhibition Effect on P. gingivalis Growth.

A new difunctional Zn(II) coordination polymer (CP) with the chemical formula of [Zn(TBTA) (L)1.5]n (1) has been synthesized hydrothermally from tetrabromoterephthalic acid (H2TBTA) and 4,4'-bis(imidazole-1-yl)-biphenyl (L) ligands. Furthermore, due to its strong intense emission and open N donor sites, complex 1 could be used as a light-emitting sensor to determine 2,4,6-trinitrophenol (TNP) which has high selectivity and sensitivity. Furthermore, the anti-bacterial effect of the compound against P. gingivalis in vitro was evaluated by measuring the P. gingivalis growth curves after compound treatment. And the RT-PCR assay was performed to detect the relative expression of ragA and ragB, which are important for the P. gingivalis growth. The potential anti-infectious mechanism was further studied by using molecular docking technique.

Intravenously administered contact allergens coupled to syngeneic erythrocytes induce in mice tolerance rather than effector immune response.

Constantly increasing prevalence of allergic diseases determines the attempts to elaborate the therapeutic strategies activating immune tolerance to particular allergen. Our current research focuses on the antigen-specific action of CD8+ suppressor T (Ts) lymphocytes induced in mice by intravenous administration of a high dose of haptenated syngeneic erythrocytes. While the regulatory activity of Ts cells mediated by exosome-delivered miRNA-150 is well de ned, the mechanism of their induction remained unclear. Therefore, the current studies investigated the immune e ects induced in mice by intravenous administration of contact allergens coupled to syngeneic erythrocytes. In mouse models of hapten-induced contact hypersensitivity (CHS) and delayed-type hypersensitivity to ovalbumin, we have shown that intravenous administration of hapten-coupled erythrocytes failed to induce CHS effector cells. Moreover, hapten-induced CHS reaction occurred to be suppressed in mice intravenously administered with syngeneic erythrocytes coupled with protein allergen. Finally, we have demonstrated that intravenously administered allergen induces immune tolerance only when bound to syngeneic erythrocytes, proving that intravenously delivered allergens are deprived of their immunizing properties when coupled with membrane of self cells. Altogether, our current studies suggest that alteration of self cell membrane by allergen binding is enough to induce Ts cell-mediated immune tolerance to nonpathogenic agents, which express a great translational potential in such conditions as allergies and hypersensitivity-related autoimmune disorders.

Cyclophosphamide-modified murine peritoneal macrophages induce CD4+ T contrasuppressor cells that protect contact sensitivity T effector cells from suppression.

BACKGROUND: Cyclophosphamide (CY) is one of the most widely used alkylating agents in the treatment of various cancers and some autoimmune diseases. Numerous reports suggest that CY exerts immunoregulatory effects. Animal studies have shown CY affects contact sensitivity (CS) response by depleting CD4+CD25+ T regulatory cells and CD8+ T suppressor (Ts) cells. In a mouse model of CS, we previously showed that in vivo treatment with CY shapes the immunogenic/immunoregulatory balance of peritoneal macrophages. The aim of the current study is to verify if macrophages (Mf) from CY-treated mice are indeed able to induce immunoregulatory cells that could protect from suppression. METHODS: Adoptive cell transfer of CS was used to examine immunomodulating properties of peritoneal Mf from CY-treated mice. Isolation of peritoneal Mf from animals that were (Mf-CY) or were not (Mf) treated with CY were cultured to identify cytokine repertoire. Further, we assessed spleen cell (SPLC) cytokine production following immunization with trinitrophenyl-conjugated Mf from donors treated (TNP-Mf-CY) or non-treated (TNP-Mf) with CY. RESULTS: In vitro experiments identified that Mf-CY produce more IL-6, TNF-α and TGF-ß than naïve Mf. Further, immunization with peritoneal TNP-Mf-CY induces CD4+ T contrasuppressor cells (Tcs) cells that protect CS-effector cells from suppression. Higher IL-17A secretion was observed from TNP-Mf-CY-treated mouse SPLC compared to SPLC from TNP-Mf injected mice suggesting that this cytokine might be important in mediating contrasuppression in this model. CONCLUSIONS: Our results show that in vivo treatment with CY influences mouse peritoneal Mf to induce CD4+ Tcs cells that protect CS-effector cells from suppressive signals of Ts cells.

Methods of Inducing Inflammatory Bowel Disease in Mice.

Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of the disease, identifying new targets for therapeutic intervention, and testing novel therapeutics. This unit provides detailed protocols for five widely used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten-induced colitis via 2,4,6-trinitrobenzene sulfonic acid (TNBS), Helicobacter-induced colitis in mdr1a(-/-) mice, the CD4(+) CD45RB(hi) SCID transfer colitis model, and the IL-10(-/-) colitis model. © 2016 by John Wiley & Sons, Inc.

Generation of reactive oxygen species in thylakoids from senescing flag leaves of the barley varieties Lomerit and Carina.

MAIN CONCLUSION: During senescence, production of reactive oxygen species increased in thylakoids. In two barley varieties, no difference in superoxide production was observed while singlet oxygen production increased only in one variety. During senescence, chlorophyll content decreased and photosynthetic electron transport was inhibited as shown for flag leaves collected from barley varieties Lomerit and Carina grown in the field. Spin trapping electron paramagnetic resonance (EPR) was used to investigate the production of reactive oxygen species in thylakoid membranes during senescence. EPR measurements were performed with specific spin traps to discriminate between singlet oxygen on one hand and reactive oxygen intermediates on the other hand. The results show that the generation of reactive oxygen intermediates increases in both varieties during senescence. Singlet oxygen increased only in the variety cv. Lomerit while it remained constant at a low level in the variety cv. Carina. Measurements in the presence of inhibitors of photosystem II and of the cytochrome b6f complex revealed that in senescing leaves reduction of oxygen at the acceptor side of photosystem I was the major, but not the only source of superoxide anions. This study shows that during senescence the production of individual reactive oxygen species varies in different barley varieties.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for peripheral B-cell development and function.

The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.

Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens.

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.

Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirin-triggered resolvin D1 and resolvin D2 prevent experimental colitis in mice.

Resolvins of the D series are generated from docosahexaenoic acid, which are enriched in fish oils and are believed to exert beneficial roles on diverse inflammatory disorders, including inflammatory bowel disease (IBD). In this study, we investigated the anti-inflammatory effects of the aspirin-triggered resolvin D1 (AT-RvD1), its precursor (17(R)-hydroxy docosahexaenoic acid [17R-HDHA]) and resolvin D2 (RvD2) in dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Our results showed that the systemic treatment with AT-RvD1, RvD2, or 17R-HDHA in a nanogram range greatly improved disease activity index, body weight loss, colonic damage, and polymorphonuclear infiltration in both colitis experimental models. Moreover, these treatments reduced colonic cytokine levels for TNF-α, IL-1ß, MIP-2, and CXCL1/KC, as well as mRNA expression of NF-κB and the adhesion molecules VCAM-1, ICAM-1, and LFA-1. Furthermore, AT-RvD1, but not RvD2 or 17R-HDHA, depended on lipoxin A4 receptor (ALX) activation to inhibit IL-6, MCP-1, IFN-γ, and TNF-α levels in bone marrow-derived macrophages stimulated with LPS. Similarly, ALX blockade reversed the beneficial effects of AT-RvD1 in DSS-induced colitis. To our knowledge, our findings showed for the first time the anti-inflammatory effects of resolvins of the D series and precursor 17R-HDHA in preventing experimental colitis. We also demonstrated the relevant role exerted by ALX activation on proresolving action of AT-RvD1. Moreover, AT-RvD1 showed a higher potency than 17R-HDHA and RvD2 in preventing DSS-induced colitis. The results suggest that these lipid mediators possess a greater efficacy when compared with other currently used IBD therapies, such as monoclonal anti-TNF, and have the potential to be used for treating IBD.

Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis.

Transforming growth factor ß (TGF-ß) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-ß signal transduction is through TGF-ß receptors, including the TGF-ß type 1 receptor. Most cell types contain a TGF-ß type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-ß/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-ß1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-ß1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-ß/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.

The murine equivalent of the A181E TACI mutation associated with common variable immunodeficiency severely impairs B-cell function.

TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NF kappaB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI(-/-) background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.

Characterization of the cell-mediated cytotoxic responses of isogeneic ginbuna crucian carp induced by oral immunisation with hapten-modified cellular antigens.

A better understanding of the immune responses in fish elicited by oral immunisation is of importance for the development of new and effective oral vaccines for cultured fish. In the present study, we characterized specific cell-mediated cytotoxic responses in isogeneic ginbuna crucian carp (Carassius auratus langsdorfii) following oral immunisation with cellular antigens. Trinitrophenyl- (TNP) or dinitrophenyl- (DNP) modified syngeneic and allogeneic cells were used for studying the fine specificity and genetic restriction of orally-induced cytotoxic cells. Hapten-specific cytotoxic responses were detected in peripheral blood leukocytes (PBLs) of fish orally immunised with haptenated syngeneic cells. PBLs from orally immunised fish had cytolytic activity for haptenated syngeneic cells, but they showed little reactivity against both haptenated and unmodified allogeneic targets. Similarly, oral immunisation of fish with hapten-modified allogeneic cells did not induce hapten-specific cytotoxic cells which can lyse haptenated syngeneic targets. Although ginbuna crucian carp possess spontaneous cytotoxic cells that are capable of killing mammalian tumour cells, cold target inhibition studies suggested that such spontaneous cytotoxic cells were not involved in the killing of haptenated syngeneic targets. Oral immunisation of fish with haptenated syngeneic cells also induced hapten-specific cytotoxic memory responses. Oral administration of haptenated fixed cells also effectively induced hapten-specific cytotoxic cells in the treated fish. These findings suggest that oral immunisation with antigens can elicit antigen-specific cytotoxic cells that are capable of recognizing antigens in an MHC-restricted manner. In addition, our results provide indirect evidence that fish possess a mechanism for taking up exogenous non-replicating antigens from the alimentary tract and generating antigen-specific cytotoxic cells.

H2O2 intensifies CN(-)-induced apoptosis in pea leaves.

H2O2 intensifies CN(-)-induced apoptosis in stoma guard cells and to lesser degree in basic epidermal cells in peels of the lower epidermis isolated from pea leaves. The maximum effect of H2O2 on guard cells was observed at 10(-4) M. By switching on non-cyclic electron transfer in chloroplasts menadione and methyl viologen intensified H2O2 generation in the light, but prevented the CN--induced apoptosis in guard cells. The light stimulation of CN- effect on guard cell apoptosis cannot be caused by disturbance of the ribulose-1,5-bisphosphate carboxylase function and associated OH* generation in chloroplasts with participation of free transition metals in the Fenton or Haber-Weiss type reactions as well as with participation of the FeS clusters of the electron acceptor side of Photosystem I. Menadione and methyl viologen did not suppress the CN(-)-induced apoptosis in epidermal cells that, unlike guard cells, contain mitochondria only, but not chloroplasts. Quinacrine and diphenylene iodonium, inhibitors of NAD(P)H oxidase of cell plasma membrane, had no effect on the respiration and photosynthetic O2 evolution by leaf slices, but prevented the CN(-)-induced guard cell death. The data suggest that NAD(P)H oxidase of guard cell plasma membrane is a source of reactive oxygen species (ROS) needed for execution of CN(-)-induced programmed cell death. Chloroplasts and mitochondria were inefficient as ROS sources in the programmed death of guard cells. When ROS generation is insufficient, exogenous H2O2 exhibits a stimulating effect on programmed cell death. H2O2 decreased the inhibitory effects of DCMU and DNP-INT on the CN(-)-induced apoptosis of guard cells. Quinacrine, DCMU, and DNP-INT had no effect on CN(-)-induced death of epidermal cells.

Ghrelin stimulates interleukin-8 gene expression through protein kinase C-mediated NF-kappaB pathway in human colonic epithelial cells.

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.

A phosphorylation site in Bruton's tyrosine kinase selectively regulates B cell calcium signaling efficiency by altering phospholipase C-gamma activation.

Loss of function of Bruton's tyrosine kinase (Btk) causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptide-specific antibodies, we identified a tyrosine phosphorylation site (Y617) near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppressed Btk-mediated phospholipase Cgamma2 activation and calcium response in DT-40 cells, whereas Akt activation was not affected. The Btk Y617E mutant could partially restore conventional B cell development and proliferation in Btk(-)/Tec(-) mice but failed to rescue CD5(+) B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway.

Participation of chloroplasts in plant apoptosis.

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN(-)-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN(-)-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN- effect. The light-dependent activation of CN(-)-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.

Cytochrome c is reduced mainly by plastoquinol and not by superoxide in thylakoid membranes at low and medium light intensities: its specific interaction with thylakoid membrane lipids.

We have found that, at low light intensity (5-10 micromol photons x m(-2) x s(-1)), photoreduction of cyt (cytochrome) c by isolated thylakoids was not inhibited by dinitrophenylether of iodonitrothymol, an inhibitor of the cyt b6- f complex, and the inhibition was only partial at medium light intensity (50-200 micromol photons x m(-2) x s(-1)). The photoreduction was not significantly influenced by superoxide dismutase. The conclusion that cyt c could be reduced directly by the plastoquinone pool was confirmed by the observation that plastoquinol-9 reduced cyt c efficiently when it was incorporated into liposome membranes prepared from thylakoid membrane lipids. It was shown that the cyt is specifically bound to thylakoid lipid liposomes owing to the presence of negatively charged lipids, phosphatidylglycerol and sulphoquinovosyldiacylglycerol, and the reduction was stimulated by the presence of monogalactosyldiacylglycerol, an inverted micelles-forming lipid, in the membranes, where the cyt c reduction by plastoquinol probably takes place. The results obtained are also discussed in terms of reliability of the method of cyt c photoreduction for determining superoxide production by illuminated thylakoids.

T-bet regulates T-independent IgG2a class switching.

The IgG2a Ig subclass plays a critical role in the pathogenesis of humoral autoimmunity and protection against pathogens. The T-box transcription factor T-bet has been implicated as a critical mediator of class-switch recombination (CSR) to IgG2a, but its relative importance to this process in various immune contexts remains incompletely defined. We report here that, surprisingly, T-bet is selectively required for IgG2a class switching in response to T-independent, but not T-dependent, stimuli. Specifically, T-dependent signaling through CD40, in contrast to T-independent signaling via lipopolysaccharide, can bypass a requirement for T-bet in IgG2a germline transcription and subsequent isotype switching. In contrast, T-bet-deficient B cells undergo class switching to other IgG isotypes at least as well as wild-type counterparts. Thus, T-bet is a class-specific regulator of IgG CSR and represents a unique regulator of B cell differentiation by participating in a T-independent, but not a T-dependent, activation pathway. T-bet-deficient B cells therefore represent a novel paradigm by which to investigate the regulation of humoral immune responses.

A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation.

S-Adenosyl-L-methionine (AdoMet) which is biologically synthesized by AdoMet synthetase bears an S configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of S and R isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R isomer is not accepted as a substrate for AdoMet-dependent methyltransferases. We report a stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (S,S)-AdoMet reacts with 2-nitro-5-thiobenzoic acid to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and S-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 microM (S,S)-AdoMet.