Efficacy of alphas1-casein hydrolysate on stress-related symptoms in women.

OBJECTIVE: To examine the effects of alpha (s1)-casein hydrolysate on females with stress-related symptoms. DESIGN: Double-blind, randomized, crossover, placebo-controlled trial. SETTING: The alpha (s1)-casein hydrolysate was manufactured by INGREDIA (Arras, France) and the placebo was manufactured by DIETAROMA (Bourg, France). Study was designed and performed at PROCLAIM (Rennes, France), and the statistical analyses were performed by D Desor (Nancy, France). SUBJECTS: A total of 63 female volunteers suffering from at least one disorder that may be related to stress such as anxiety, sleep problems and general fatigue. INTERVENTIONS: A total of 63 volunteers participated in a double-blind, randomized, crossover, placebo-controlled study. Subjects were randomly allocated to receive either tablets containing alpha (s1)-casein hydrolysate or placebo at the dose of 150 mg/day for 30 days. After a 3 weeks washout period, they were crossed over for a new 30-day period of tablets intake. The outcome measure was a questionnaire including 44 items of symptoms that may be related stress in which the severity of each sign was evaluated using a 10-degree scale. These measures were studied repeatedly at the day of 0, 15 and 30 after the start of each interventional period. RESULTS: The 30-day treatment by alpha (s1)-casein hydrolysate in females with stress-related symptoms reduced their symptoms, particularly in digestion (P<0.01), cardiovascular (P<0.05), intellectual (P<0.01), emotional (P<0.05) and social problems (P<0.05). CONCLUSION: This study showed that a 30-day ingestion of alpha (s1)-casein hydrolysate decreased the stress-related symptoms in females suggesting that this product may be used as an effective functional ingredient alleviating such symptoms. SPONSORSHIP: This study was partially supported by the INGREDIA of France and Neurobiology Research Program from the Korea Ministry of Science and Technology (2004-01757) of Korea.

Capsazepine, a vanilloid receptor antagonist, inhibits the expression of inducible nitric oxide synthase gene in lipopolysaccharide-stimulated RAW264.7 macrophages through the inactivation of nuclear transcription factor-kappa B.

High amounts of nitric oxide (NO) production following the induction of inducible NO synthase (iNOS) gene expression has been implicated in the pathogenesis of inflammatory diseases. Capsaicin, a vanilloid receptor agonist, is known to have an inhibitory effect on NO production in macrophages. In the present study, we have found that capsazepine (CAPZ), a vanilloid receptor antagonist, also inhibited NO and iNOS protein syntheses induced by lipopolysaccharide in RAW264.7 macrophages via the suppression of iNOS mRNA. The mechanistic studies showed that CAPZ inhibited the expression of iNOS mRNA through the inactivation of nuclear transcription factor-kappa B (NF-kappa B). Thus, capsazepine may be a useful candidate for the development of a drug to treat inflammatory diseases related to iNOS gene overexpression.

Octamer binding protein-1 is involved in inhibition of inducible nitric oxide synthase expression by exogenous nitric oxide in murine liver cells.

Nitric oxide (NO) has diverse effects on immune responses and hepatic functions. In BNL CL.2 cells, the murine embryonic liver cells, inducible nitric oxide synthase (iNOS) mRNA expression appeared after 3 h of treatment with IFN-gamma and LPS. Interestingly, mRNA and protein expression of iNOS was down-regulated by sodium nitroprusside (SNP) and diethylamine dinitric oxide in a time- and dose-dependent manner, but not by H2O2. TNF-alpha gene expression was also dramatically reduced by SNP, but IL-6 gene expression was inhibited much less. IFN-gamma and LPS-induced chloramphenicol acetyltransferase activity of iNOS promoter constructs was inhibited by SNP. Electrophoretic mobility shift assay showed that SNP inhibited IFN-gamma plus LPS-induced Oct-1 binding activity, and the inhibition was reversed by DTT. Mutation in the Oct-1 site completely abolished iNOS promoter activity. In addition, supershift assay and Southwestern analysis demonstrated that the Oct-1 binding activity was inhibited by SNP. Taken together, these results indicate that NO suppresses IFN-gamma plus LPS-induced iNOS expression, and that Oct-1 is an important element in this process.

Endogenous interleukin-18 modulates immune escape of murine melanoma cells by regulating the expression of Fas ligand and reactive oxygen intermediates.

It has been known that melanoma cells can suppress the immune system by the Fas ligand. The present study investigated whether interleukin (IL)-18, which can enhance Fas ligand expression, is produced by B16F10 melanoma cells and is involved in immune escape of tumor cells. Immunohistology, reverse transcription-PCR, intracellular fluorescence-activated cell-sorting analysis, and immunoblotting demonstrated that melanoma cells express IL-18. C57BL/6 splenocytes cultured with culture supernatants of B16F10 melanoma cells enhanced IFN-gamma production, which was blocked by anti-IL-18 antibody, indicating that IL-18 in the culture supernatants is functional. In addition to IL-18, the IL-18 receptor was also detected in B16F10 melanoma cells, suggesting a role of this cytokine in regulating the functions of B16F10 melanoma cells. The functional effect of IL-18 on B16F10 melanoma cells was shown by reduction of Fas ligand expression in cells treated with anti-IL-18 antibody or transfected with IL-18 antisense cDNA. In addition, the same treatments decreased intracellular reactive oxygen intermediate levels in B16F10 melanoma cells, indicating that IL-18 regulates reactive oxygen intermediate production, which is involved in Fas ligand expression. Furthermore, transfection of IL-18 antisense cDNA into melanoma cells increased the susceptibility of tumor cells to natural killer cells in vitro. When IL-18 antisense transfectants were implanted into syngeneic mice, severe reduction of tumor cell growth was observed with concomitant infiltrated natural killer cells in the tumor area. Taken together, these results demonstrate that IL-18 has a critical role as a survival factor for B16F10 melanoma cells.

Ligation of ICAM-1 molecules inhibits target cell-induced granule exocytosis of IL-12-activated natural killer cells.

The importance of cell adhesion molecules such as ICAM-1 is emphasized in cell-to-cell interactions that are critical in the generation of effective immune reactions. In this study, the involvement of ICAM-1 in natural killer (NK) cell activities was characterized in IL-12-activated human NK cells. To address the question of whether ligation of ICAM-1 molecules can modulate NK cell cytolytic activities, a 4-h (51)Cr-release assay was performed after pretreatment of NK cells with R6.5 mAb (anti-human ICAM-1 mAb). Ligation of membrane ICAM-1 molecules significantly inhibited IL-12-enhanced NK cytotoxicity against K562, and the pretreatment of neutralizing soluble ICAM-1 with R6.5 mAb blocked this inhibitory effect. The involvement of Ca(2+)-dependent granular exocytosis was evaluated. BLT esterase assay demonstrated that the ligation of ICAM-1 molecules inhibited granular exocytosis of NK cells. Additionally, the ICAM-1-mediated inhibition of Ca(2+) flux in NK cells was detected using Fluo-3AM, while the pretreatment of NK cells with R6.5 mAb did not affect conjugate formation between NK and K562 cells. Collectively, these results suggest that the signals transduced from ICAM-1 molecules might be sufficient to induce inhibitory effects on NK cells.

Roles of IFN consensus sequence binding protein and PU.1 in regulating IL-18 gene expression.

IL-18 is expressed from a variety of cell types. Two promoters located upstream of exon 1 (5'-flanking region) and upstream of exon 2 (intron 1) regulate its expression. Both promoter regions were cloned into pCAT-Basic plasmid to yield p1-2686 for the 5'-flanking promoter and p2-2.3 for the intron 1 promoter. Both promoters showed basal constitutive activity and LPS inducibility when transfected into RAW 264.7 macrophages. To learn the regulatory elements of both promoters, 5'-serial deletion and site-directed mutants were prepared. For the activity of the p1-2686 promoter, the IFN consensus sequence binding protein (ICSBP) binding site between -39 and -22 was critical. EMSA using an oligonucleotide probe encompassing the ICSBP binding site showed that LPS treatment increased the formation of DNA binding complex. In addition, when supershift assays were performed, retardation of the protein-DNA complex was seen after the addition of anti-ICSBP Ab. For the activity of the p2-2.3 promoter, the PU.1 binding site between -31 and -13 was important. EMSA using a PU.1-specific oligonucleotide demonstrated that LPS treatment increased PU.1 binding activity. The addition of PU.1-specific Ab to LPS-treated nuclear extracts resulted in the formation of a supershifted complex. Furthermore, cotransfection of ICSBP or PU.1 expression vector increased p1 promoter activity or IL-18 expression, respectively. Taken together, these results indicate that ICSBP and PU.1 are critical elements for IL-18 gene expression.

Up-regulation of interleukin-4 and CD23/FcepsilonRII in minimal change nephrotic syndrome.

Although the pathogenesis of childhood minimal change nephrotic syndrome (MCNS) has not been clearly defined, the current hypothesis favors an involvement of T cell dysfunction. The symptom onset and the relapse of MCNS are frequently associated with allergy and increased IgE levels in sera. Since a T cell-derived cytokine interleukin-4 (IL-4) plays a key role in the regulation of IgE production and allergic response, we investigated the role of IL-4 in the pathophysiology of MCNS. Using fluorescence-activated cell scanning we observed a significantly higher expression of CD23, the type II IgE receptor (FcepsilonRII), on fresh B cells from active MCNS patients (n=22) compared with age-matched healthy normal controls (n=12). The upregulation of CD23 correlates with greater IL-4 activity in the culture supernatant of MCNS peripheral blood lymphocytes (PBLs) than normal PBLs stimulated by mitogens, as assessed by the CD23-inducing effect of the PBL supernatant on tonsillar B cells. Furthermore, Northern blot and reverse transcription-based polymerase chain reaction analysis have revealed significantly elevated levels of IL-4 mRNAs both in mitogen-stimulated and unstimulated MCNS PBLs, compared with healthy normals or disease controls with other renal disorders. Together these results strongly suggest that the upregulation of IL-4 in T cells may be part of the T cell dysfunction involved in MCNS.

Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells.

Glutamic acid decarboxylase (GAD) is a pancreatic beta cell autoantigen in humans and nonobese diabetic (NOD) mice. beta Cell-specific suppression of GAD expression in two lines of antisense GAD transgenic NOD mice prevented autoimmune diabetes, whereas persistent GAD expression in the beta cells in the other four lines of antisense GAD transgenic NOD mice resulted in diabetes, similar to that seen in transgene-negative NOD mice. Complete suppression of beta cell GAD expression blocked the generation of diabetogenic T cells and protected islet grafts from autoimmune injury. Thus, beta cell-specific GAD expression is required for the development of autoimmune diabetes in NOD mice, and modulation of GAD might, therefore, have therapeutic value in type 1 diabetes.

Antitumor effects of IL-6 on murine liver tumor cells in vivo.

IL-6 is a pleiotropic cytokine that is capable of modulating the diverse functions of hepatocytes such as acute phase responses and inflammation in the liver. To learn its antitumor effects in vivo, the cDNA of IL-6 was transfected into murine liver cells, TIB cells. IL-6-transfected TIB cells (TIB73-IL-6 or TIB75-IL-6) produced much higher levels of IL-6 compared with vector-transfected TIB cells (TIB73-vec or TIB75-vec). To investigate the effects of IL-6 on TIB tumor growth in vivo, IL-6-transfected TIB cells or vector-transfected TIB cells were injected subcutaneously into syngeneic mice. Vector-transfected TIB cells grew rapidly 3 weeks after injection, but IL-6-transfected TIB cells did not grow at all for up to 6 weeks. Pathologically, IL-6-transfected TIB cells demonstrated a severe necrosis and apoptotic pattern. Taken together, these results indicate that IL-6 functions as a growth inhibiting factor in vivo, and another biological role of IL-6 in the liver is suggested.

Effects of acanthoic acid on TNF-alpha gene expression and haptoglobin synthesis.

Tumour necrosis factor-alpha (TNF-alpha) is a major pro-inflammatory cytokine inducing the synthesis and release of many inflammatory mediators. It is involved in immune regulation, autoimmune diseases, and inflammation. Our previous study demonstrated that acanthoic acid, (-)-pimara-9(11), 15-dien-19-oic acid, a pimaradiene diterpene isolated from Acanthopanax koreanum, inhibited TNF-alpha production. To extend our understanding of inhibitory effects of acanthoic acid on TNF-alpha production, its effects on TNF-alpha gene expression was tested. Based on the results from RT-PCR and promoter analysis of TNF-alpha, it was found that acanthoic acid suppressed TNF-alpha gene expression. But the same concentration of acanthoic acid had no effect on IL-6 gene expression. Haptoglobin is an acute phase protein which is induced by TNF-alpha. When liver cells were treated with acanthoic acid, haptoglobin synthesis was blocked by acanthoic acid. These data confirmed that acanthoic acid inhibited gene expression and biological function of TNF-alpha.

Roles of protein phosphatase 2A in IL-6 signal transduction in Hep3B cells.

IL-6 is a pleiotropic cytokine that modulates the diverse functions of hepatocytes such as acute phase responses and inflammation. When human hepatoma cells, Hep3B cells, were treated with IL-6, p140 was phosphorylated rapidly and reached its maximal rate at 1 min after treatment. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, affected IL-6-induced p140 phosphorylation. Interferon regulatory factor-1 (IRF-1) is a transcription factor on the enhancer of type I interferons, and its gene expression is induced by IL-6. When IRF-1 promoter-luciferase construct was transfected into Hep3B cells, okadaic acid increased IL-6- induced IRF-1 promoter activity. In addition, co-transfection of protein phosphatase 2A (PP2A) antisense constructs further increased IL-6-induced IRF-1 promoter activity, suggesting that PP2A is involved in IL-6 signaling. In addition, IL-6 directly induced the PP2A phosphorylation. PP2A phosphorylation was maximal at 1 min after IL-6 stimulation, but it was not induced by other inflammatory cytokines such as TNF-alpha or TGF-beta. Furthermore, IL-6 activated PP2A activity simultaneously. Taken together, these data indicate that IL-6 modulates the functions of PP2A which is involved in downstream events of IL-6 signaling in Hep3B.

Modulation of NK-target cell interaction by a monoclonal antibody to K562 cells.

In order to identify the target cell recognition molecules involved in the interaction between natural killer (NK) cells and target cells, we have generated monoclonal antibodies to K562, NK-sensitive target cells. After screening by FACScan for the reactivity to K562, one monoclonal antibody (mAb), 4A60, was selected. MAb 4A60 was found to inhibit the proliferation of NK cells induced by IL-2 and K562 cells. However, this monoclonal antibody could not significantly block the conjugate formation between NK and target cells. Moreover, mAb 4A60 only slightly inhibited the cytotoxicity of NK cells induced by IL-2. Protein analysis showed that mAb 4A60 recognized a 53-kDa protein of K562 cells. Taken together, these data suggest that mAb 4A60 inhibits the proliferation of NK cells induced by IL-2 and target cells, and the 53-kDa protein, a tentative ligand of this mAb of K562, may be involved in this process.

Roles of tyrosine kinases in the regulation of nitric oxide synthesis in murine liver cells: modulation of NF-kappa B activity by tyrosine kinases.

Nitric oxide (NO) synthesis is upregulated during chronic hepatic inflammation. The present study characterized the mechanisms involved in the induction of NO production and inducible NO synthase (iNOS) messenger RNA (mRNA) expression in murine embryonic liver cell line, BNL CL.2 cells. No production by BNL CL.2 cells was induced by interferon-r (IFN-r) plus lipopolysaccharide (LPS). However, other inflammatory cytokines such as interleukin (IL)-beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 had no additional effects on it. The stimulatory effects of IFN-r and LPS were time- and dose-dependent. NO secretion was inhibited by treatment with inducible NOS inhibitors such as NG-monomethyl L-arginine, NG-amino-L-arginine, and diphenylene iodonium. iNOS mRNA was induced 3 hours after IFN-r plus LPS treatment, and iNOS expression was maximal in the presence of IFN-r and LPS. The protein tyrosine kinase inhibitors such as genistein and tyrphostin reduced IFN-r plus LPS-induced iNOS mRNA expression and NO production. In contrast, the inhibitors of protein kinase C, protein kinase A, and protein phosphatases did not affect iNOS expression induced by IFN-r plus LPS. In addition, iNOS mRNA expression was completely blocked by treatment with tyrphostin. However, mRNA expression of an early response gene, JunB, and constitutively expressed genes beta-actin and GAPDH were not inhibited by tyrphostin. Furthermore, tyrphostin inhibited the promoter activation of iNOS gene induced by IFN-gamma plus LPS, and it also suppressed IFN-gamma plus LPS-induced nuclear factor-kappa B-binding activity but not AP-1-binding activity. These results suggest that NO production and iNOS mRNA expression in this cell line is dependent on protein tyrosine kinases but does not require protein kinase C, protein kinase A, or protein phosphatases.

Molecular cloning and chromosomal localization of a human gene homologous to the murine R-PTP-kappa, a receptor-type protein tyrosine phosphatase.

Tyrosine phosphorylation of proteins plays an important role in cellular signaling and many cellular activities. The levels of cellular phosphorylation are reversibly controlled by protein tyrosine kinases and protein tyrosine phosphatases. The murine R-PTP-kappa, a receptor-type protein tyrosine phosphatase, has recently been cloned (Jiang et al. (1993) Mol. Cell. Biol. 13, 2942-2951). In order to identify the protein tyrosine phosphatases critical to the cellular signal transduction in human keratinocytes, a polymerase chain reaction (PCR)-based strategy was employed, and we have cloned a human homologue of the murine R-PTP-kappa. Here, we report the isolation of a complementary DNA encoding a human R-PTP-kappa. Of the several overlapping cDNA clones, one clone, which we originally termed p55-7, was found to encode a transmembrane protein of 1440 amino acids and was highly conserved with murine R-PTP-kappa with 98% identity at the amino-acid levels. The human R-PTP-kappa gene was localized to chromosome 6 by southern hybridization of DNA from a rodent/human somatic cell mapping panel. Northern blot analysis of RNA from several human tissues revealed, like the murine R-PTP-kappa, the presence of a major mRNA of approx. 7.0 kb and a minor mRNA of approx. 5.3 kb. In contrast to the expression of murine R-PTP-kappa which was highly expressed in liver and kidney, the human R-PTP-kappa was predominantly expressed in spleen, prostate, and ovary. However, the transcripts were detectable at various levels in all examined tissues (thymus, testis, small intestine, and colon) except for PBL (peripheral blood leukocytes). In addition, human R-PTP-kappa displayed a restricted pattern of expression among a series of cell lines, and was apparently expressed in an epidermal cells and cell lines (human normal keratinocytes, HaCaT, and A431), but was not detectable in other cell lines tested after longer exposure.

IL-6 Undergoes Transition from in vitro Autocrine Growth Factor toin vivo Growth Inhibitor of B Lymphoma Cells.

IL-6 is a multifunctional cytokine involved in differentiation and proliferation of immune cells. Moreover, it has diverse effects on the proliferation of tumor cells in vivo and in vitro. Although stimulating cell growth of multiple myeloma cells, it inhibits the proliferation of B16 melanoma cells and lung cancer cells. B9.55 cells, B-cell lymphoma, are IL-6-dependent cells, definitely requiring exogenous IL-6 for growth. When the cDNA for IL-6 was transfected into B9.55 cells, they began growing in an autocrine pattern without exogenous IL-6. To investigate the effects of IL-6 on B9.55 lymphoma in vivo, IL-6-transfected B9.55 cells (B9.G7) or neotransfected B9.55 cells (B9.vec) were injected subcutaneously into syngeneic mice. Initially, B9.G7 outgrew B9.vec, but after 3 weeks, B9.G7 grew slower than B9.vec. In addition, 5 micro g of recombinant human IL-6 was injected daily into the tumor site. Reduced tumor sizes of IL-6-treated rats, similar to those observed in mice which received B9.G7, indicated that IL-6 itself is the mediator of tumor regression. When B9.G7 cells were injected into the irradiated normal mice, tumor regression was released compared with the untreated normal control, suggesting that radiosensitive host components were involved in the regression of B9.G7 cell growth. However, the tumor regression of B9.G7 cells was not released in SCID mice. Histologically, B9.G7 tumor demonstrated severe necrosis and apoptotic cells with infiltration of host inflammatory cells. Above data indicate that IL-6 functions as an autocrine growth factor for B9.G7 cells in vitro, but behaves as an autocrine inhibiting factor in vivo. These contrasting effects of IL-6 on tumor cells in vitro and in vivo will be facilitative in understanding the interaction of cytokines and host immune systems.

Transforming growth factor-beta1 inhibits human keratinocyte proliferation by upregulation of a receptor-type tyrosine phosphatase R-PTP-kappa gene expression.

We studied regulation of a receptor-type tyrosine phosphatase R-PTP-kappa gene expression in a human keratinocyte cell line, HaCaT. Addition of TGF-beta 1 to the HaCaT cells markedly induced the expression of R-PTP-kappa mRNA in a time- and dose-dependent manner. The induction of R-PTP-kappa mRNA expression was observed at a dose as low as 0.02 ng/ml TGF-beta1 and reached a peak at 2 ng/ml TGF-beta 1 after 6 h treatment. The TGF-beta 1-induced R-PTP-kappa mRNA expression was suppressed by sodium orthovanadate, a tyrosine phosphatase inhibitor, and H7, a serine/threonine kinase inhibitor, but not by genistein, a tyrosine kinase inhibitor. In addition, the inducing effect is not dependent on de novo protein synthesis. Taken together, these results suggest that TGF-beta 1 inhibits the human keratinocyte proliferation in vitro, possibly through induction of R-PTP-kappa gene expression.

Interleukin 4-induced proliferation in normal human keratinocytes is associated with c-myc gene expression and inhibited by genistein.

We studied the effect of IL-4 on the proliferation of cultured normal human keratinocytes. Keratinocyte proliferation was stimulated by IL-4 and inhibited by anti-IL-4 antibody in a concentration-dependent manner. Anti-IL-6 antibody did not inhibit normal human keratinocyte proliferation, suggesting that the IL-4 could directly induce proliferation of these cells. IL-4 significantly induced cell cycle G0/G1 to S phase progression. The keratinocyte proliferation by IL-4 was mediated through one of the growth control genes, c-myc protooncogene. The expression of c-myc mRNA was significantly increased after IL-4 treatment of the keratinocytes, suggesting that c-myc plays a key role in the control of proliferation. The signal transduction pathways induced by IL-4 in the keratinocytes were studied with inhibitors of signal transduction. Genistein, a tyrosine kinase inhibitor, suppressed the level of the induced c-myc mRNA expression, but H7, a serine/threonine kinase inhibitor, and okadaic acid, a protein phosphatase 1 and 2A inhibitor, did not block the induced c-myc gene expression. Taken together, these results suggest that IL-4 stimulates the proliferation of keratinocytes in vitro by promoting a transition from G0/G1 to S phase of the cell cycle. Induction of c-myc after IL-4 treatment could indicate an important role for c-myc in the proliferation of keratinocytes. Our observations also suggest that tyrosine kinases may be involved in IL-4-induced proliferation.

Suppression of interleukin-1 and tumor necrosis factor-alpha production by acanthoic acid, (-)-pimara-9(11),15-dien-19-oic acid, and it antifibrotic effects in vivo.

Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are major proinflammatory cytokines inducing the synthesis and release of many inflammatory mediators. They are involved in immune regulation, autoimmune diseases, and inflammation. Acanthoic acid, (-)-pimara-9(11),15-dien-19-oic acid, is a pimaradiene diterpene isolated from the Korean medicinal plant, Acanthopanax koreanum. When human monocytes/macrophages stimulated with silica were treated with 0.1-10 microg/ml acanthoic acid, the production of IL-1 and TNF-alpha was inhibited up to 90%, but the production of interleukin-6 (IL-6) was not inhibited at all. At these concentrations, it had no cytotoxic effect on human monocytes/macrophages. It also suppressed the production of TNF-alpha by alveolar macrophages and lymphocytes stimulated with silica. In addition, acanthoic acid inhibited the release of superoxide anion and hydrogen peroxide from human monocytes/macrophages and neutrophils. To know the antifibrotic effects of acanthoic acid, its effects on fibroblast proliferation and collagen synthesis were tested. The proliferation of NIH3T3 cells was inhibited almost completely by the addition of the culture supernatants of human monocytes/macrophages treated with acanthoic acid, but not by the addition of acanthoic acid only. In vitro and in vivo treatment with acanthoic acid reduced collagen production by rat lung fibroblasts and lung tissue. Furthermore, acanthoic acid suppressed granuloma formation and fibrosis in the experimental silicosis. Acanthoic acid reduced serum GOT and GPT in the rats with cirrhosis induced by CCl4, and it was effective in reducing hepatic fibrosis and nodular formation. Taken together, these data indicate that acanthoic acid has a potent anti-inflammatory and antifibrosis effect by reducing IL-1 and TNF-alpha production.

Roles of protein phosphatase 1 and 2A in an IL-6-mediated autocrine growth loop of human myeloma cells.

Deregulation of IL-6 production is one of the major causes for human multiple myeloma. Exogenous IL-6 stimulated the proliferation of fresh human myeloma cells and the myeloma cell line, U266, which produced IL-6 spontaneously. Anti-IL-6 antibody and IL-6 antisense oligonucleotide suppressed the IL-6 stimulated myeloma cell proliferation, indicating that IL-6 induced the myeloma cell proliferation via an autocrine loop. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, inhibited the U266 cell proliferation at a concentration of less than 1 ng/ml. At this concentration, okadaic acid suppressed the IL-6-induced IL-6 gene expression of myeloma cells. It seems that the okadaic acid blocked the myeloma cell proliferation by reducing IL-6 synthesis in myeloma cells. In addition, IL-6 itself also regulated IL-6 receptor expression. Analysis by FACScan and RT-PCR showed that anti-IL-6 antibody treatment up-regulated IL-6 receptor expression. Interestingly, the presence of okadaic acid induced the up-regulation of IL-6 receptor expression as well as the down-regulation of IL-6-induced gp130 phosphorylation in the myeloma cells. Taken together, these data suggest that protein phosphatase 1 and 2A are involved in IL-6-mediated autocrine growth of human myeloma cells by modulating IL-6 signaling and IL-6 receptor expression in myeloma cells.

Anti-inflammatory effects of Stephania tetrandra S. Moore on interleukin-6 production and experimental inflammatory disease models.

Deregulation of interleukin-6 (IL-6) expression caused the synthesis and release of many inflammatory mediators. It is involved in chronic inflammation, autoimmune diseases, and malignancy. Stephania tetrandra S. Moore is a Chinese medicinal herb which has been used traditionary as a remedy for neuralgia and arthritis in China. To investigate the anti-inflammatory effects of S. tetrandra S. Moore in vitro and in vivo, its effects on the production of IL-6 and inflammatory mediators were analysed. When human monocytes/macrophages stimulated with silica were treated with 0.1-10 mug/ml S. tetranda S. Moore, the production of IL-6 was inhibited up to 50%. At these concentrations, it had no cytotoxicity effect on these cells. It also suppressed the production of IL-6 by alveolar macrophages stimulated with silica. In addition, it inhibited the release of superoxide anion and hydrogen peroxide from human monocytes/macrophages. To assess the anti-fibrosis effects of S. tetrandra S. Moore, its effects on in vivo experimental inflammatory models were evaluated. In the experimental silicosis model, IL-6 activities in the sera and in the culture supernatants of pulmonary fibroblasts were also inhibited by it. In vitro and in vivo treatment of S. tetrandra S. Moore reduced collagen production by rat lung fibroblasts and lung tissue. Also, S. tetrandra S. Moore reduced the levels of serum GOT and GPT in the rat cirrhosis model induced by CCL(4), and it was effective in reducing hepatic fibrosis and nodular formation. Taken together, these data indicate that it has a potent anti-inflammatory and antifibrosis effect by reducing IL-6 production.