Preventive effect of Coriandrum sativum (Chinese parsley) on localized lead deposition in ICR mice.

The preventive effect of Coriandrum sativum, Fam. UMBELLIFERAE (Chinese parsley) on lead deposition was investigated in male ICR mice given lead (1000 ppm) as lead acetate trihydrate in drinking water for 32 days. Administration of Chinese parsley to mice by gastric intubation was performed for 25 days from day 7 after the start of lead exposure up to the end of the experiment. The mice were then sacrificed for comparison of lead distribution. The lead reached its highest concentration in the femur but localized lead deposition in the femur was significantly decreased by meso-2,3-dimercaptosuccinic acid (DMSA), a chelating agent used as a positive control to validate this experimental model. Administration of Chinese parsley also significantly decreased lead deposition in the femur and severe lead-induced injury in the kidneys. In addition, urinary excretion of delta-aminolevulinic acid (ALA) which is known to increase with lead intake was significantly decreased after administration of Chinese parsley. The MeOH extract of Chinese parsley also reduced lead-induced inhibition of delta-aminolevulinic acid dehydratase (ALAD) activity in vitro. These results suggest that Chinese parsley has suppressive activity on lead deposition, probably resulting from the chelation of lead by some substances contained in Chinese parsley.

Cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (U-937 and HL-60) by tryptanthrin, an active ingredient of Polygonum tinctorium Lour.

Tryptanthrin, a bioactive ingredient of Polygonum tinctorium Lour., is a member of the Indigo plant family and has potent cytocidal effects on various human leukemia cells in vitro. At low concentrations, tryptanthrin enhanced the expression of cell differentiation (CD) markers in human monocytic (U-937) and promyelocytic (HL-60) leukemia cells indicative of differentiation to monocytes/macrophages. Furthermore, nitroblue tetrazolium (NBT) reductive and alpha-naphthyl butyrate esterase (NBE) activities were markedly increased after treatment. Tryptanthrin was more potent than dimethyl sulfoxide (DMSO) at inducing U-937 cell differentiation into monocytes/macrophages. After treatment with higher concentrations of tryptanthrin for 24 h, cytoplasmic vacuolation and destruction of mitochondria were observed. The leukemia cells died via apoptosis 48 h after treatment. Cytoplasmic vacuolation and apoptotic changes correlated with the dysfunction of mitochondria. Electron microscopic observations revealed marked swelling and destruction of mitochondria after exposure of the leukemia cells to tryptanthrin. Exposure to tryptanthrin enhanced Fas-induced apoptosis and increased caspase-3 activity before induction of apoptosis. These results show that low concentrations of tryptanthrin can induce differentiation of leukemia cells but higher concentrations will kill leukemia cells through apoptosis, possibly through a caspase-3/Fas antigen pathway.

Antibacterial action of tryptanthrin and kaempferol, isolated from the indigo plant (Polygonum tinctorium Lour.), against Helicobacter pylori-infected Mongolian gerbils.

We evaluated the effect of tryptanthrin and kaempferol, both isolated from Polygonum tinctorium Lour., against Helicobacter pylori colony formation in vitro and in H. pylori-infected Mongolian gerbils. H. pylori suspension was mixed with solution of tryptanthrin and/or kaempferol and placed onto agar plates. These plates were incubated at 37 degrees C, under 10% CO2 for 5 days, and the H. pylori colonies were counted. For the in vivo experiment, Mongolian gerbils were inoculated with H. pylori ATCC 43504 orally. After 4 weeks, the infected gerbils were given tryptanthrin and/or kaempferol, administered orally, twice a day for 10 days. The animals were killed and the number of live H. pylori in their stomachs was determined. In vitro both tryptanthrin and kaempferol significantly decreased the numbers of H. pylori colonies a dose-dependent manner. An additive effect on colony formation was observed with the combined use. In the in vivo experiment, oral administration of tryptanthrin and/or kaempferol significantly decreased the numbers of colonies in the gerbils' stomachs. We concluded that tryptanthrin and kaempferol were effective against H. pylori in vivo.

Prevention of azoxymethane-induced intestinal tumors by a crude ethyl acetate-extract and tryptanthrin extracted from Polygonum tinctorium Lour.

The effect of a crude ethyl acetate (AcOEt)-extract and tryptanthrin extracted from the Indigo plant (Polygonum tinctorium Lour.) on azoxymethane (AOM)-induced intestinal tumors was examined in F344 rats. The rats were given subcutaneous (s.c.) injections of either AOM (15 mg/kg body weight (b.w.)) once a week for 3 weeks to induce atypical crypt foci (ACF) as a known cancer precursor, or AOM (7.5 mg/kg b.w.) once a week for 10 weeks to induce intestinal tumors. The rats were also administered the AcOEt-extract (500 mg/kg b.w.) or tryptanthrin (50 mg/kg b.w.) orally, 5 days a week, for 7 or 30 weeks, starting two days before the first administration of AOM. All rats were killed 4 or 20 weeks after the last treatment. In the short-term experiment, the incidence of ACE and atypical crypts (AC) in the groups receiving the AcOEt-extract and tryptanthrin was significantly lower than in the control group. In the tumor-inducing experiment, intestinal tumor incidence in the tryptanthrin group was lower than in the AOM-control group (5% versus 26%), and small intestine tumor incidence in the AcOEt-extract and tryptanthrin groups were lower than in the AOM-control group (0% and 0% versus 23%). These results show that the AcOEt-extract of Indigo and tryptanthrin have cancer chemopreventive activity.

Tryptanthrin inhibits nitric oxide and prostaglandin E(2) synthesis by murine macrophages.

Nitric oxide (NO) and prostaglandins have been implicated in the pathogenesis of several inflammatory diseases. In this study, we investigated the effect of tryptanthrin (6,12-dihydro-6, 12-dioxoindolo-(2,1-b)-quinazoline), an antimicrobial and antitumoral plant compound isolated from Porigonum tinctorium, on NO and prostaglandin E(2) production by interferon-gamma and lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells. Tryptanthrin markedly inhibited both NO and prostaglandin E(2) production in a dose-dependent manner. Tryptanthrin at 20 microM fully inhibited expression of inducible NO synthase, suggesting that the inhibitory effect on NO synthesis was mediated by inhibited expression of the enzyme. On the other hand, tryptanthrin had no effect on the levels of cyclooxygenase-2 protein, but inhibited cyclooxygenase enzyme activity with a ICM(50) value of 1.5 microM. Thus, tryptanthrin has the dual functions of inhibiting both NO and prostaglandin E(2) production by activated macrophages, suggesting that tryptanthrin exhibits anti-inflammatory properties.

Cloning and expression of interleukin-18 binding protein.

Interleukin-18 binding protein is a novel glycoprotein that we successfully cloned and expressed. First, murine interleukin-18 binding protein was purified from the sera of mice with endotoxin shock using ligand affinity chromatography. The murine interleukin-18 binding protein cDNA was cloned after RT-PCR using mixed primer pair sequences based on partial murine interleukin-18 binding protein amino acid sequence analysis. Subsequently, human interleukin-18 binding protein cDNA was cloned from cDNA libraries of normal human liver using murine interleukin-18 binding protein cDNA as a probe. Next, we transiently expressed recombinant human and murine interleukin-18 binding proteins in COS-1 cells and purified them from culture supernatants. Both recombinant interleukin-18 binding proteins did not exhibit species specificity and prevented interleukin-18 binding to its receptor. In addition, they inhibited interleukine-18 dependent IFN-gamma production from KG-1 cells effectively. These results suggest that the interleukin-18 binding protein may possess interleukine-18 antagonist activity.

Constitutive and induced IL-18 receptor expression by various peripheral blood cell subsets as determined by anti-hIL-18R monoclonal antibody.

Interleukin-18 (IL-18) was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. Characterization of the receptor for human IL-18 (hIL-18R) is important for investigating the physiological role of IL-18 in nature. In the present study, we describe a monoclonal antibody (mAb) against hIL-18R (mAb No. 117-10C). This mAb inhibited the binding of 125I-labeled hIL-18 to IL-18R-expressing L428 cells. This mAb also neutralized hIL-18-induced T helper 1 type cytokine (IFN-gamma and GM-CSF) production by Con A-stimulated PBMC. PBMC were examined for the expression of IL-18R by two-color flow cytometry. Most CD19(+) B cells and a percentage of CD8(+) T cells were found to constitutively express IL-18R. Treatment of PBMC with IL-12 preferentially induced IL-18R expression on CD56(+) NK cells regardless of costimulation with mitogen. IL-18R expression on CD4(+) T cells was induced weakly by IL-12 treatment and moderately by PHA stimulation. However, neither could IL-12 treatment nor PHA stimulation induce IL-18R expression on CD8(+) T cells. Costimulation with both IL-12 and PHA was necessary for optimal IL-18R expression on CD8(+) T cells as well as on CD56(+) NK cells, CD4(+) T cells, and CD19(+) B cells. These results support the growing number of reports showing that IL-18 has modulatory effects on T, B, and NK cells.

Establishment of the cells useful for murine interleukin-18 bioassay by introducing murine interleukin-18 receptor cDNA into human myelomonocytic KG-1 cells.

We genetically engineered human myelomonocytic KG-I cells by introducing cDNA of murine interleukin-18 receptor (MuIL-18R) and established human cells which were capable of responding to MuIL-18. These cells expressed larger number of MuIL-18R (> 13,000 sites/cell) than intrinsic human IL-18 receptor (HuIL-18R) (< 2,500 sites/cell). And the cells responded to MuIL-18 as well as to HuIL-18 in a dose-dependent manner, and produced large amounts of interferon-gamma (IFN-gamma). We could estimate the amount of murine IL-18 based on the amounts of IFN-gamma produced by these cells. The stoichiometry was observed up to 150 ng/ml of MuIL-18. By using these cells, a large amount of MuIL-18 (448 +/- 89.2 ng/ml) was detected in sera of Propionibacterium acnes (P. acnes)/lipopolysaccharide (LPS)-treated endotoxic mice (the same conditions in which IL-18 was first identified). These cells provide us with a useful tool for determining the bioactivity of MuIL-18.

Purification and characterization of the human interleukin-18 receptor.

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.

Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production.

The novel cytokine interferon-gamma-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-gamma had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-gamma; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-gamma production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-gamma, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-gamma production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.

Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.

We have recently reported that a novel molecule, murine IFN-gamma-inducing factor (IGIF) produced by mouse liver cells, possesses potent biologic activities, including the induction of IFN-gamma production by spleen cells and the enhancement of NK cell cytotoxicity. In this paper, we report on the isolation of human IGIF cDNA clones from normal human liver cDNA libraries using murine IGIF cDNA as a probe. The amino acid sequence deduced from the human cDNA clones indicated a 193-amino acid precursor peptide and revealed 65% homology with that of murine IGIF. The amino acid sequence of IGIF also included an IL-1 signature-like sequence. Subsequently, the cloned cDNA was expressed in Escherichia coli, and preliminary studies on the biologic activities of the recombinant protein were performed. The recombinant human IGIF induced IFN-gamma production by mitogen-stimulated PBMC and enhanced NK cell cytotoxicity, in a manner similar to murine IGIF. In addition, recombinant human IGIF also augmented granulocyte-macrophage-CSF production and decreased IL-10 production, but had no effect on IL-4 production by Con A-stimulated PBMC. Based on these pleiotropic effects of IGIF, we propose that this novel cytokine be designated as IL-18.

Metastasis-promoting activity of a novel molecule, Ag 243-5, derived from mycoplasma, and the complete nucleotide sequence.

We found that mycoplasma-infected cells have a higher ability to metastasize in vivo than non-mycoplasma-infected cells. To investigate this phenomenon, we obtained a monoclonal antibody, MAb 243-5, by immunization with Mycoplasma arginini-infected RPMI 4788 cells. This MAb recognized a mycoplasmal protein with an MW of 47 kDa and completely inhibited the experimental metastasis of M. arginini-infected RPMI 4788 cells using a nude mouse model. Using this MAb, we purified a molecule called Ag 243-5 and determined the N-terminal amino acid sequence and clarified the entire nucleotide sequence of the Ag 243-5 gene. PCR analysis showed the existence of a homologous gene in Mycoplasma hyorhinis. Four sequential injections of Ag 243-5 (30 micrograms/shot) promoted the experimental metastasis of non-mycoplasma-infected RPMI 4788 cells more than 10-fold using a nude mouse model. Ag 243-5 also promoted the experimental metastasis of the non-mycoplasma-infected mouse colon cancer cell line colon 26. This metastasis-promoting effect was neutralized by MAb 243-5.