OBJECTIVETo study the relationship between polymorphism of inducible Nitric Oxide Synthase (iNOS) gene and the susceptibility of intestinal type stomach cancer and stomach cardia cancer in Chinese people.

METHODSA community-based case-control study was designed. Ninety-three intestinal type of stomach cancer and 50 stomach cardia cancer patients with endoscopy and pathology diagnosis were identified as cases. Two hundred and forty-six controls served as controls.

RESULTSC-->T polymorphism was found in exon 16 of iNOS gene, which changed the coding amino acid from serine to leucine, and formed a recognition site identified by Tsp 509 I restriction enzyme (we called it C-->T polymorphism). The T allele gene frequency in the control group was 13.21%. No statistically significant difference was found between C-->T polymorphism alone and the increased susceptibility to intestinal stomach cancer or stomach cardia cancer. A significant type 2 multiplicative interaction was found in increasing both the risk of intestinal stomach cancer and stomach cardia cancer when both C-->T polymorphism and tobacco smoking exposure existed. An additive interaction model, which showed statistically significant difference, was found to increase only the risk of stomach cardia cancer when CagA antibody shared negative but C-->T polymorphism occurred.

CONCLUSIONC-->T polymorphism of iNOS gene was considered as one of the possible susceptible genes, which specifically increased the risk of tobacco-related but CagA negative types of intestinal stomach cancer and stomach cardia cancer.

Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Bacterial Proteins ; immunology ; Genetic Predisposition to Disease ; Humans ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type II ; Polymorphism, Genetic ; Stomach Neoplasms ; genetics

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.
Anti-Inflammatory Agents/immunology/pharmacology ; Cell Line, Tumor ; Cyclic GMP/analogs & derivatives/immunology/metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 2/antagonists & inhibitors/metabolism ; Humans ; Interleukin-1beta/*metabolism ; Male ; Nitric Oxide/*biosynthesis/genetics/immunology ; Nitric Oxide Synthase Type II/*biosynthesis/genetics/immunology ; Phosphodiesterase Inhibitors/immunology/*pharmacology ; Piperazines/immunology/*pharmacology ; Purines/immunology/pharmacology ; Signal Transduction/drug effects/genetics/immunology ; Sulfones/immunology/*pharmacology ; Synovial Membrane/enzymology/immunology

OBJECTIVETo investigate the effect of activated greater omental milky spots and peritoneal macrophages in mice on tumoricidal activity against gastric carcinoma SGC-7901, following intraperitoneal (i.p.) injection of INF-gamma, staphylococcin aureus or NDV-L.

METHODSThe quantitative changes of milky spots were determined by activated carbon, the number of the macrophage in milky spots was assessed by nonspecific esterase stain and the number of peritoneal macrophages was counted by trypan blue exclusion. The morphology of peritoneal macrophages was observed by scanning electron microscope, the amount of TNF-alpha and iNOS mRNA expressed by peritoneal macrophages was measured by fluorescence quantitative PCR and the cytotoxicity of peritoneal macrophages supernatant against SGC-7901 was evaluated by MTT assay.

RESULTSIt was found in the treated groups that: 1. The amount of greater omental milky spots and the macrophages in milky spots increased, 2. The number of peritoneal macrophages increased. The peritoneal macrophages were in activated status. The effect TNF-alpha and iNOS mRNA expression increased and 3. The cytotoxicity against in vitro SGC-7901 increased.

CONCLUSIONIntraperitoneal injection of IFN-gamma, staphylococcin aureus or NDV-L could activate the milky spots of the greater omentum and the macrophages in peritoneal cavity in mice, with IFN-gamma being the best. The supernatant of activated peritoneal macrophages has cytotoxicity against SGC-7901. Administration of LPS to macrophages cultured in vitro could amplify the activation and enhance the cytotoxicity of the supernatant against SGC-7901.

Animals ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Female ; Interferon-gamma ; pharmacology ; Macrophages, Peritoneal ; drug effects ; immunology ; ultrastructure ; Mice ; Nitric Oxide Synthase Type II ; genetics ; Omentum ; drug effects ; immunology ; ultrastructure ; RNA, Messenger ; analysis ; Stomach Neoplasms ; immunology ; Tumor Necrosis Factor-alpha ; genetics

Scutellariae Radix (SR), the root of Scutellaria baicalensis Georgi, is used as an antipyretic drug and has been demonstrated to have anti-inflammatory activity. SR is divided into two specifications, "Ku Qin" (KQ) and "Zi Qin" (ZQ), for use against different symptoms (upper energizer heat or lower portion of the triple energizer), according to the theory of traditional Chinese medicine (TCM). However, differences in the efficacies of these two specifications have not been determined. In the present study, we aimed to characterize the differences in the anti-inflammatory activities between KQ and ZQ and to explore how their differences are manifested in lipopolysaccharide (LPS)-induced macrophages. Our results showed that, in RAW264.7 cells (a mouse macrophage cell line derived from ascites), KQ and ZQ displayed anti-inflammatory effects by inhibiting the release of nitric oxide (NO), inducible NOS (iNOS), and nuclear factor-κB (NF-κB) in a dose-dependent manner without distinction. In NR8383 cells (a rat alveolar macrophage cell line), KQ and ZQ displayed similar effects on NO, iNOS, and NF-κB as seen in RAW264.7 cells, but KQ showed a higher inhibition rate for NO and iNOS than that shown by ZQ at the same concentration. These results indicated that there were differences in efficacy between KQ and ZQ in treating lung inflammation. Our findings provided an experimental evidence supporting the different uses of KQ and ZQ in clinic, as noted in ancient herbal records.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; RAW 264.7 Cells ; Rats ; Scutellaria baicalensis ; chemistry

OBJECTIVETo observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.

METHODThe effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.

RESULTThe safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.

CONCLUSIONThis study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; Panax ; chemistry ; Protective Agents ; pharmacology ; Saponins ; pharmacology

OBJECTIVEObjective To construct recombinant Mycobacterium smegmatis expressing ESAT-6 of the human pathogen Mycobacterium tuberculosis.

METHODSESAT-6 gene was amplified from M. tuberculosis genomic DNA and inserted into an E.coli-mycobacterium shuttle vector under the control of HSP60 promoter. The recombinant vector was transformed into M. smegmatis by electroporation. To assess the ability of recombinant M. smegmatis to activate macrophage, mouse macrophage ANA-1 was cocultured with recombinant M. smegmatis. The apoptosis of ANA-1 cells was detected by flow cytometry and iNOS mRNA expression of the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The survival of M. smegmatis strains in ANA-1 cells was evaluated.

RESULTSThe recombinant vector was verified by restriction endonuclease digestion and DNA sequencing. ESAT-6 protein was expressed in M. smegmatis in response to heat shock and the molecular weight of the expression product was identical to the expected value. The growth curve of the new recombinant M. smegmatis was consistent with that of the wild-type strain, suggesting the absence of ESAT-6 protein toxicity against M. smegmatis. The recombinant M. smegmatis did not induce significant changes in mouse macrophage ANA-1 apoptosis. Coculture of the macrophages with recombinant M. smegmatis for 4 to 24 h could induce iNOS expression in the former, and the CFU of recombination M. smegmatis grown in ANA-1 cells was much less than that of the control bacteria.

CONCLUSIONThe recombinant M. smegmatis expressing M. tuberculosis ESAT-6 gene possess immunogenicity, which provides experimental evidence for the development of novel M. smegmatis-based vaccine against tuberculosis.

Animals ; Antigens, Bacterial ; biosynthesis ; genetics ; immunology ; Apoptosis ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Flow Cytometry ; Genetic Vectors ; Humans ; Macrophage Activation ; immunology ; Macrophages ; cytology ; immunology ; metabolism ; Mice ; Mycobacterium smegmatis ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transformation, Genetic

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.
*Adjuvants, Immunologic ; Animals ; Antibodies, Protozoan/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Cytokines/genetics/immunology ; Euphorbiaceae/*chemistry ; Hypersensitivity, Delayed/immunology ; Immunization ; Immunoglobulin G/immunology ; Latex/chemistry ; Leishmania/immunology ; Leishmaniasis, Cutaneous/*immunology/pathology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II/genetics/immunology ; Plant Lectins/*immunology/isolation & purification ; Protozoan Vaccines/immunology/pharmacology ; Skin/pathology

The effect of extracellular beta-(1-->3), (1-->6)-glucan, produced by Paenibacillus polymyxa JB115, on nitric oxide (NO) production in RAW264.7 macrophages was investigated. beta-glucan induced the production of NO by RAW264.7 macrophages in a concentration- and time-dependent manner. Moreover, beta-glucan stimulation increased the mRNA expression of iNOS, COX-2 and IL-6 in RAW264.7 macrophages in a concentration-dependent manner.
Animals ; Bacillus/*metabolism ; Cell Line ; Cyclooxygenase 2/biosynthesis/genetics ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/*drug effects/enzymology/immunology ; Mice ; Nitric Oxide/*biosynthesis/immunology ; Nitric Oxide Synthase Type II/biosynthesis/genetics/metabolism ; RNA, Messenger/biosynthesis/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; beta-Glucans/metabolism/*pharmacology

To investigate the purging effect of CD3AK/iNOS on primary leukemic cells from chronic myeloid leukemia patients in vitro, amphotropic packaging cell line PA317 transfected with the whole length of iNOS gene was cultivated, amplified and screened by G418. The viral titer was determined by the NIH3T3 cells. Human peripheral blood mononuclear cells were isolated and activated by anti-CD3 monoclonal antibody in vitro. CD3AK cells were incubated with viral supernatant and selected by G418. Resistant clones were assayed for iNOS gene expression by RT-RCR. The content of nitric oxide and the activity of iNOS in the culture supernatant of CD3AK/iNOS were evaluated by the method of Griess. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene was detected by serial dilution semi-quantitative net RT-PCR assay. The results showed that anti-G418 positive packaging cell line PA317 transfected with the whole length of iNOS gene clones could stably synthesize and excrete recombinant retroviral vectors. The titer of recombinant retroviral vectors was 1.0 x 10(5) CFU/ml. After being transfected by recombinant retroviral supernatant, the iNOS cDNA was expressed in CD3AK/iNOS. The content of NO and activity of iNOS that synthesized and excreted by CD3AK/iNOS were notably increased, compared with those of CD3AK. There were statistically significant differences in NO content and iNOS activity between two groups. After BMMNC or PBMNC from CML patients were co-cultured with CD3AK/iNOS, CD3AK/Neo and CD3AK/iNOS respectively, the expression of bcr/abl fusion gene in all of them was down-regulated by serial dilution semi-quantitative RT-PCR assay. It is concluded that construction of CD3AK/iNOS can markedly increase the content of NO and the activity of iNOS, which can be more efficient in in vitro purging leukemia cells for autologous hematopoietic stem cell transplantation.
Adult ; Aged ; Animals ; CD3 Complex ; immunology ; Cell Line ; Cytotoxicity, Immunologic ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; immunology ; pathology ; Male ; Mice ; Middle Aged ; NIH 3T3 Cells ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE2 production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-kappaB DNA binding activity, IkappaBalpha degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.
Animals ; Cell Line ; Cyclooxygenase 2/*genetics/metabolism ; Cytokines/immunology ; *Gene Expression Regulation ; Lipopolysaccharides/immunology/metabolism ; Mice ; Mitogen-Activated Protein Kinase Kinases/metabolism ; NF-kappa B/metabolism ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type II/*genetics/metabolism ; Reactive Oxygen Species/metabolism ; Superoxide Dismutase/*metabolism