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This study aimed to investigate the effects of gastric cancer interstitial fluid (GCIF) on tumors and explore the possible mechanism of Xiaotan Sanjie decoction (XTSJ) on treatment of gastric cancer from the view of regulating microRNA-21 (miR-21) expression. The GCIF was extracted and identified by measuring the levels of interleukin-8 (IL-8), intercellular adhesion molecule 1 (ICAM-1) and miR-21. The effects of GCIF on the proliferation of SGC-7901 cells and tumor growing were assessed by cell counting kit-8 (CCK-8) assay and subcutaneously transplanted tumor-bearing nude mice model, respectively. Additionally, inhibition effect of XTSJ decoction on proliferation of SGC-7901 cells intervened by GCIF were assessed in vitro and anti-cancer effect of it was further assessed using orthotopic transplanted tumor-bearing nude mice model. The concentration of SGC-7901 gastric cancer cells were dependent on the concentration of the added GCIF. After 72 hours of continuous culture, the interstitial fluid had an obvious proliferative effect on the SGC-7901 tumor cells, which was the most significant in the high concentration group. XTSJ decoction could inhibit the growth-promoting effect (P < 0.01) of GCIF on gastric cancer cells. Intervention of the GCIF might promote the growth (P < 0.05) of the subcutaneously transplanted tumors in nude mice and decrease the net weight of the tumor-bearing nude mice (P < 0.05) after tumor removal. The GCIF was able to up-regulate the expression (P < 0.001) of miR-21 in the subcutaneously transplanted tumors. XTSJ decoction could downregulate the expression (P < 0.05) of miR-21 in SGC-7901 orthotopically transplanted tumors. XTSJ decoction can inhibit the multiplicative effect of GCIF on gastric cancer cells, growth of gastric tumor and promotion effect of GCIF on tumors, probably due to the down-regulating miR-21 expression in tumor tissues. [ABSTRACT FROM AUTHOR]

Antitumor effects of combined transfer of suicide and cytokine genes were investigated in this study. Adenovirus harboring E. coli cytosine deaminase gene (AdCD) and adenovirus harboring murine granulocyte–macrophage colony-stimulating factor gene (AdGMCSF) were used simultaneously for in vivo gene transfer in melanoma-bearing mice. Growth inhibition of established tumors and prolongation of survival period were observed more significantly in tumor-bearing mice after transfection with AdGMCSF and AdCD followed by continuous injection of prodrug 5-fluorocytosine (5FC) when compared with mice treated with control adenovirus AdlacZ/5FC, AdCD/5FC or AdGMCSF alone (P < 0.01). after combined therapy the expression of mhc-i (h-2db) and B7–1 molecules on freshly isolated tumor cells increased greatly and more dendritic cells and CD8+ T cells infiltrated into the tumor mass. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy. Further experiments showed that apoptosis of tumor cells and induction of antitumor immune response might be involved in the mechanisms of the tumor cell killing by the combined therapy. Our results demonstrated that combined transfer of the GM-CSF and CD suicide genes, being able to inhibit the growth of melanoma synergistically and induce specific antitumor immune response efficiently, thus addressing the drawbacks of suicide gene therapy or cytokine gene therapy which were proved to be not satisfactory when used alone, might be of therapeutic potential for gene therapy of cancer. [ABSTRACT FROM AUTHOR]

Chemokine gene transfer represents a promising approach in the treatment of malignancies. Macrophage-derived chemokine (MDC) (CCL22) belongs to the CC chemokine family and is a strong chemoattractant for dendritic cells (DC), NK cells and T cells. Using adenoviral vectors, human MDC gene was transferred in vivo to investigate its efficacy to induce an antitumor response and to determine the immunologic mechanisms involved. We observed that intratumoral injection of recombinant adenovirus encoding human MDC (AdMDC) resulted in marked tumor regression in a murine model with pre-established subcutaneous 3LL lung carcinoma and induced significant CTL activity. The antitumor response was demonstrated to be CD4+ T cell- and CD8+ T cell-dependent. Administration of AdMDC induced chemoattraction of DC to the tumor site, facilitated DC migration to draining lymph nodes or spleen, and finally activated DC to produce high levels of IL-12. Furthermore, a significant increase of IL-4 production within the tumors was observed early after the AdMDC administration and was followed by the increase of IL-12 and IL-2 production. The levels of IL-2, IL-12 and IFN-gamma in serum, lymph nodes and spleen were also found to be higher in mice treated with AdMDC as compared with that in AdLacZ- or PBS-treated mice. The antitumor response induced by AdMDC was markedly impaired in IL-4 knockout mice, suggesting an important role of IL-4 in the induction of antitumor immunity by MDC. These results suggest that MDC gene transfer might elicit significant antitumor effects through efficient induction of antitumor immunity and might be of therapeutic potentials for cancer.

Dendritic cell (DC)-based tumor vaccine represents a promising approach to the immunotherapy of malignant tumors. We prepared a novel type of DC-based vaccine, stable conjugates of DCs and EL4 cells transduced with cDNA of OVA (E.G7). Immunization with DC-E.G7 conjugates led to generation of T helper (Th) 1 cytokine-producing cells, antigen-specific CD8(+) T cells, and strong antitumor immunity that is dependent on both CD4(+) T cells and CD8(+) T cells. To further increase the potency of the vaccine, interleukin 18-transfected DCs were used to prepare the IL18DC-E.G7 conjugates. Immunization with such conjugates significantly increased the production of Th1 cytokine-producing cells and the number of antigen-specific CD8(+) T cells, as well as stronger antitumor immunity. Furthermore, the increased Th1 cytokine production and stronger antitumor effect were not observed in mice depleted of IFN-gamma. These data indicated that DC-tumor cell conjugates are a potent tumor vaccine. Interleukin 18 can be administrated using gene-transfected cells and enhances antitumor immunity, which is mainly mediated by IFN-gamma.

Purpose: Tumor suppressor gene therapy and cytokine gene therapy have limited antitumor effects when used alone. Thus, in the present study, we investigated the antitumor potentials of the combined transfer of the p16 tumor suppressor gene and the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene., Methods: The adenovirus-harboring p16 gene (Adp16) and adenovirus-harboring GM-CSF (AdGMCSF) gene were utilized for the treatment of established tumors in vivo. The mice were inoculated s.c. with Renca renal carcinoma cells and 3 days later received an intratumoral injection of Adp16 in combination with AdGMCSF., Results: The results demonstrated that tumor-bearing mice treated with Adp16 and Ad-GMCSF showed more potent inhibition of tumor growth and a prolonged survival period than mice treated with Adp16. AdGMCSF, adenovirus-expressing beta-galactosidase or PBS (P<0.01). Treatments of the mice with Adp16 alone or AdGMCSF alone also showed obvious antitumor effects as compared with those mice treated with PBS (P<0.05). After combined p16 and AdGMCSF gene therapy, the expression of H2Kd and Fas molecules on freshly isolated tumor cells increased markedly, and more CD(4)+ T cells and CD(8)+ T cells infiltrated in the tumor sites. The cytotoxicity of natural killer cells and specific cytotoxic T lymphocytes increased more significantly after the combined therapy., Conclusions: Our results demonstrated that combination p16 gene and GM-CSF gene therapy could inhibit the growth of established tumors in mice more significantly through efficient induction of antitumor immunity.

Suicide gene therapy has been studied intensively for the treatment of cancer. A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC). To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy. We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma. AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration. The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently. A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P < .01). Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs. Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy.

Interleukin 2 (IL-2) expressing plasmid and interleukin 6 (IL-6)-expressing plasmid were encapsulated in liposome and administrated intratumoraly into tumor-bearing mice 4 days after subcutaneous inoculation of B16F10 melanoma cells. The results showed that treatment of tumor-bearing mice with IL-2 gene or IL-6 gene transfer inhibited the growth of subcutaneous tumor and prolonged the survival of tumor-bearing mice significantly when compared with the treatment of PBS or control gene transfer mediated by liposome (P < 0.01). Combined transfer of IL-2 gene and IL-6 gene was found to elicit inhibitory effects on the growth of B16F10 tumor more significantly and prolonged the survival period of tumor-bearing mice more obviously. We investigated the local immunity in tumor microenvironment and found that IL-2 and IL-6 gene transfer could significantly increase the expression of lymphocyte function-associated antigen-1 on tumor infiltrating lymphocytes (TIL) and MHC-I molecule on tumor cells freshly isolated from the tumor mass. The NK and CTL activity of TIL increased markedly after the combined transfer of these two cytokine genes. We also observed the systemic antitumor immune response in the tumor-bearing mice and demonstrated that NK and CTL activity of splenocytes and the production of IL-2, tumor necrosis factor and interferon-gamma from splenocytes increased obviously in mice after the combined transfer of IL-2 and IL-6 gene. In conclusion, local and systemic antitumor immunity of the tumor-bearing host could be induced efficiently after the combined gene transfer. The enhanced specific and non-specific antitumor immunity might be responsible for the more potent antitumor effects of the combined gene therapy.

Background: Bone marrow transplantation (BMT) and gene therapy are potent approaches to the recovery of bone marrow depression and induction of antitumor immunity after chemotherapy for the treatment of malignancies. In the present study, enhanced antitumor effect of BMT in combination with fibroblast-mediated interleukin (IL)-2 and IL-3 gene therapy was observed in tumor-bearing mice after chemotherapy., Methods: BALB/c mice were inoculated s.c. with J558L plasmacytoma cells and injected i.p. with cyclophosphamide 300 mg/kg 3 days later. 24 hours after chemotherapy syngeneic bone marrow cells in combination with NIH3T3 fibroblast cells engineered to produce IL-2 (NIH3T3-IL-2) and/or NIH3T3 cells engineered to produce IL-3 (NIH3T3-IL-3) were implanted into the tumor-bearing mice., Results: BMT in combination with implantation of either NIH3T3-IL-2 or NIH3T3-IL-3 cells exerted significant inhibition on the growth of J558L tumors and prolonged the survival period of the tumor-bearing mice as compared with the treatments with Hanks solution, BMT alone, or BMT plus implantation of NIH3T3 cells transduced with Neo gene. Synergistic antitumor effect was observed in mice after combined BMT and cytokine gene therapy. The cytotoxicities of natural killer cells, cytotoxic T lymphocytes, and macrophages in mice increased markedly after the combined treatment. Recovery of CFU-GM, CFU-MK and CFU-E formation in mice after combined therapy was accelerated obviously in mice after combined therapy., Conclusions: BMT in combination with fibroblast-mediated IL-2 and IL-3 gene therapy elicited augmented antitumor effects synergistically in tumor-bearing mice after chemotherapy mainly through induction of antitumor immune response and accelerated recovery of hematopoiesis.

Objective: To investigate the antitumor effect of combined adenovirus encoding E. coli cytosine deaminase (AdCD) and adenovirus encoding murine interleukin 2 (AdIL-2) on murine melanoma., Methods: C57BL/6 mice were inoculated s.c. with B16F10 melanoma cells and 3 days later received injections of AdCD and/or AdIL-2 at the site of tumor inoculation followed by administration of 5-flurocytosine (5FC) 300 mg/kg per day for 10 days., Results: Mice receiving AdCD/5FC/AdIL2 therapy developed tumors more slowly and survived much longer when compared with mice treated with AdCD/5FC, AdIL2, AdlacZ/5FC, or PBS. Immunological analysis illustrated that combined treatment could enhance NK activity and CTL activity. Flow cytometry demonstrated that AdCD/5FC/AdIL2 therapy increased the expression of MHC-1 and CD80 molecules on freshly isolated tumor cells. The CD4+ and CD8+ T cell infiltration in the tumor increased significantly after the combined therapy., Conclusions: Our data showed that combined transfer of CD suicide gene and IL-2 gene could inhibit the tumor growth more significantly. The increased specific and non-specific antitumor immunity might be responsible for the enhanced therapeutic effect.

Fibroblast-mediated cytokine gene therapy has proven to be a promising strategy for restoring hematopoiesis following repeated chemotherapy. Interleukin 3 (IL-3) and interleukin 6 (IL-6) can synergistically promote the recovery of hematopoiesis following chemotherapy. In this investigation, combined use of fibroblast-mediated IL-3 and IL-6 gene therapy was tested for hematopoietic effects on mice with or without 5-fluorouracil administration. The results demonstrated that combined therapy with IL-3 gene-modified NIH3T3 cell (NIH3T3-IL-3) and IL-6 gene-modified fibroblast NIH3T3 cell (NIH3T3-IL-6) implantation achieves obvious stimulation of hematopoiesis in normal mice and accelerates recovery of hematopoiesis. In normal mice the quantities of platelets, neutrophils, and total white blood cells in peripheral blood increased significantly after the combined implantation of NIH3T3-IL-3 and NIH3T3-IL-6 cells. The numbers of colony-forming unit (CFU) granulocyte/macrophage (CFU-GM) and CFU megakaryocyte (CFU-MK) formed by stem cells in bone marrow was significantly higher after the combined implantation of NIH3T3-IL-3 and NIH3T3-IL-6 cells than after the implantation of NIH3T3-IL-3 alone, NIH3T3-IL-6 alone, or neomycin gene-modified NIH3T3 cells. In hematopoiesis-depressed mice induced by preinjection with 5-fluorouracil at the dose of 150 mg/kg before cell implantation, the platelets, neutrophils, and white blood cells showed accelerated recovery, and the numbers of CFU-GM and CFU-MK formed by bone marrow cells were also markedly higher after the combined implantation of NIH3T3-IL-3 and NIH3T3-IL-6 cells than in control groups. Our data show that combined use of fibroblast-mediated IL-3 and IL-6 gene therapy may be of clinical relevance for the recovery of hematopoietic depression for patients after chemotherapy.

Esculentoside A, a kind of saponin isolated from the root of the Chinese herb Phytolaca esculenta, is reported to possess potent anti-inflammatory effects in acute and chronic experimental models. In the present study, we investigated the effects of esculentoside A on the production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in mice. In vitro experiments demonstrated that esculentoside A (0.1-10 mumol/l) significantly reduced the release of TNF from the peritoneal macrophages derived from mice pretreated with thioglycolate. IL-1 and IL-6 secretion was also obviously inhibited in a concentration-dependent manner by esculentoside A from 0.01 to 10 mumol/l. In vivo experiments demonstrated that detectable TNF was observed 0.25 h after injection, was maximal at 0.5 h, and returned to baseline at 4 h. Maximal production of IL-1 and IL-6 were observed to be 1 and 2 h, respectively, after injection of LPS. Pretreatment of mice with 5, 10, or 20 mg/kg esculentoside A once a day for 7 consecutive days dose-dependently decreased the TNF, IL-1 and IL-6 levels in the sera of mice following LPS challenge. TNF, IL-1, and IL-6 are important cytokines involved in the pathogenesis of inflammatory lesions. Inhibition of inflammatory cytokine production may contribute to the anti-inflammatory effects of esculentoside A.

The antitumor effect of the combined transfer of a suicide gene and a cytokine gene was evaluated in the present study. Adenoviruses expressing Escherichia coli cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin-2 (AdIL-2) were utilized for the treatment of established tumors. The mice were inoculated s.c. with FBL-3 erythroleukemia cells and 3 days later received an intratumoral injection of AdCD in the presence or absence of AdIL-2 followed by intraperitoneal 5-fluorocytosine (5-FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5-FC in combination with AdIL-2 showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdCD/5-FC, AdIL-2, adenovirus expressing beta-galactosidase/5-FC or phosphate-buffered saline. The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrating the tumor after combined therapy. The splenic natural killer and cytotoxic T lymphocyte activities increased significantly in the mice after combined therapy with AdCD/5-FC/AdIL-2. Our results demonstrate that therapy combining a suicide gene and IL-2 gene can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.

A recombinant vaccinia virus containing and expressing the gene for murine granulocyte-macrophage colony-stimulating factor (VVGM-CSF) was constructed and tested for its antitumor activity. A murine tumor model was established by injecting 10(5) B16F10 melanoma cells into the right rear leg of C57BL/6 mice. Three days after B16F10 inoculation, VVGM-CSF or a thymidine kinase gene-deficient vaccinia virus (VVTK) were injected intratumorly twice weekly for 3 weeks. The results showed that VVGM-CSF treatment significantly inhibited the growth of subcutaneous tumor and delayed the survival period of tumor-bearing mice. Splenic lymphokine-activated killer cell, natural killer cell, and cytotoxic T lymphocyte activities were not found to be altered after VVGM-CSF or VVTK therapy. Cytotoxic and phagocytic activity of peritoneal macrophages were found to be greatly elevated in mice treated with VVGM-CSF. Nitric oxide released from the macrophages was also increased. Considering these data, we may speculate that continuous secretion of GM-CSF and activation of macrophages may contribute to the antitumor effects of VVGM-CSF injected intratumorally.

Vaccinia melanoma oncolysate (VMO) prepared with recombinant vaccinia virus encoding the gene of murine granulocyte/macrophage-colony-stimulating factor (GM-CSF) was tested for its therapeutic effect on melanoma pulmonary metastasis. The murine pulmonary metastasis model was established by injecting 2 x 10(5) B16F10 melanoma cells into the tail vein of a C57BL/6 mouse. Intraperitoneal injection of VMO was performed in tumor-bearing mice 3 and 10 days after B16F10 cell inoculation. The results showed that treatment with VMO prepared with GM-CSF-gene-encoded vaccinia virus (GM-CSFVMO) significantly decreased the number of murine pulmonary metastases and prolonged the survival of the tumor-bearing mice. Lymphocytes isolated from fresh blood and spleen of GM-CSFVMO-treated mice showed higher cytolytic activity against B16F10 melanoma cells when compared with lymphocytes from the mice of other treatment groups. Natural killer activity remained unchanged in the GM-CSFVMO-treated group. Cytotoxic activities of peritoneal macrophages were found to be greatly elevated in mice treated with GM-CSFVMO. Further study illustrated that the increased tumor necrosis factor and nitric oxide release from macrophages may contribute to their cytotoxic effects. These results suggest that the tumor oncolysate vaccine prepared with GM-CSF-gene-encoded vaccinia virus has a potent therapeutic effect on tumor metastasis through the efficient induction of antitumor immunity of the host, mainly through the cytotoxic effects of cytotoxic T lymphocytes and macrophages.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates the immune response, acute phase anaphylactic reaction, and haematopoiesis. Lipopolysaccharide (6-24 mug/ml) significantly induced IL-6 release from murine spleen cells. In cultured rabbit synovial cells interleukin-1 (IL-1, 1-10 U/ml) induced IL-6 production in a concentration-dependent manner. Triazolodiazepine (Tri) is a hetrazepine platelet-activating factor antagonist. In this study we found that Tri (0.1-10 mumol/l) exerted strong inhibitory effects on LPS stimulated IL-6 production in murine spleen cells. Kinetic studies showed that the inhibition of IL-6 release was time-independent. In rabbit synovial cells Tri also reduced IL-6 release induced by IL-1 and tumour necrosis factor. Inhibition of cytokine production by Tri may partially explain its wide and strong anti-inflammatory effects.

The effects of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on DNA synthesis of rabbit synovial cells were studied. IL-1 beta 1000-10,000 U.ml-1, IL-6 10-1000 U.ml-1, TNF alpha 0.5-50 U.ml-1 and GM-CSF 1-100 ng.ml-1 concentration-dependently stimulated DNA synthesis in rabbit synovial cells in culture. Leflunomide (LFM) and its metabolite A77 1726 elicited an inhibitory effect on such cytokine-induced DNA synthesis of synovial cells. These results suggested that IL-1 beta, IL-6, TNF alpha and GM-CSF play a key role in the pathogenesis of rheumatoid arthritis. Inhibition of cytokine-induced proliferation of synovial cells by LFM may partially explain its antirheumatic activity.

Leflunomide (LFM, HWA 486) is an isoxazol derivative with antiphogistic and novel immunomodulating properties. It has been shown to be very effective in preventing and curing arthritis. In this report we found that platelet-activating factor (PAF) at 0.1-10 micrograms.ml-1 significantly stimulated DNA synthesis in cultured rabbit synovial cells. While LFM and its metabolite A771726 elicited inhibitory effects on this action of PAF. These two agents were also shown to markedly inhibit A23187 induced PAF production from rat peritoneal macrophages. The inhibition was dose and time-dependent. The inhibitory effects of LFM and A771726 on DNA synthesis in synovial cells and PAF production from macrophages may play an important role in the antiinflammatory effects of LFM.

Effects of triazolodiazepine (Tri) on mouse splenocyte proliferation and macrophage phagocytosis were studied. Tri 0.1-10 mumol.L-1 significantly inhibited concanavalin A (Con A) 5 micrograms.ml-1 and lipopolysaccharides (LPS) 10 micrograms.ml-1-induced splenocyte proliferation and phagocytic activity of activated peritoneal macrophages. Tri 10 mumol.L-1 reduced Con A-induced colony stimulating factor release from mouse splenocytes and LPS-challenged intracellular and extracellular interleukin-1 production from peritoneal macrophages. These results partially explain the wide and strong anti-inflammatory effects of Tri.

Esculentoside A (EsA) is a kind of saponin isolated from Phytolacca esculenta Van Houtte. It was shown to significantly inhibit phagocytic activity, intracellular and extracellular production of interleukin-1 by thioglycollate primed murine peritoneal macrophages in vitro at the concentration 0.01-1.0 mumol.L-1. In vivo experiments showed that EsA 2.5-5 mg.kg-1 markedly decreased serum hemolysin concentration in sensitized mice challenged with sheep red blood cells. Inhibition of antibody production by B lymphocytes, phagocytosis and the production of inflammatory mediators by macrophages may partially explain the wide and strong anti-inflammatory effect of EsA.

The effects of leukotrienes (LT) and platelet activating factor (PAF) on DNA synthesis and proliferation of bovine cerebral microvascular smooth muscle cells (BCMSMC) were studied. At 100 pmol.L-1, LTB4, LTC4, LTD4, and PAF promoted the DNA synthesis by 44%, 50%, 48%, and 57%, and enhanced the cell proliferation by 33%, 47%, 27%, and 40%, respectively. Dauricine and anisodamine inhibited the DNA synthesis of the cells induced by LT and PAF (0.1-100 mumol.L-1). These results indicate the bright future of the 2 drugs in the prevention and treatment of cerebral vascular diseases.

Radioactivities of [3H]TdR uptaken by splenocytes and released from [3H]TdR-labeled YAC-1 cell line were measured to determine the degree of lymphocyte proliferation and natural killer (NK) cell activity. Seven days after mice treated with Phytolacca acinosa polysaccharides I (PAP-I) 5-50 mg.kg-1, the NK cell activity, and lymphocyte proliferation induced by Con A 5 micrograms.ml-1 or lipopolysaccharides 10 micrograms.ml-1 were significantly augmented. Splenocytes from mice treated with ip PAP-I 5-50 mg.kg-1 were incubated with Con A 5 micrograms.ml-1 for 24 h to induce interleukin-2 (IL-2) and for 40 h to induce NK cytotoxic factor (NKCF). Radioactivities of [3H]TdR uptaken by CTLL-2 cell line and YAC-1 cell line were used to measure the IL-2 and NKCF activities, respectively. PAP-I enhanced the production of IL-2 and NKCF. These results suggest that PAP-I augments the immunological functions in vivo.

Phytolacca acinosa polysaccharides II (PAP-II), a kind of polysaccharides isolated from Phytolacca acinosa Roxb with MW 40 kDa, on lymphocyte proliferation and colony stimulating factor (CSF) production from splenocytes in vitro, was studied. The radioactivities of [3H] TdR uptake by lymphocyte and bone marrow cells were used to determine the ability of lymphocyte proliferation and CSF production respectively. PAP-II was found to significantly augment splenocyte proliferation in a dose-dependent fashion, and significantly enhance Con A (1, 2.8 micrograms.ml-1) and LPS (3, 10, 30 micrograms.ml-1) induced lymphocyte proliferation at concentration of 31-125 micrograms.ml-1. As the concentration of PAP-II increased, significant suppression of Con A-induced lymphocyte proliferation was observed. Induction of CSF by PAP-II from splenocyte was confirmed at the present study. The optimal dosage was 100 micrograms.ml-1 and the optimal effect occurred on day 5. These results suggest that PAP-II can augment immunological function and enhance hematopoiesis.

In the present study the effect of platelet activating factor (PAF) antagonist WEB 2086 on the production of tumor necrosis factor (TNF) from primed murine peritoneal macrophages was investigated. At 10(-6) and 10(-5) mol/L, WEB 2086 was found to significantly inhibit LPS induced TNF production from macrophages by activated thioglycollate solution. WEB 2086 inhibition of TNF release began 4 h after LPS stimulation and lasted 22 h with a peek at 16 h. The results showed that PAF might play an important role in the production of TNF. Four methods were compared in the bioassay of TNF. The data demonstrated that L-929 cells treated with actinomycin D and sodium fluoride were the most sensitive for the assay of TNF. This method was employed in this study.

The effects of Phytolacca acinosa polysaccharides I (PAP-I), a polysaccharide extracted from Phytolacca acinosa Roxb: on splenic lymphocyte proliferation and cytokines production from splenic lymphocyte and macrophage were studied. Lipopolysaccharides (LPS) and PAP-I were found to significantly augment splenic lymphocyte proliferation of normal BALB/c, nude BALB/c and NC mice in vitro, but concanavalin A (Con A) was shown to stimulate only normal BALB/c and nude BALB/c splenic lymphocyte proliferation. Also, PAP-I significantly enhanced Con A or LPS-induced lymphocyte proliferation and mixed lymphocyte reaction. Significant enhancement of colony stimulating factor (CSF) production was observed from splenic lymphocyte of normal BALB/c and nude BALB/c mice but not from NC mice when treated with PAP-I for 5 d. PAP-I was shown to significantly enhance interleukin-2 (IL-2) production from normal mice splenocyte and Con A stimulated normal mice splenocyte in a concentration-dependent fashion. Supernatant of PAP-treated macrophage (M phi) were collected and CSF activity was tested. The results confirmed that PAP-I can significantly stimulate M phi to secret CSF activity on d 1. The supernatant also contained a cytokine which exhibited a synergistic action with recombinant murine granular-macrophage CSF (RMGM-CSF) to stimulate mice bone marrow cell proliferation. PAP-I, 5-50 mg.kg-1, ip can enhanced splenic lymphocyte proliferation and IL-2 production. These findings indicate that PAP-I can augment immunologic function in vitro and in vivo.

By using [3H] WEB 2086, a PAF antagonist, specific binding sites of PAF on bovine anterior cerebral arterial smooth muscle cells was identified. Two populations of binding sites with different dissociation constants on the cells were found. The Kd-1 = 22.8 +/- 5.0 nmol.L-1, Kd-2 = 186 +/- 20.5 nmol.L-1 at 25 C. The total number of binding sites were Bmax-1 = 2.1 +/- 0.3 pmol/10(6) cells and Bmax-2 = 12.1 +/- 1.5 pmol/10(6) cells. Dauricine and tetrandrine, two active compounds with similar chemical structure extracted from traditional Chinese herbs, were found to inhibit [3H] WEB 2086 specific binding significantly in culture cells.

The effects of leukotrienes (LTs) and platelet activating factor (PAF) on DNA synthesis in bovine anterior cerebral arterial smooth muscle cells were studied. The results showed that LTB4, LTD4 and PAF were all stimulatory to the cells. The maximal rates of stimulation were 32%, 29% and 77%, respectively, for LTB4, LTD4 and PAF at 10(-7) mol/L. Both dauricine and anisodamine at concentrations of 10(-7) to 10(-4) mol/L exhibited strong inhibitory effects on LTs and PAF induced DNA synthesis.

The action of platelet activating factor in the rat brain was detected by Evans blue staining after injection of PAF into the rat brain. The results show that PAF increased the Evans blue staining of the brain, but no staining was observed without prior injection of PAF. Meanwhile, PAF was shown to stimulate the release of 14C-arachidonic acid (14C-AA) in the cerebral microvascular smooth muscle cells (CMSMC). SZ-1, a new synthetic drug, dose-dependently inhibited the Evans blue staining of the rat brain and the PAF induced 14C-AA release in CMSMC. These results indicate that the action of PAF in the rat brain might be related to the stimulation of AA release. SZ-1 may antagonize the PAF receptor and protect the brain from PAF induced damage.