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Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy that causes a poor survival. We aimed to identify its prognostic factors and to develop a nomogram that will predict survival of ICC patients among all stages., Methods: A total of 442 patients with pathology-proven ICC registered at the Fifth Medical Center of PLA General Hospital between July 2007 and December 2019 were enrolled. Subjects were followed for survival status until June 30, 2020. A prognostic model visualized as a nomogram was constructed in the training cohort using multivariate cox model, and was then validated in the validation cohort., Results: The median age was 55 years. With a median follow-up of 50.4 months, 337 patients died. The median survival was 11.6 months, with 1-, 3- and 5-year survival rates of 48.3%, 22.7% and 16.2%, respectively. Factors associated with overall survival were multiple tumors, lymph node involvement, vascular invasion, distant metastasis, decreased albumin, elevated lactate dehydrogenase (LDH), decreased iron, elevated fibrinogen, elevated CA125 and elevated CA19-9. A nomogram predicting survival of ICC patients at the time of diagnosis achieved a Harrel's c-statistic of 0.758, significantly higher than the 0.582 of the TNM stage alone. Predicted median survivals of those within the low, mid and high-risk subgroups were 35.6, 12.1 and 6.2 months, respectively., Conclusions: A nomogram based on imaging data and serum biomarkers at diagnosis showed good ability to predict survival in patients with all stages of ICC. Further studies are needed to validate the prognostic capability of our new model., Competing Interests: The authors have no conflict of interests related to this publication., (© 2022 Authors.)

A phytochemical investigation of an extract of the leaves of Piper betle , guided by a synergistic antibacterial screen, led to the isolation and structural elucidation of 10 new neolignans, Pibeneolignan A-J ( 1 - 10 ), together with 11 known compounds. The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic data, single-crystal X-ray diffraction analysis, and experimental and calculated ECD investigations. Compounds 1 and 2 are new naturally occurring neolignan skeletons, based on the cyclohept-2-ene-1,4-dione framework. We propose that these natural products are biosynthetically formed from bicyclic [3.2.1] neolignans by oxidative cleavage and ring opening at C-1' and C-2'. Among these compounds, 9 , 13 , 15 , and 16 , in combination with norfloxacin against an effluxing S. aureus strain (SA1199B), exhibited significant synergistic activity with fractional inhibitory concentration indices (FICIs) of 0.078, 0.156, 0.125, and 0.25, respectively. Bacterial growth curves, ethidium bromide (EtBr) efflux, and qRt-PCR were further employed to verify their synergistic antibacterial mechanism. Furthermore, computational molecular modeling suggested the binding of compounds 14 - 17 and 19 to the active site of the modeled structure of the NorA efflux pump, which is the main efflux pump in SA1199B.

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.

Objective: To investigate the preventive effects of Qiangzhi Decoction (, QZD) on influenza A pneumonia through inhibition of inflammatory cytokine storm in vivo and in vitro., Methods: One hundred ICR mice were randomly divided into the virus control, the Tamiflu control and the QZD high-, medium-, and low-dose groups. Mice were infected intranasally with influenza virus (H1N1) at 10 median lethal dose (LD50). QZD and Tamiflu were administered intragastrically twice daily from day 0 to day 7 after infection. The virus control group was treated with distilled water alone under the same condition. The number of surviving mice was recorded daily for 14 days after viral infection. The histological damage and viral replication and the expression of inflammatory cytokines were monitored. Additionally, the suppression capacity on the secretion of regulated on activation normal T cells expressed and secreted (RANTES) and tumor necrosis factor-α (TNF-α) in epithelial and macrophage cell-lines were evaluated., Results: Compared with the virus control group, the survival rate of the QZD groups significantly improved in a dose-dependent manner (P<0.05), the viral titers in lung tissue was inhibited (P<0.05), and the production of inflammatory cytokines interferon-γ (IFN-γ), interleukin-6 (IL-6), TNF-α, and intercellular adhesion molecule-1 (ICAM-1) were suppressed (P<0.05). Meanwhile, the secretion of RANTETS and TNF-α by epithelial and macrophage cell-lines was inhibited with the treatment of QZD respectively in vitro (p<0.05) CONCLUSIONS: The preventive effects of QZD on influenza virus infection might be due to its unique cytokine inhibition mechanism. QZD may have significant therapeutic potential in combination with antiviral drugs.

Baicalin, a natural product isolated from Scutellariaradix, has been reported to have significant in vivo and in vitro anti-influenza virus activity, but the underlying mechanism remains poorly understood. In this study, we found that baicalin inhibited autophagy induced by influenza virus A3/Beijing/30/95 (H3N2) in both A549 and Ana-1 cells. The results showed that H3N2 induced autophagy by suppressing mTOR signaling pathway, which however could be significantly inhibited by baicalin. Baicalin could suppress the expression of Atg5-Atg12 complex and LC3-II, and attenuate autophagy induced by starvation. Thus, the inhibition of autophagy induced by virus may account for the antiviral activities of baicalin against H3N2. Autophagy may be a potential marker in developing novel anti-influenza drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this report, single crystalline rutile TiO2 nanoparticles and nanorods were synthesized via the hydrothermal method using titanium tetra-isopropoxide as a precursor then, these were coated on top of a fluorine-doped tin oxide (FTO) substrate by using a doctor blade and direct deposition, respectively. Consequently, TiO2 nanorods-based dye-sensitized solar cells (DSSC) exhibit a J(sc) of 3.37 mA/cm2, a V(oc) of 0.82 V and fill factor of 60.1% with an overall conversion efficiency of 1.66%. This result shows an increase of around 38% for current density and 35% for conversion efficiency. Also, with respect to the impedance data, TiO2 nanorods-based DSSCs had smaller semicircles than did the nanoparticles-based DSSCs. These results demonstrate that the nanorod structure can have fast electron transport and reduced charge recombination.

Objective: To extract tumor interstitial fluid (TIF) from MKN-45 gastric cancer which is similar to "muddy phlegm" in Chinese medicine and observe influences of MKN-45 tumor interstitial fluid (MKN-45 TIF) intervention on metastasis of gastric cancer and on the expressions of vascular endothelial growth factor (VEGF), kinase insert domain containing receptor (KDR), epithelial-cadherin (E-cad), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1) and telomerase genes and proteins in primary tumor tissue., Methods: An MKN-45 tumor-bearing model was established in 50 nude mice. The modeled animals were equally randomized to 5 groups: the simple tumor-bearing group (model group), the normal saline (NS) via tail vein injection (i.v.) group (NS i.v. group), MKN-45 TIF i.v. group (TIF i.v. group), NS intraperitoneal injection (i.p.) group (NS i.p. group), and MKN-45 TIF i.p. group (TIF i.p. group). The TIF and NS intervention groups received injection (i.p. or i.v.) of MKN-45 TIF or NS twice a week, 0.2 mL at a time. After 8 weeks, the primary tumors were removed, weighed and HE stained to observe tumor metastasis. The primary tumor tissues were analyzed by immunohistochemistry and real-time quantitative PCR to detect expressions of VEGF, KDR, E-cad, COX-2, ICAM-1, and telomerase genes and proteins in different groups., Results: There were significant differences in tumor weight between TIF intervention groups and the model and NS intervention groups. Tumor metastasis was observed in all 5 groups, but the tumor metastasis rate in TIF intervention groups was significantly higher than those in the model and NS intervention groups. The gene and protein expressions of gastric cancer-related factors VEGF, KDR, COX-2, ICAM-1 and telomerase were unregulated while the gene and protein expressions of E-cad were downregulated in TIF intervention groups., Conclusions: TIF promotes tumor growth, invasion and metastasis of gastric cancer. These findings provide preliminary experimental clues for verifying the hypothesis of "tumor-phlegm microenvironment".

Objective: To explore a method of extracting tumor interstitial fluid (TIF) which is similar to muddy phlegm in Chinese medicine (CM), interleukin-8 (IL-8) in concentration was taken as the representative of the content of TIF, analyzed in the extracted TIF and the original tumor tissue, and examined to see whether TIF has an interfering effect on tumor recurrence., Methods: Tumor tissue was ground, centrifuged, and filtered for intercellular substances. Tumor-bearing Kunming S180 mice were raised for 21 days and then the tumors were removed to observe the influence of intervention with TIF, normal saline (NS) and a blank control on tumor recurrence., Results: The content of IL-8 in the filtered and unfiltered tumor tissue was not significantly different (P>0.05). Postoperative tumor recurrence in TIF intervention group was significantly higher than that in the NS intervention and control groups (60%, 12/20 vs. 20%, 4/20. vs. 15%, 3/20, χ(2) =11.058, P<0.01). Tumor cells grew vigorously and infiltrated to muscular tissue in TIF intervention group. Large numbers of tumor cells were seen necrotic in the NS intervention group, and small numbers of tumor cells were seen necrotic in the blank control group., Conclusions: TIF can be effectively extracted by the means described. It does not contain tumor cells, but its contents such as IL-8 may stimulate tumor cell growth and promote postoperative tumor recurrence, which provided preliminary experimental basis for hypothesis of "tumor-phlegm microenvironment".

Background: Pain has a substantial impact on patients' activities and overall quality of life, but current conventional drugs have debilitating side effects, including gastrointestinal disorders. Thus there is a pressing need for new therapies with fewer side effects to alleviate cancer pain. We recently developed a topical herbal formula Xiaotan Tongluo analgesic gel (XTTL gel) based on the principles of traditional Chinese herbalism, and we have received positive feedback from bone cancer pain patients. The aim of this study was to determine the analgesic effects and explore the mechanisms of XTTL gel in a rat model of bone cancer pain., Methods: The rat model of bone cancer pain was established by inoculating Walker-256 rat carcinoma cells directly into the right tibial medullary cavity of Wistar rats. The rats were randomly assigned to three groups (n = 10 per group): (1) sham bone cancer control (sham group): vehicle (PBS) inoculation without carcinoma cells plus topical administration of blank gel; (2) Sham treatment control (vehicle group): Walker-256 cell inoculation plus topical administration of blank gel; (3) XTTL gel treatment (treatment group): Walker-256 cell inoculation plus topical administration of XTTL gel. XTTL gel treatments were applied daily for 7 days starting on day 14 following inoculation. Outcomes were assessed 21 days after inoculation by mechanical allodynia, histological staining, and by measuring concentrations of type I collagen carboxy-terminal telopeptide (ICTP) and bone-specific alkaline phosphatase (BAP) in serum., Results: Fourteen days after cancer cell incubation, significant mechanical allodynia in the ipsilateral hind paw and tumor growth in proximal end of the tibia were observed in the vehicle and treatment groups but not in the sham group. At day 21, mechanical withdrawal thresholds in treatment group rats were significantly higher ((4.8557 +/- 0.8336) g) compared with those of the vehicle group ((1.8630 +/- 1.4369) g, P < 0.05). ICTP and BAP levels increased significantly in vehicle group rats ((101.5176+/- 11.0694) U/L and (370.7838 +/- 12.8273) U/L, respectively) compared with those of the sham group ((11.7553 +/- 1.1885) U/L and (185.7338 +/- 3.6761) U/L, respectively; P < 0.05). XTTL gel decreased the level of blood serum ICTP ((41.8998 +/- 6.4970) U/L, P < 0.05) but had little effect on blood serum BAP ((365.5338 +/- 18.5361) U/L, P > 0.05)., Conclusion: Topical use of XTTL gel may have an analgesic effect on bone cancer pain, an effect mediated by lowering of ICTP levels and inhibiting bone resorption.

Unlabelled: The treatment of advanced lung cancer remains a major challenge in clinical medicine, justifying an urgent need for new therapeutic approaches. In a rather unique international collaboration, 43 patients with advanced lung cancer were treated using iodine-131-labeled tumor necrosis therapy chimeric antibody (131I-chTNT)., Methods: Patients were treated either with intravenous (i.v.) infusion (n = 22), intratumoral injection using a computer tomography (CT)-guided catheter (n = 16), or combination i.v. and intratumoral infusion (n = 5). All patients, regardless of route of administration, received 2 doses of 131I-chTNT on days 1 and 14., Results: The results showed that of those patients receiving i.v. injection alone, 2 achieved partial response (PR) (9%), 16 had stable disease (73%), and 4 progressed (18%). Of those patients receiving intratumoral injection only, 1 had a complete response (CR) (6%), 8 achieved PR (50%), 7 had stable disease (44%), and none (0%) progressed. Finally, of those patients receiving both i.v. and intratumoral administration, 1 had a CR (20%), 1 achieved PR (20%), 2 had stable disease (40%), and 1 (20%) showed progression., Conclusions: These promising results demonstrate that sufficient doses of radiolabeled antibody can be safely delivered to tumors to cause significant therapeutic effects in advanced lung cancer.

In this work, the recovery rate (RR) of preconcentration technique was examined using a combination of the Peltier cooling (PC) and thermal desorption (TD) system for the gas chromatographic (GC) analysis of reduced sulfur compounds (RSC) in air. The possible loss or gain of analytes resulting from the use of the PC/TD system was estimated by analyzing equimolar standards (10ppm) of four S compounds including H(2)S, CH(3)SH, DMS, and DMDS in two different manners: (1) by injecting directly the four S compounds into the GC via injector and (2) by introducing them through the PC/TD system. When a series of tests were conducted on different types of gas media (ultrapure air versus N(2)) and across varying relative humidity (RH), it was found that the RR values for the four S compounds vary from 80 to 110% range. The overall results of our study thus indicate that the RR for the PC/TD system is fairly good and that subtle differences in their RR values may reflect the combined effects of different factors investigated in this study such as types of gas media, RH change, and properties of target analytes (e.g., recovery rate of the least (H(2)S) versus the highest compound (DMDS)).

Purpose: Tumor necrosis treatment (TNT) uses degenerating tumor cells and necrotic regions of tumors as targets for radioimmunotherapy. Previous studies in animal tumor models and clinical trials have demonstrated that when linked to the therapeutic radionuclide iodine-131, recombinant chimeric TNT antibody ((131)I-chTNT) can deliver therapeutic doses to tumors regardless of the location or type of malignancy. Therapeutic efficacy and toxicity of (131)I-chTNT in advanced lung cancer patients were studied in this pivotal registration trial., Patients and Methods: Patients with advanced lung cancer were treated with systemic or intratumoral injection of (131)I-chTNT in eight oncology centers in China. The objective response rate (ORR) was assessed as the primary end point., Results: All 107 patients who were entered onto the study and completed therapy had experienced treatment failure after prior radiotherapy or chemotherapy a mean of three times. The results showed an ORR of 34.6% (complete response, 3.7%; partial response, 30.8%; no change, 55.1%; and progressive disease, 10.3%) in all patients and 33% in 97 non-small-cell lung cancer patients. A biodistribution study demonstrated excellent localization of the radioactivity in tumors in both systemically and intratumorally injected patients. The most obvious adverse side effect was mild and reversible bone marrow suppression., Conclusion: Radioimmunotherapy with (131)I-chTNT was well tolerated and can be used systemically or locally to treat refractory tumors of the lung.

Based on the fact that aberrant overexpression of some growth factor receptors was observed in a variety of human cancer cells, a novel nonviral gene delivery system GE7, which contains a 16-amino-acid ligand for identifying EGF receptor was constructed for tumor-targeted gene therapy. Intravenous administration of GE7 system revealed that it has the ability to target beta-galactosidase (beta-gal) reporter gene into murine hepatoma (Hepa) cells. Owing to the limited antitumor effects elicited by a single-gene transfer, recent efforts to treat malignancy using combined gene therapy have been accomplished with varying degrees of success. In this study, the human cyclin-dependent kinase inhibitor gene p21(WAF-1) and the murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injection of the EGF R targeted GE7/DNA complex into murine hepatoma-bearing mice. The results demonstrated that combined administration of p21(WAF-1) and GM-CSF could remarkably inhibit the growth of subcutaneously transplanted hepatoma Hepa cells, and significantly increase the survival rate of tumor-bearing mice. The activities of natural killer (NK) cells and specific cytotoxic T lymphocytes (CTL) were clearly enhanced after combined gene therapy. In vitro experiments showed that p21(WAF-1) gene transfer exhibited a suppressive function on the growth of Hepa cells and the expression of H-2K(b) and B7-1 molecules on Hepa cells increased significantly after combined genes delivery. All these results suggested that the GE7 system was able to target therapeutic genes efficiently to cancer cells, which showed high EGF R expression. The cotransfer of p21(WAF-1) and GM-CSF genes apparently inhibited the growth of tumors through (a) the arrest of tumor cell growth and (b) the enhancement of systemic antitumor immunity.

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.

Antibody-targeted superantigen C215Fab-SEA is a fusion protein of staphylococcal enterotoxin A (SEA) and the Fab region of the tumor-reactive C215 mAb. It can trigger CTL against C215 antigen-positive tumor cells and induce tumor-suppressive cytokines. However, the antitumor effect of C215Fab-SEA is not satisfactory because of suboptimal production of Th1 cytokines after repeated administration. Interleukin 18 (IL-18) is a novel cytokine with profound effects on Th1 cellular response. In this study, we showed that adenovirus-mediated intratumoral IL-18 gene transfer strongly improved the therapeutic efficacy of C215Fab-SEA in the pre-established C215 antigen-expressing B16 melanoma murine model. More significant tumor inhibition and prolonged survival time were observed in tumor-bearing mice received combined therapy of C215Fab-SEA and Ad IL-18 than those of mice treated with C215Fab-SEA or AdIL-18 alone. Combination therapy augmented NK and CTL activities of tumor-bearing mice more markedly. The production of IL-2 and IFN-gamma also increased more significantly. More potent antitumor effect of combined therapy was observed in IL-10 KO mice with enhanced Th1 response. Our data demonstrated that the antitumor effect of C215Fab-SEA immunotherapy could be potentiated significantly by combination with intratumoral IL-18 gene transfer through more efficient activation of Th1 immune responses.

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.

To increase the antitumor effects of cytosine deaminase (AdCD) gene therapy and induce more potent antitumor immunity, Th1 cytokine interleukin-18 encoded adenovirus (AdIL18) was combined with adenovirus encoding CD (AdCD) for the therapy of established murine B16 melanoma. Combination therapy of the tumor-bearing mice with AdIL 18 and AdCD/5FC inhibited the growth of the subcutaneous B16 tumors more significantly, compared with AdIL 18 or AdCD/5FC alone. In vivo depletion analysis with anti-CD4, anti-CD8 or anti-NK 1.1 McAb illustrated that both CD8+ T cells and CD4+ T cells played key roles in the augmented antitumor response of the combined therapy. Peptide/MHC tetramer represents a powerful and general tool for rapid, highly sensitive, and direct analysis of antigen-specific T cells. In this study, we prepared H-2Kb/TRP-2180-188 tetramer, which was demonstrated to bind H-2Kb-restricted, B16 melanoma-specific CD8+ T cells. B16 specific H-2Kb/TRP2180-188 tetramer was used to stain the tumor-specific CD8+ T cells and the results showed that CD8+ tetramer+ T cells were about 3-5% of the splenic CD8+ T cells derived from tumor-bearing mice after combined therapy. The CTL cytotoxicity was markedly induced in mice after combined therapy, suggesting efficient induction of tumor-specific CD8+ T cells after combined gene therapy with AdCD/5FC/AdIL18. IL-18 gene transfer could significantly augment the cytotoxicity of NK cells and macrophages, and increase the production of interleukin-2 and interferon-gamma, as compared with treatments with AdCD/5FC, AdlacZ/5FC or PBS. These data suggested that in vivo IL-18 gene transfer could augment the antitumor effects of CD suicide gene therapy through efficient induction of antitumor immunity.

Adoptive transfer of activated macrophages, being both effector cells and antigen-presenting cells, represents a promising approach to immunotherapy of cancer. In order to get activated macrophages with increased antitumor potential, in the present study, murine peritoneal macrophages were transduced with human macrophage colony-stimulating factor (M-CSF) and murine interferon-gamma (IFNgamma) by recombinant adenovirus infection. The results demonstrate that M-CSF and IFNgamma gene-modified macrophages exhibited higher expression of MHC-II, B7.1 and ICAM-1, increased antigen-presenting activity and cytotoxicity. It was also shown that they secreted more tumor necrosis factor, interleukin-1 and nitric oxide. In vivo experiments showed that in previously initiated murine pulmonary metastatic melanoma, tumor lysate-pulsed, M-CSF and IFNgamma gene-modified macrophages elicited more potent antitumor effects than tumor lysate pulsed M-CSF or IFNgamma gene-modified macrophages. Cytotoxic T lymphocyte (CTL) activity, IFNgamma and tumor-necrosis factor production of the splenocytes increased significantly in mice after intravenous injection of the gene-modified macrophages. M-CSF and IFNgamma gene-modified macrophages may act as activated effector and antigen-presenting cells, thus eliciting a more potent antitumor response.

Lymphotactin (Ltn) is the sole member of C chemokines which attracts T cells and NK cells specially. Ltn gene was transferred in vivo to improve the antitumor efficacy of cytosine deaminase (CD) gene therapy. Upregulation of CD80 and CD54 on murine CT26 colon carcinoma cells was observed after combined transfection with adenovirus encoding CD (AdCD) and adenovirus encoding murine Ltn (AdLtn) followed by administration of 5-fluorocytosine (5FC) in vitro. AdCD/5FC treatment also increased the expression of CD95 and induced obvious apoptosis of CT26 cells. After combined treatment with AdLtn and AdCD/5FC, the pre-established murine model with subcutaneous CT26 colon carcinoma exhibited most significant tumor growth inhibition, and four of eight tumor-bearing mice were tumor free, while tumors in other mice grew more progressively. Examination of lymphocyte infiltration and cytokine gene expression in tumor tissue revealed that tumors from AdLtn/AdCD/5FC-or AdLtn-treated mice were heavily infiltrated with CD4+, CD8+ T cells and NK cells, and IL-2 and IFN-gamma mRNA expression were present in parallel with T cell and NK cell infiltration. Splenic NK and CTL activities increased significantly after the combination therapy. In vivo depletion analysis showed that NK cells, CD4+ T cells and CD8+T cells participated in the antitumor effect of the host with CD8+T cells being the main T cell subset responsible for the enhanced antitumor immune response. These findings suggested that increased immunogenicity and induction of apoptosis of the tumor cells, and efficient induction of local and systemic antitumor immunity of the host might contribute to the enhanced antitumor effects of the combined Ltn and CD suicide therapy. Gene Therapy (2000) 7, 329-338.

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.

Murine fibroblast NIH3T3 cells engineered to secrete interleukin-6 (NIH3T3-IL-6) were implanted intraperitoneally into mice and observed for their hematopoietic effects with or without 5-fluorouracil (5-FU) administration. In normal mice, the platelet and neutrophil counts in peripheral blood increased significantly after implantation of NIH3T3-IL-6 cells, but the total white blood cell numbers showed no obvious elevation. The granulocyte-macrophage colony-forming unit (CFU-GM) and megakaryocyte colony-forming unit (CFU-MK) numbers formed by stem cells in bone marrow and spleen were also found to be significantly increased after implantation of NIH3T3-IL-6 cells when compared with those in mice after implantation of NIH3T3 cells transduced with neomycin gene (NIH3T3-Neo). To observe the protective effects of NIH3T3-IL-6 cells on hematopoietic depression in chemotherapy-treated mice, the mice were preinjected with 5-FU at a dosage of 150 mg/kg before implantation of NIH3T3-IL-6 cells. The platelet and neutrophil counts showed accelerated recovery after implantation of NIH3T3-IL-6 cells. The numbers of CFU-GM and CFU-MK in bone marrow and spleen were also found to be markedly increased in hematopoietic-depressed mice when compared with those in mice implanted with NIH3T3-Neo cells. These data suggest that fibroblast-mediated IL-6 gene therapy, which can augment hematopoiesis in mice with or without chemotherapy, will be of great help in the recovery from hematopoietic depression 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.

Background: Both fibroblast-mediated cytokine gene therapy and bone marrow transplantation (BMT) have proven to be efficient protocols for the recovery of bone marrow depression. In this report, the effects of fibroblast-mediated interleukin (IL)-6 gene therapy, in combination with BMT, on the recovery of irradiation-induced bone marrow depression were investigated., Methods: NIH3T3 fibroblast cells engineered to secrete IL-6 (NIH3T3-IL-6) or NIH3T3 cells transduced with the neomycin gene (NIH3T3-Neo), in combination with 10(7), 10(6), or 10(5) syngeneic bone marrow cells, were implanted into irradiated mice., Results: The platelets and white blood cells in the peripheral blood of the irradiated mice increased greatly 12 days after implantation of NIH3T3-IL-6 cells and BMT, the white blood cell counts were restored to a normal level 32 days after the combined therapy, and the platelet number was obviously higher than that in mice implanted with NIH3T3-Neo and BMT. Twenty and 25 days after the combined therapy, the mice showed accelerated recovery of colony-forming unit (CFU)-granulocyte/macrophages and CFU-megakaryocytes when compared with the mice implanted with NIH3T3-Neo cells and BMT. Ten days after lethal irradiation with gamma rays, the spleens formed more CFU-spleen in mice implanted with NIH3T3-IL-6 cells and BMT than in mice injected with phosphate-buffered saline or NIH3T3-Neo cells. Combined therapy with NIH3T3-IL-6 cell implantation and BMT delayed the survival period of the hematopoietic-depressed mice significantly when compared with therapy with NIH3T3-Neo cell implantation and BMT., Conclusions: These data demonstrated that the combined therapy of fibroblast-mediated IL-6 gene therapy and BMT could significantly promote the recovery of irradiation-induced hematopoietic depression.

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.

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.

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.

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.

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.