Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages.

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-10, and TGF-ß1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Enhancing effect of chitosan nanoparticles on the immune efficacy of Bordetella bronchiseptica outer membrane vesicles.

The outer membrane vesicle (OMV) of bacteria is a bilayer membrane vesicle with a diameter of about 10-300 nm that is secreted during the growth of Gram-negative bacteria. OMV is considered as a high-quality vaccine candidate antigen because of its natural immunogenicity and non-replicability. Although the excellent antigenicity of OMV has been widely confirmed, its instability and heterogeneity greatly affect its immune effect. Many studies have demonstrated that in combination with nanoparticles can enhance the stability of OMV. In this study, OMVs were used to coat chitosan nanoparticles (CNPs) and obtain a stable OMV vaccine. The characteristics, including morphology, hydrodynamic size, and zeta potential were evaluated. The immune protection of CNP-OMV and anti-infection efficacy were examined and compared in vivo and in vitro. The results showed that the CNP-OMV were homogenous with a size of 139 nm and a stable core-shell structure. And CNP-OMV could significantly increase the cell proliferation, phagocytosis and TNF-α, IL-6 and IL-10 secretion of RAW264.7 in vitro. In vivo, CNP-OMV could significantly increase the levels of anti-Bb and OMV IgG antibodies. Levels of blood lymphocyte, and Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-5), and Th17 (IL-17, TNF-α) type cytokines in the serum were all significantly increased. At the same time, CNP-OMV could significantly reduce the bacterial invading the lungs of challenged rabbits. And CNP-OMV could largely protect the lungs from injury. The above results showed that CNP-OMV had a good immune efficacy and could resist the infection of Bordetella bronchiseptica. This study provided a scientific basis for the development of novel effective and safe vaccine against Bordetella bronchiseptica, and also provided a new idea for the development of new bacterial vaccine.

Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Safety and Efficacy of the Bordetella bronchiseptica Vaccine Combined with a Vegetable Oil Adjuvant and Multi-Omics Analysis of Its Potential Role in the Protective Response of Rabbits.

Infectious respiratory diseases caused by Bordetella bronchiseptica (Bb) are seriously endangering the development of the rabbit industry in China. Unfortunately, no licensed vaccines are available for this pathogen. The present study was designed to determine whether the inactivated Bb antigen formulated with vegetable oil adjuvant (named E515) which contains soybean oil, vitamin E, and ginseng saponins, functions as a safe and effective vaccine (E515-Bb) against Bb infection in rabbits. Based on local and systemic reactions, both the E515 adjuvant alone and the E515-Bb vaccine exhibited good safety in rabbits. Immune response analysis implies that rabbits immunized with the E515-Bb vaccine produced significantly higher, earlier, and longer-lasting specific antibody responses and activated Th1/Th2/Th17 cell responses than those immunized with the aluminum hydroxide (Alum)-adjuvanted Bb vaccine (Alum-Bb) or Bb antigen alone. Moreover, the E515-Bb vaccine effectively protected rabbits from Bb infection. Additionally, integrated multi-omics analysis revealed that the immunoprotective effect of the E515-Bb vaccine was achieved through upregulation of the complement and coagulation cascades and cell adhesion molecule (CAM) pathways, and the downregulation of the P53 pathway. Overall, these results indicate that the E515-Bb vaccine is safe, elicits an efficient immune response and provides good protection against Bb infection in rabbits. Thus, the E515-adjuvanted Bb vaccine can be considered a promising candidate vaccine for preventing Bb infection.

Sunflower seed oil combined with ginseng stem-leaf saponins as an adjuvant to enhance the immune response elicited by Newcastle disease vaccine in chickens.

The present study was to evaluate the adjuvant effect of sunflower seed oil containing saponins extracted from the stem and leaf of Panax ginseng C.A. Meyer (E515-D) on the immune response induced by an inactivated Newcastle disease virus (NDV) in chickens. The results showed that E515-D promoted significantly higher serum NDV-specific HI and neutralizing antibody responses, IFN-γ and IL-4 levels, and lymphocyte proliferative responses to Con A, LPS, and NDV antigen than the conventional adjuvant Marcol 52. Different adjuvant effect between E515-D and Marcol 52 may be attributed to different genes expressed in two groups. Transcriptome analysis of splenocytes showed that there were 1198 differentially expressed genes (DEGs) with 539 up and 659 down regulated in E515-D group while 1395 DEGs with 697 up and 698 down regulated in Marcol 52 group in comparison with the control group. Analysis of gene ontology (GO) term and kyoto encyclopedia of Genes and Genomes (KEGG) pathways showed that the predominant immune related pathways included "Toll-like receptor signaling pathway", "NOD-like receptor signaling pathway", "C-type lectin receptor signaling pathway", and "Phosphatidylinositol signaling system" in E515-D group while Marcol 52 were "NOD-like receptor signaling pathway", "Phagosome", and "Lysosome", and the most relevant DEGs in E515-D group were STAT1, STAT2, PI3K, and IL-6. Considering the excellent adjuvant activity and vegetable origin, E515-D deserves further study as an adjuvant for vaccines used in food animals.

Enhanced Immune Responses with Serum Proteomic Analysis of Hu Sheep to Foot-and-Mouth Disease Vaccine Emulsified in a Vegetable Oil Adjuvant.

Our previous study demonstrated that a vegetable oil consisting of soybean oil, vitamin E, and ginseng saponins (SO-VE-GS) had an adjuvant effect on a foot-and-mouth disease (FMD) vaccine in a mouse model. The present study was to compare the adjuvant effects of SO-VE-GS and the conventional ISA 206 on an FMD vaccine in Hu sheep. Animals were intramuscularly (i.m.) immunized twice at a 3-week interval with 1 mL of an FMD vaccine adjuvanted with SO-VE-GS (n = 10) or ISA 206 (n = 9). Animals without immunization served as control (n = 10). Blood was sampled prior to vaccination and at 2, 4, 6, and 8 weeks post the booster immunization to detect FMD virus (FMDV)-specific IgG. Blood collected at 8 weeks after the booster was used for the analyses of IgG1 and IgG2, serum neutralizing (SN) antibody, IL-4 and IFN-γ production, and proteomic profiles. The results showed that IgG titers rose above the protection level (1:128) in SO-VE-GS and ISA 206 groups after 2 and 4 weeks post the booster immunization. At 6 weeks post the booster, the ISA 206 group had 1 animal with IgG titer less than 1:128 while all the animals in the SO-VE-GS group retained IgG titers of more than 1:128. At 8 weeks post the booster, 6 of 9 animals had IgG titers less than 1:128 with a protective rate of 33.3% in the ISA 206 group, while only 1 of 10 animals had IgG titer less than 1:128 with a protective rate of 90% in the SO-VE-GS group, with statistical significance. In addition, IgG1, IgG2, SN antibodies, IL-4, and IFN-γ in the SO-VE-GS group were significantly higher than those of the ISA 206 group. Different adjuvant effects of SO-VE-GS and ISA 206 may be explained by the different proteomic profiles in the two groups. There were 39 and 47 differentially expressed proteins (DEPs) identified in SO-VE-GS compared to the control or ISA 206 groups, respectively. In SO-VE-GS vs. control, 3 immune related gene ontology (GO) terms and 8 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were detected, while 2 immune related GO terms and 5 KEGG pathways were found in ISA 206 vs. control. GO and KEGG analyses indicated that 'positive regulation of cytokine secretion', 'Th1/Th2 cell differentiation', and 'Toll-like receptor signaling pathways', were obviously enriched in the SO-VE-GS group compared to the other groups. Coupled with protein-protein interaction (PPI) analysis, we found that B7TJ15 (MAPK14) was a key DEP for SO-VE-GS to activate the immune responses in Hu sheep. Therefore, SO-VE-GS might be a promising adjuvant for an FMD vaccine in Hu sheep.

Early IgG Response to Foot and Mouth Disease Vaccine Formulated with a Vegetable Oil Adjuvant.

The present study evaluated soybean oil (SO) containing vitamin E (VE) and ginseng saponins (GS) (SO-VE-GS) for their adjuvant effect on foot-and-mouth disease (FMD) vaccine. Since mineral oil ISA 206 is a common adjuvant used in the FMD vaccine, it was used as a control adjuvant in this study. VE and GS were found to have a synergistic adjuvant effect. When mice were immunized with the FMD vaccine emulsified in SO with VE and GS, significantly higher serum IgG, IgG1, and IgG2a were found than VE and GS used alone. SO-VE-GS and ISA 206 behaved differently in adjuvant activities. When mice were immunized with the FMD vaccine adjuvanted with SO-VE-GS, significantly higher and earlier production of serum IgG was found than that adjuvanted with ISA 206. Although both adjuvants significantly increased the number of bone marrow plasma cells, a stimulation index of lymphocytes (SI) as well as the production of IL-4 and IL-6, SO-VE-GS promoted significantly higher SI and the ratio of CD4+/CD8+ T cells with production of increased IFN-γ and decreased TGF-ß1 as compared with the ISA 206 group. The data suggested that SO-VE-GS activated Th1/Th2 immune responses. Transcriptome analysis of splenocytes showed that differentially expressed genes (DEGs), immune-related gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the SO-VE-GS group. Therefore, the potent adjuvant effect of SO-VE-GS on the FMD vaccine may be attributed to the immune-related gene profile expressed in lymphocytes. Due to its plant origin and due to being much cheaper than imported mineral oil ISA 206, SO-VE-GS deserves further study in relation to vaccines used in food animals.

Signaling pathway underlying splenocytes activation by polysaccharides from Atractylodis macrocephalae Koidz.

Previous study demonstrated that total polysaccharides isolated from Atractylodis macrocephalae Koidz. (RAMPtp) were effective to eliminate intramammary infection in cows. The present study was designed to investigate the immunomodulatory activity of RAMPtp in mouse splenocytes. Splenocyte proliferation, natural killer (NK) cytotoxicity, productions of NO and cytokines, transcription factor activity as well as the signal pathways and receptor were examined. The results showed that RAMPtp significantly promoted splenocyte proliferation and made the cells enter S and G2/M phases, increased ratios of T/B cells, boosted NK cytotoxicity, enhanced transcriptional activities of nuclear factor of activated T cells (NFAT), nuclear factor κB (NF-κB) and activator protein 1 (AP-1), and stimulated secretions of NO, immunoglobulin G (IgG) and multiple cytokine families (IL-1α, IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, IL-13, IFN-γ, TNF-α, G-CSF, GM-CSF, KC, MIP-1α, MIP-1ß, RANTES and Eotaxin). In addition, all the specific inhibitors against the mitogen-activated protein kinases (MAPKs) and NF-κB significantly suppressed the IL-6 production induced by RAMPtp. Moreover, splenocytes from Toll-like receptor 4 (TLR4) deficient mouse responded equally to RAMPtp stimulation as the wild-type. Therefore, RAMPtp might induce splenocytes activation at least in part via the TLR4-independent MAPKs and NF-κB signaling pathways. The present results would be useful to further understand the immunomodulatory mechanisms of RAMPtp in elimination of intramammary infection in cows.

Soybean oil containing ginseng saponins as adjuvants promotes production of cytokines and enhances immune responses to foot-and-mouth disease vaccine.

In the present study, the adjuvant effect of soybean oil containing ginseng root saponins (SO-GS-R) on the immune response to foot-and-mouth disease vaccine (FMDV) in mice was investigated. When immunized with FMDV antigen emulsified in an SO-GS-R formulation, mice generated remarkably higher serum antibody and cytokine responses than mice immunized with FMDV antigen alone. To elucidate the mechanisms underlying the adjuvant effect of SO-GS-R, we measured cytokines in serum and muscle tissue after intramuscular injection of SO-GS-R. The results showed that injection of SO-GS-R significantly increased the levels of IL-1ß, IL-5, IL-6, G-CSF, KC, MCP-1, MIP-1α, and MIP-1ß in both serum and muscle. These results suggested that SO-GS-R recruits neutrophils, eosinophils, T cells and macrophages, causing immune cell recruitment at the injection site, driving antigen-presenting cells to actively participate in the onset of immunity, and amplifying the immune responses. Considering its adjuvant activity and plant-derived properties, SO-GS-R should be further studied for its adjuvant effect on vaccines used in food animals.

Prophylactic Antitumor Effect of Mixed Heat Shock Proteins/Peptides in Mouse Sarcoma.

BACKGROUND: To develop a vaccine-based immunotherapy for sarcoma, we evaluated a mixture of heat shock proteins (mHSPs) as a vaccine for sarcoma treatment in a mouse model. Heat shock protein/peptides (HSP/Ps) are autoimmune factors that can induce both adaptive and innate immune responses; HSP/Ps isolated from tumors can induce antitumor immune activity when used as vaccines. METHODS: In this study, we evaluated the effects of mHSP/Ps on prophylactic antitumor immunity. We extracted mHSP/Ps, including HSP60, HSP70, GP96, and HSP110, from the mouse sarcoma cell lines S180 and MCA207 using chromatography. The immunity induced by mHSP/Ps was assessed using flow cytometry, ELISPOT, lactate dehydrogenase release, and enzyme-linked immunosorbent assay. RESULTS: Of S180 sarcoma-bearing mice immunized with mHSP/Ps isolated from S180 cells, 41.2% showed tumor regression and long-term survival, with a tumor growth inhibition rate of 82.3% at 30 days. Of MCA207 sarcoma-bearing mice immunized with mHSP/Ps isolated from MCA207 cells, 50% showed tumor regression and long-term survival with a tumor growth inhibition rate of 79.3%. All control mice died within 40 days. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells and tumor-specific cytotoxic T-lymphocyte activity were increased in the immunized group. CONCLUSIONS: Vaccination with a polyvalent mHSP/P cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors. This mHSP/P vaccine exerted greater antitumor effects than did HSP70, HSP60, or tumor lysates alone.

Activation of the phase II enzymes for neuroprotection by ginger active constituent 6-dehydrogingerdione in PC12 cells.

The cellular endogenous antioxidant system plays pivotal roles in counteracting or retarding the pathogenesis of many neurodegenerative diseases. Molecules with the ability to enhance the antioxidant defense thus are promising candidates for neuroprotective drugs. 6-Dehydrogingerdione (6-DG), one of the major components of dietary ginger, has received increasing attention due to its multiple pharmacological activities. However, how this pleiotropic molecule works on the neuronal system has not been studied. This paper reports that 6-DG efficiently scavenges various free radicals in vitro and displays remarkable cytoprotection against oxidative stress-induced neuronal cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-DG significantly up-regulates a panel of phase II genes as well as the corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H: quinone oxidoreductase, and thioredoxin reductase. Mechanistic study indicates that activation of the Keap1-Nrf2-ARE pathway is the molecular basis for the cytoprotection of 6-DG. This is the first revelation of this novel mechanism of 6-DG as an Nrf2 activator against oxidative injury, providing the potential therapeutic use of 6-DG as neuroprotective agent.

Co-encapsulation of magnetic Fe3O4 nanoparticles and doxorubicin into biodegradable PLGA nanocarriers for intratumoral drug delivery.

In this study, the authors constructed a novel PLGA [poly(D,L-lactic-co-glycolic acid)]-based polymeric nanocarrier co-encapsulated with doxorubicin (DOX) and magnetic Fe(3)O(4) nanoparticles (MNPs) using a single emulsion evaporation method. The DOX-MNPs showed high entrapment efficiency, and they supported a sustained and steady release of DOX. Moreover, the drug release was pH sensitive, with a faster release rate in an acidic environment than in a neutral environment. In vitro, the DOX-MNPs were easily internalized into murine Lewis lung carcinoma cells and they induced apoptosis. In vivo, the DOX-MNPs showed higher antitumor activity than free DOX solution. Furthermore, the antitumor activity of the DOX-MNPs was higher with than without an external magnetic field; they were also associated with smaller tumor volume and a lower metastases incidence rate. This work may provide a new modality for developing an effective drug delivery system.

Synthesis and cytotoxic evaluation of quinazolin-4(3H)-one derivatives bearing thiocarbamate, thiourea or N-methyldithiocarbamate side chains.

We have previously found that the dithiocarbamate derivatives of quinazolin-4(3H)-one could act as cytotoxic agents against a panel of human tumor cell lines. To investigate the contribution of dithiocarbamate moiety to the cytotoxic activity, three series of novel quinazolin-4(3H)-one derivatives bearing thiocarbamate, thiourea or Nmethyldithiocarbamate side chains were synthesized and tested for their cytotoxic activity against human cancer cell lines A549, MCF-7, HeLa, HT29 and HCT-116 by MTT assay. The results showed that transformation of the dithiocarbamate moiety in lead compound I to thiocarbamate or thiourea led to a decrease or loss of cytotoxic activity. Some N-alkylated analogs of lead compound II preferentially inhibited the proliferation of A549 cells, although their potencies were not improved in comparison with the unalkylated counterparts. The structure-activity relationship obtained in this research will be beneficial for further synthesis and discovery of effective cytotoxic agents.

Small molecule inhibitors of mammalian thioredoxin reductase.

Mammalian thioredoxin reductases (TrxRs) are a family of NADPH-dependent flavoproteins with a penultimate selenocysteine residue at the carboxy-terminus. Besides their native substrate thioredoxins (Trx), the enzymes show a broad substrate specificity, at least partially, because of the C-terminal redox-active site that is easily accessible in the reduced form. TrxRs are ubiquitous in all kinds of cells and have a critical role in regulating intracellular redox signaling. In recent years, a wealth of evidence has revealed that overactivation/dysfunction of TrxRs is closely related to various diseases, especially in tumor development, and thus the past decades have witnessed an expanding interest in finding TrxRs inhibitors, which might be promising agents for cancer chemotherapy. Herein we reviewed the small molecule inhibitors of mammalian TrxRs, with an emphasis on those that have potential anticancer activity. This review includes the nonpatent references up to 2010 that deal with mammalian TrxR inhibitors.

3,4,4'-Trihydroxy-trans-stilbene, an analogue of resveratrol, is a potent antioxidant and cytotoxic agent.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally occurring polyphenol widely distributed in food and dietary plants. This phytochemical has been intensively studied as an efficient antioxidant and anticancer agent, and a variety of substituted stilbenes have been developed in order to improve the potency of resveratrol. In this work, we described the synthesis of 3,4,4 -trihydroxy-trans-stilbene (3,4,4'-THS), an analogue of resveratrol, and studied its antioxidant and cytotoxic activity in vitro. 3,4,4 -THS was much more efficient than resveratrol in protecting against free radical-induced lipid peroxidation, photo-sensitized DNA oxidative damage, and free radical-induced hemolysis of human red blood cells. More potent growth inhibition in cultured human leukemia cells (HL-60) was also observed for 3,4,4 -THS. The relationship between the antioxidant efficiency and cytotoxic activity was discussed, with the emphasis on inhibition of the free radical enzyme ribonucleotide reductase by antioxidants. The result that this subtle structure modification of resveratrol drastically improves its bioactivity provides important strategy to develop novel resveratrol-based molecules.

Antitumor activity of mixed heat shock protein/peptide vaccine and cyclophosphamide plus interleukin-12 in mice sarcoma.

BACKGROUND: The immune factors heat shock protein (HSP)/peptides (HSP/Ps) can induce both adaptive and innate immune responses. Treatment with HSP/Ps in cancer cell-bearing mice and cancer patients revealed antitumor immune activity. We aimed to develop immunotherapy strategies by vaccination with a mixture of HSP/Ps (mHSP/Ps, HSP60, HSP70, Gp96 and HSP110) enhanced with cyclophosphamide (CY) and interleukin-12 (IL-12). METHODS: We extracted mHSP/Ps from the mouse sarcoma cell line S180 using chromatography. The identity of proteins in this mHSP/Ps was assayed using SDS-PAGE and Western blot analysis with antibodies specific to various HSPs. BALB/C mice bearing S180 cells were vaccinated with mHSP/Ps ×3, then were injected intraperitoneally with low-dose CY and subcutaneously with IL-12, 100 µg/day, ×5. After vaccination, T lymphocytes in the peripheral blood were analyzed using FACScan and Cytotoxicity (CTL) was analyzed using lactate dehydrogenase assay. ELISPOT assay was used to evaluate interferon γ (IFN-γ), and immune cell infiltration in tumors was examined in the sections of tumor specimen. RESULTS: In mice vaccinated with enhanced vaccine (mHSP/Ps and CY plus IL-12), 80% showed tumor regression and long-term survival, and tumor growth inhibition rate was 82.3% (30 days), all controls died within 40 days. After vaccination, lymphocytes and polymorphonuclear leukocytes infiltrated into the tumors of treated animals, but no leukocytes infiltrated into the tumors of control mice. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells were all increased in the immune group, and tumor-specific cytotoxic T lymphocyte activity was increased. CONCLUSIONS: In this mice tumor model, vaccination with mHSP/Ps combined with low-dose CY plus IL-12 induced an immunologic response and a marked antitumor response to autologous tumors. The regimen may be a promising therapeutic agent against tumors.

[Biological characterization of rabbit's articular chondrocytes by confluent culture in vitro].

OBJECTIVE: To obtain large amount of differentiated chondrocytes in vitro, examine and compare the biological characterization of rabbits' articular chondrocyte cultured in different density in vitvo. METHODS: From November 2001 to June 2004, articulate tissues were obtained from the joints of the adult rabbits. Chondrocytes were isolated from the cartilage tissue with type II collagenase digestion and cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). The chondrocytes were cultured with low density of monolayer culture and high density of confluent culture respectively. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. RESULTS: When chondrocytes cultured in monolayer and in low density, it proliferated rapidly during the three generations, but with the same time, dedifferentiation was also rapid. After the third passage, most of the passage cells lost the phenotype, and the proliferation also stagnated. While chondrocytes cultured in high density, dedifferentiation slowed down. And even the phenotypes of the dedifferentiated chondrocyte which were cultured in low density could reduced partly by followed high density culture. CONCLUSIONS: Culture chondrocytes by high density in vitro can effectively maintain the differentiated phenotype of chondrocyte. It also keeps the proliferation character as monolayer culture. The dedifferentiated chondrocyte caused by many passages could redifferentiate partly. So it is indicated that confluent culture of original or expanded chondrocytes in high density is a better culture methods than culture in low density.

[Experimental study on preparation of decellularized artery vascular graft matrix and explantation of carotid artery allografts].

OBJECTIVE: To set up a new process to access the preparation of decellularized artery grafts. And to evaluate the feasibility of decellularized artery allografts was evaluated. METHODS: This study compared the effects of four extraction chemicals [1% t-octyl-phenoxypolyethoxyethanol (Triton X-100), 1% tri (n-butyl) phosphate (TnBP), and 1% sodium dodecyl sulfate (SDS) and trypsin (0.125, 0.25%) on thoracic artery vascular for 24 h (except trypsin for 2 h). At the base of it, a four-step process, including hypotonic, hypertonic solutions and combining with 1% Triton X-100 and 1% SDS detergents, were performed in rabbit thoracic artery vascular. Histological examination, tensile tests and expanding-burst tests were done on the samples. The decellularized carotid artery allografts were transplanted in other rabbits. RESULTS: Treatment with 1% SDS or 1% Triton X-100 for 24 h could remove most cells with retention of near normal structure. A four-step process could remove all cells with the extracellular matrix well conserved. The pulling mechanical properties and burst pressure of decellularized carotid artery were similar to the control. The decellularized carotid artery allografts (diameter of 2 mm) were patent at explanting up to 2 months. CONCLUSIONS: The acellular artery vascular graft matrix is well prepared with four-step process including detergents, such as TritonX-100, SDS without compromising the graft structure or mechanical properties significantly. The carotid artery allografts (diameter of 2 mm) decellularized by the process are patent at explanting up to 2 months.

Identification of specific peptide ligands for B-lymphoma cell and its effect on tyrosine phosphorylation and cell apoptosis.

OBJECTIVE: To search novel method for diagnosis and therapy of B-lymphoma, specific small molecular peptide ligands against binding site of tumor cells were screened and its effects on signal transduction and cell apoptosis were tested. METHODS: Specific peptide ligands were screened by binding with site of human B lymphoma cell (OC1LY8) using peptide-bead libraries. The identified peptides were characterized with responsible cells by rebinding test. The role of tyrosine phosphorylation of peptide ligand was tested by Western blot; and its apoptosispromoting role was observed by confocal fluorescent microscope. RESULTS: Specific peptide ligand was able to bind specifically to site on cell surface and enter into cytoplasm. Tetrameric peptide ligand was able to strongly trigger signal transduction resulting in tyrosine phosphorylation and cellular apoptosis in OC1LY8 cell line. CONCLUSION: Screened peptide ligand can effectively bind with OC1LY8 cell, stimulate cellular tyrosine phosphorylation and induce cellular apoptosis.

Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats.

OBJECTIVE: To study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats. METHODS: Apoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. RESULTS: (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively. CONCLUSIONS: Bax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.