Ginsenoside Rb1, Compound K and 20(S)-Protopanaxadiol Attenuate High-Fat Diet-Induced Hyperlipidemia in Rats via Modulation of Gut Microbiota and Bile Acid Metabolism.

Hyperlipidemia, characterized by elevated serum lipid concentrations resulting from lipid metabolism dysfunction, represents a prevalent global health concern. Ginsenoside Rb1, compound K (CK), and 20(S)-protopanaxadiol (PPD), bioactive constituents derived from Panax ginseng, have shown promise in mitigating lipid metabolism disorders. However, the comparative efficacy and underlying mechanisms of these compounds in hyperlipidemia prevention remain inadequately explored. This study investigates the impact of ginsenoside Rb1, CK, and PPD supplementation on hyperlipidemia in rats induced by a high-fat diet. Our findings demonstrate that ginsenoside Rb1 significantly decreased body weight and body weight gain, ameliorated hepatic steatosis, and improved dyslipidemia in HFD-fed rats, outperforming CK and PPD. Moreover, ginsenoside Rb1, CK, and PPD distinctly modified gut microbiota composition and function. Ginsenoside Rb1 increased the relative abundance of Blautia and Eubacterium, while PPD elevated Akkermansia levels. Both CK and PPD increased Prevotella and Bacteroides, whereas Clostridium-sensu-stricto and Lactobacillus were reduced following treatment with all three compounds. Notably, only ginsenoside Rb1 enhanced lipid metabolism by modulating the PPARγ/ACC/FAS signaling pathway and promoting fatty acid ß-oxidation. Additionally, all three ginsenosides markedly improved bile acid enterohepatic circulation via the FXR/CYP7A1 pathway, reducing hepatic and serum total bile acids and modulating bile acid pool composition by decreasing primary/unconjugated bile acids (CA, CDCA, and ß-MCA) and increasing conjugated bile acids (TCDCA, GCDCA, GDCA, and TUDCA), correlated with gut microbiota changes. In conclusion, our results suggest that ginsenoside Rb1, CK, and PPD supplementation offer promising prebiotic interventions for managing HFD-induced hyperlipidemia in rats, with ginsenoside Rb1 demonstrating superior efficacy.

Local radiotherapy for murine breast cancer increases risk of metastasis by promoting the recruitment of M-MDSCs in lung.

BACKGROUND: Radiotherapy is one of the effective methods for treatment of breast cancer; however, controversies still exist with respect to radiotherapy for patients with TNBC. Here, we intend to explore the mechanism by which local radiotherapy promotes the recruitment of M-MDSCs in the lung and increases the risk of lung metastasis in TNBC tumor-bearing mice. METHODS: A single dose of 20 Gy X-ray was used to locally irradiate the primary tumor of 4T1 tumor-bearing mice. Tumor growth, the number of pulmonary metastatic nodules, and the frequency of MDSCs were monitored in the mice. Antibody microarray and ELISA methods were used to analyze the cytokines in exosomes released by irradiated (IR) or non-IR 4T1 cells. The effects of the exosomes on recruitment of MDSCs and colonization of 4T1 cells in the lung of normal BALB/c mice were observed with the methods of FCM and pathological section staining. T lymphocytes or 4T1 cells co-cultured with MDSCs were performed to demonstrate the inhibitory effect on T lymphocytes or accelerative migration effect on 4T1 cells. Finally, a series of in vitro experiments demonstrated how the exosomes promote the recruitment of M-MDSCs in lung of mice. RESULTS: Even though radiotherapy reduced the burden of primary tumors and larger lung metastatic nodules (≥ 0.4 mm2), the number of smaller metastases (< 0.4 mm2) significantly increased. Consistently, radiotherapy markedly potentiated M-MDSCs and decreased PMN-MDSCs recruitment to lung of tumor-bearing mice. Moreover, the frequency of M-MDSCs of lung was positively correlated with the number of lung metastatic nodules. Further, M-MDSCs markedly inhibited T cell function, while there was no difference between M-MDSCs and PMN-MDSCs in promoting 4T1 cell migration. X-ray irradiation promoted the release of G-CSF, GM-CSF and CXCl1-rich exosomes, and facilitated the migration of M-MDSCs and PMN-MDSCs into the lung through CXCL1/CXCR2 signaling. While irradiated mouse lung extracts or ir/4T1-exo treated macrophage culture medium showed obvious selective chemotaxis to M-MDSCs. Mechanistically, ir/4T1-exo induce macrophage to produce GM-CSF, which further promoted CCL2 release in an autocrine manner to recruit M-MDSCs via CCL2/CCR2 axis. CONCLUSIONS: Our work has identified an undesired effect of radiotherapy that may promote immunosuppressive premetastatic niches formation by recruiting M-MDSCs to lung. Further studies on radiotherapy combined CXCR2 or CCR2 signals inhibitors were necessary.

Improving the ability of CAR-T cells to hit solid tumors: Challenges and strategies.

Chimeric antigen receptor T cell (CAR-T) therapy is a late-model of immune cell therapy that has been shown to be effective in refractory/recurrent B-cell leukemia and lymphoma. Compared with the traditional anti-tumor methods, CAR-T cell therapy has the advantages of higher specificity, stronger lethality and longer-lasting efficacy. Although CAR-T cells have made significant progress in the treatment of hematologic malignancies, diverse difficulties remain in the treatment of solid tumors, including immune escape due to tumor antigen heterogeneity, preventing entry or limiting the persistence of CAR-T cells by physical or cytokine barriers and along with other immunosuppressive molecule and cells in the tumor microenvironment (TME). Otherwise, the intracellular signaling of CAR also impact on CAR-T cells persistence. Appropriate modification of intracellular costimulatory molecular signal in the structure of CAR or coexpression of CAR and cytokines can provide a way to enhance CAR-T cells activity. Additionally, CAR-T cells dysfunction due to T cell exhaustion is associated with multi-factors, especially transcription factors, such as c-Jun, NR4A. Engineering CAR-T cells to coexpress or knockout transcription factors in favor of TCM memory CAR-T cells differentiation was proved to prolonged the survival of CAR-T cells. Finally, combination of CAR-T cells with oncolytic viruses, nanoparticles or immune checkpoint inhibitors provides an effective measure to improve CAR-T cells function. Here, we discuss all of these advances and challenges and review promising strategies for treating solid tumors. In particular, we also highlight that CAR-T cells have enormous potential to be used in combination with other immunotherapies.

Incorporation of the YRHQ motif into CD3ζ chain enhances the antitumor activity of HER2-targeted CAR-T cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨在靶向HER2的CAR的CD3ζ链胞内区引入YRHQ基序对CAR-T细胞的特异性杀伤活性及免疫记忆形成的影响。方法：通过DNA合成获得包含靶向HER2的编码抗原受体H28ζ或H28ζ(YRHQ)的DNA片段，通过慢病毒载体将不同CAR的DNA片段分别转导健康人外周血T细胞，制备靶向HER2的H28ζ-CAR-T及H28ζ(YRHQ)-CAR-T细胞。扩增过程中对不同CAR-T细胞进行计数，FCM检测CAR的表达率。将CAR-T细胞分别与HER2阳性的SKOV3、MDA-MB-453或HER2阴性的MCF-7细胞共培养，LDH释放法检测其杀伤活性，ELISA法检测IL-2、IFN-γ和颗粒酶B的水平，WB法检测STAT3磷酸化水平及免疫检查点分子TIM-3和PD-1的表达，通过FCM检测CCR7、CD45RO的表达，分析CAR-T细胞的表型。结果：H28ζ-CAR-T和H28ζ(YRHQ)-CAR-T细胞扩增能力较好，体外培养7 d时扩增4~5倍。H28ζ-CAR和H28ζ(YRHQ)-CAR表达率分别为（33.3±2.85）%和（28.30±3.2）%。H28ξ(YRHQ)-CAR-T细胞的杀伤活性较H28ζ-CAR-T细胞更高（P<0.05）。经HER2抗原刺激后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞的STAT3磷酸化水平较H28ξ-CAR-T细胞明显升高（P<0.01）；而两者间PD-1和TIM-3的表达无明显差异。未经抗原刺激的CAR-T细胞CCR7和CD45RO表达与正常T细胞比较差异无统计学意义（均P>0.05），与SKOV3细胞共培养后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞中TEM细胞比例明显增加、TCM细胞比例明显减少（均P<0.05）。结论：在CD3胞内区引入YRHQ基序可在一定程度上提高CAR-T细胞的杀伤潜力。

Irradiation enhances dendritic cell potential antitumor activity by inducing tumor cell expressing TNF-α.

Dendritic cells (DCs)-based tumor vaccines have shown to be the promising methods for inducing therapeutic antitumor response. However, DCs alone rarely carry curative antitumor activity, and the immunosuppressive microenvironment may contribute to this defect of DC vaccinal function. Irradiation in combination with DCs has been shown to promote immune-mediated tumor destruction in preclinical studies. However, little is known about how irradiation alters the tumor microenvironment, and what host pathways modulate the activity of administrated DCs. In this study, BALB/c mice and the 4T1 breast cancer cell line were used in a tumor-bearing model. The tumor-bearing mice were irradiated locally up to 10 Gy for 3 consecutive days or a single dose of 30 Gy using a cesium source. Studies of dynamic change of the tumor microenvironment in irradiated versus untreated tumors revealed that there was no obvious change on IL-10, IL-6 and TGF-ß expression or production, whereas increased TNF-α level within the first 2 weeks of irradiation. The increased TNF-α level is exactly right timing window for DCs injection, corresponding to the significant elevation of intratumoral CD8+ T infiltration and the regression of tumor size. With attention to scheduling, combination X-ray with DCs i.t. injection may offer a practical strategy to improve treatment outcomes.

The protective effects of chronic intermittent hypobaric hypoxia pretreatment against collagen-induced arthritis in rats.

OBJECTIVE: To explore the immunological mechanisms underlying the effect of chronic intermittent hypobaric hypoxia (CIHH) pretreatment on collagen-induced arthritis (CIA) in rat. METHODS: Fifty-four adult male Sprague-Dawley rats were used in the experiment. Arthritis in CIA rats (n=18) was induced by injection of collagen. The CIHH+CIA rats (n=18) were treated with CIHH (simulated 3000 m altitude, 5 hours per day for 28 days, PO2=108.8 mmHg) before CIA. The control rats (n=18) were not given any treatment. RESULTS: (1) Incidence rate of CIA in CIHH+CIA rats was significantly lower than that in CIA rats (P<0.05). (2) The paw thickness and arthritis index (AI) value in CIHH+CIA rats were lower than those in CIA rats (P<0.05). (3) The hyperplasia with inflammatory infiltration in synovial tissue of joints in CIHH+CIA rats was much alleviative compared with CIA rats. (4) TNF-α, IFN-γ, IL-4 and IL-17 in synovial tissue of joint and serum in CIHH+CIA rats were decreased compared with CIA rats (P<0.05). (5) The number of CD4-positive T-lymphocytes and the ratio of CD4/CD8 T-lymphocytes in peripheral blood in CIHH+CIA rats were lower than those in CIA rats (P<0.05). (6) The protein expression of HIF-1α and NF-κB in synovial tissue of joint in CIHH+CIA rats was decreased compared with CIA rats (P<0.05). CONCLUSION: CIHH pretreatment has a protective effect against collagen-induced arthritis in rat through down-regulation of HIF-1α and NF-κB, inhibition of inflammatory cytokines TNF-α and IL-17, and balance in CD4/CD8 and Th1/Th2 T lymphocytes.

[Inhibiting effect of IL-10 in tumor microenvironment on anti-tumor activity of SOCS1-silenced DC vaccine].

OBJECTIVE: To observe the anti-tumor effect of suppressors of cytokine signaling 1(SOCS1)-silenced dentritic cell (DC) vaccines in melanoma-bearing mice, and the influence of IL-10 in the tumor microenvironment on DC vaccine action. METHODS: To obtain SOCS1-silenced DCs, DCs derived from mouse bone marrow cells ex vivo were induced to differentiation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, and then transduced with Len-SOCS1-shRNA or control Len-GFP lentiviruses. The SOCS1-silenced DCs were loaded by TRP2 peptide to prepare the DC vaccine, which was induced to mature by LPS. The DCs were analyzed by flow cytometry (FCM) for surface expressions of MHCII and CD86 and by real-time PCR for the expressions of SOCS1, IL-10 as well as IL-12p40. B16 or IL-10-silenced B16 (IL-10(-/-);) cells were inoculated into C57BL/6 mice. Five days later, the mice were randomly divided into 3 groups (PBS-DC, Len-DC and SOCS1-shRNA-DC groups) and injected with 1×106;/100 µL per mouse of the transduced DCs or PBS-DCs. We observed the tumor growth and the survival of the tumor-bearing mice. Tumor-infiltrating leukocytes (TIL) were isolated from tumor tissues using the discontinuous gradient centrifugation and the distribution of CD8⁺;T was analyzed by FCM; IFN-γ secretion and CTL activity were detected by the ELISpot and the standard microcytotoxicity assay, respectively. RESULTS: SOCS1 expression in DCs was down-regulated by 80% after Len-SOCS1-shRNA lentivirus infection. In the DCs with down-regulated SOCS1 expression, the expressions of MHCII and CD86 increased a little, which did not differ significantly from the control DCs, and IL-10 level dropped and IL-12p40 went up significantly compared with the control DCs. There was no any effect of SOCS1-silenced DCs on the survival of melanoma-bearing mice, however, the survival of B16-IL-10(-/-);-bearing mice was promoted(P<0.05). The further investigation showed that SOCS1-shRNA DCs raised the number of CD8⁺;T lymphocytes, promoted the TRP2-specific IFN-γ production and CTL responses in B16-IL-10(-/-);-bearing mice. CONCLUSION: The activity of the DC vaccine could be enhanced by silencing SOCS1 expression; however, the anti-tumor activity of SOCS1-silenced DC vaccine could be inhibited by IL-10 in tumor microenvironment.

Plasminogen activator inhibitor-1 promotes the proliferation and inhibits the apoptosis of pulmonary fibroblasts by Ca(2+) signaling.

AIM: Our previous investigation demonstrated that plasminogen activator inhibitor-1 (PAI-1) siRNA ameliorated bleomycin (BLM)-induced rat lung fibrosis. The present study was undertaken to explore the effect and the mechanism of PAI-1 siRNA and plasmid pcDNA on the proliferation and apoptosis of cultured fibroblasts from BLM-induced fibrotic lung tissues. MATERIALS AND METHODS: The fibroblasts from BLM-induced fibrotic lung tissue were isolated and transfected using PAI-1 siRNA and plasmid pcDNA-PAI-1. The techniques of real time RT-PCR and/or western blot were used to determine the expression of PAI-1, α-smooth muscle actin (α-SMA) (real time RT-PCR only), collagen type-1 and type-3 (real time RT-PCR only), and the levels of caspase-3, ERK and AKT signal molecules. The proliferation of fibroblasts was measured by cell cycle with flow cytometry. The intracellular concentration of Ca(2+) was examined by confocal laser microscopy. RESULTS: PAI-1 siRNA downregulated the PAI-1 mRNA expression by 70%±7% at 24h and protein expression by 73.5%±10% and 42%±3% at 48h and 72h compared to Non-specific siRNA group. Flow cytometry showed that the fibroblasts at the G(2)M+S phase were significantly reduced by 20.56±1.03% after transfecting PAI-1 siRNA and were significantly increased by 43.8±1.21% after transfecting plasmid pcDNA-PAI-1. The mRNA expressions of α-SMA, collagen type-1and type-3 were downregulated after transfecting the PAI-1 siRNA, while upregulated after the transfection of pcDNA-PAI-1. PAI-1 siRNA increased the level of caspase-3, inhibited the expressions of p-ERK and p-AKT protein molecules, while the pcDNA-PAI-1 transfection showed a reversal effect on these expressions. Intracellular Ca(2+) concentration was decreased after transfecting PAI-1 siRNA, whereas increased after transfecting pcDNA-PAI-1. CONCLUSION: PAI-1 promotes the proliferation, transforming into myofibroblasts, collagen synthesis, and inhibits apoptosis of pulmonary fibroblasts by activating Ca(2+), ERK and AKT signaling pathway. Decreasing PAI-1 expression is an available strategy in inhibiting the progression of pulmonary fibrosis.

[Anti-tumor effect of cisplatin combined with DC vaccine on tumor-bearing mice].

OBJECTIVE: To explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice. METHODS: B16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-ß were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay. RESULTS: Cisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01). CONCLUSION: Cisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.

siRNA against plasminogen activator inhibitor-1 ameliorates bleomycin-induced lung fibrosis in rats.

AIM: Plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. The present study was undertaken to examine the effects on pulmonary fibrosis of silencing PAI-1 expression with small interfering RNA (siRNA) and to assess the possible underlying mechanisms. METHODS: Male Wistar rats were subjected to intratracheal injection of bleomycin (BLM, 5 mg/kg, 0.2 mL) to induce pulmonary fibrosis. Histopathological changes of lung tissue were examined with HE or Masson's trichrome staining. The expression levels of α-smooth muscle actin (α-SMA), collagen type-I and type-III, caspase-3, as well as p-ERK1/2 and PI3K/Akt in the lung tissue were evaluated using imunohistochemistry and Western blot analyses. The fibroblasts isolated from BLM-induced fibrotic lung tissue were cultured and transfected with pcDNA-PAI-1 or PAI-1siRNA. The expression level of PAI-1 in the fibroblasts was measured using real time RT-PCR and Western blot analysis. The fibroblast proliferation was evaluated using MTT assay. RESULTS: Intratracheal injection of PAI-1-siRNA (7.5 nmoL/0.2 mL) significantly alleviated alveolitis and collagen deposition, reduced the expression of PAI-1, α-SMA, collagen type-I and collagen type-III, and increased the expression of caspase-3 in BLM-induced fibrotic lung tissue. In consistence with the in vivo results, the proliferation of the cultured fibroblasts from BLM-induced fibrotic lung tissue was inhibited by transfection with PAI-1-siRNA, and accelerated by overexpression of PAI-1 by transfection with pcDNA-PAI-1. The expression of caspase-3 was increased as a result of PAI-1 siRNA transfection, and decreased after transfection with pcDNA-PAI-1. In addition, the levels of p-ERK1/2 and PI3K/Akt in the fibrogenic lung tissue were reduced after treatment with PAI-1siRNA. CONCLUSION: The data demonstrate that PAI-1 siRNA inhibits alveolitis and pulmonary fibrosis in BLM-treated rats via inhibiting the proliferation and promoting the apoptosis of fibroblasts. Suppression ERK and AKT signalling pathways might have at least partly contributed to this process. Targeting PAI-1 is a promising therapeutic strategy for pulmonary fibrosis.

Tumour-derived IL-10 within tumour microenvironment represses the antitumour immunity of Socs1-silenced and sustained antigen expressing DCs.

It has been shown that silencing of suppressor of cytokine signalling 1 (Socs1) or stably expressing transgenic protein Ags in antigen-presenting dentritic cells (DCs) strongly enhances antigen-specific anti-tumour immunity. However, whether the strong and long-lasting T cell responses induced by the modified DCs could modulate the immunosuppressive tumour microenvironment has not been clarified. In this study, we explored the anti-tumour immunity of DCs modified by Socs1-shRNA lentiviral transduction combined with sustained expression of TRP2 in different tumour models. We showed that transfer Socs1-silenced or tumour antigen TRP2 persistent expressed DCs, or DCs modified by combination of Socs1-silencing and sustaining TRP2 expression prior to inoculation of tumour cells delayed B16 tumour cell growth, prolonged mouse survival and increased the ratio of CD8+ T/Treg as well as the CTL activity in tumours. However, there was no significant effect on tumour growth and mouse survival rate upon tumour established. Further, we showed that tumour cell secreted IL-10 counteracted the immunity of modified DCs in established tumour model, injection of Socs1-shRNA and TRP2 antigen modified significantly inhibited growth of the established B16-IL-10(-/-) tumours. These data indicated that the high level of IL-10 within tumour microenvironment is one of factors that compromise DC vaccine functions.

Augmented induction of antigen-specific cytotoxic T cell responses against canine hepatitis by co-immunization with pVAX1-CpG-Loop and adjuvants in BALB/c mice.

The objective of this study was to obtain better antigen specific cytotoxic T cell responses in vivo. We examined the augmented induction of antigen-specific cytotoxic T cell responses to co-administration of oligonucleotides (CpG-ODN), dimethyl dioctadecyl ammonium bromide (DDA), and Lipofectamine™ 2000 with a DNA vaccine (pVAX1-CpG-Loop) and boosting with pVAX1-CpG-Loop in BALB/c mice. The results show that Loop protein-specific T cell proliferation, cytotoxic T cell activity, and the production of CD8+ T cells and IFN-γ were enhanced after co-immunization of mice with adjuvants and pVAX1-CpG-Loop. We demonstrated that significant T cell-mediated immune responses were induced in the mice with the help of DDA, CpG-ODN and Lipofectamine™ 2000.

A preliminary study on the activation and antigen presentation of hepatitis B virus core protein virus-like particle-pulsed bone marrow-derived dendritic cells.

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.

Molecular mechanism of cell apoptosis by paclitaxel and pirarubicin in a human osteosarcoma cell line.

BACKGROUND: Paclitaxel and pirarubicin exhibit cytotoxic and antitumor activities. However, little is known about the apoptosis-inducing effects of paclitaxel and pirarubicin on human osteosarcoma MG-63 cells. METHODS: The effects of paclitaxel and pirarubicin on cell cycle arrest and apoptosis were studied in MG-63 cells using flow cytometry. PCNA, Bcl-2, Bax, cyclin D1 and cyclin E expression was assessed by Western blotting. RESULTS: Paclitaxel and pirarubicin caused G2/M and G0/G1 cell cycle arrest in MG-63 cells, respectively. Apoptosis of MG-63 cells mediated by paclitaxel was dependent on treatment duration. Interestingly, in cells treated with pirarubicin, apoptosis was related to treatment duration at concentrations of 10(2)-10(3) nM, whereas the effect of treatment duration was less marked at concentrations >10(4)-10(5) nM. Furthermore, paclitaxel and pirarubicin suppressed the expression of PCNA, cyclin D1, cyclin E and Bcl-2, and increased Bax expression. CONCLUSION: These results suggest that the G2/M or G0/G1 cell cycle arrest and apoptosis induced by paclitaxel and pirarubicin are Bcl-2/Bax dependent, suggesting favorable effects of combination therapy with paclitaxel and pirarubicin in the treatment of osteosarcoma.

[Immunoadjuvant effect of Hsp70L1 in tumor vaccine].

AIM: To explore the immune enhancement of Hsp70L1 in the tumor cell vaccines. METHODS: TRP2(153-243) and Hsp70L1 genes were obtained by RT-PCR from B16 cells in murine melanoma and from spleens of C57BL/6 mice and then were inserted into pcDNA3.1/V5-His eukaryotic expression vectors respectively. The recombinants of pTRP2, pHSP70L1 or pTRP2-Hsp fusion gene were obtained and transfected into B16 cells respectively. TRP2(153-243), HSP70L1 or TRP2-Hsp fusion gene-expressing B16 cells were then induced to necrosis by freezing-thawing or to apoptosis by mitomycin C. C57BL/6 mice were immunized with the necrotic or apoptotic B16 cells twice, then the live B16 tumor cells were transplanted into the immunized mice and the tumor growth was observed in some tumor-bearing mice. IFN-gamma-producing cells in splenocytes were measured by flow cytometry and the CTL activity of spleno-lymphocyte was detected by LDH release assay. RESULTS: After the normal mice were immunized with the necrotic or apoptotic tumor vaccines modified with TRP2(153-243), Hsp70L1 or TRP2-Hsp fusion genes, CTL lysis activity and IFN-gamma production from the splenic lymphocytes were promoted in the groups of Hsp70L1 and TRP2-Hsp modified tumor vaccines (P<0.05 or P<0.01). Additionally, the tumor growth was inhibited obviously in the groups of mice immunized with necrotic tumor vaccines (P<0.05 or P<0.01). However, no marked inhibition of tumor growth was observed in the groups of mice immunized with apoptotic tumor vaccines (P>0.05). CONCLUSION: Hsp70L1 remarkably improves the immunogenicity of B16 tumor vaccines, especially that of necrotic tumor vaccines.

A novel mouse model for immunogenic evaluation of human HBV vaccines.

To prepare a human HBV vaccine, investigators need an animal model that can help them screen and prioritize vaccine candidates. In this study, HBV/HLA-A2.1 double transgenic mice (dTg), confirmed by analysis of genomic integration and by observation of long-term expression of HBV and HLA genes, were generated for the first time. The HBV/HLA-A2.1 dTg not only display tolerance to HBV antigens (Ags), but also have the ability to process and present HLA-A2 restricted antigenic epitopes. The animals vaccinated with HLA-A2 restricted HBc(18-27) or HBs(183-191) epitope peptide vaccine induced effective HLA-A2 restricted peptide-specific cytolytic T lymphocyte responses. This was supported by the evidence of cytotoxicity assay, ELISPOT and tetramer staining analysis. Furthermore, T cell tolerance against HBV Ags in HBV/HLA-A2.1 dTg was broken by the HBc(18-27) or HBs(183-191) peptide vaccine. In conclusion, HBV/HLA-A2.1 dTg was demonstrated to be useful model for in vivo immunogenicity testing of human HBV-based vaccines.

Vaccination with combination of Fit3L and RANTES in a DNA prime-protein boost regimen elicits strong cell-mediated immunity and antitumor effect.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in the antitumor response, strategies are being pursued to elicit augmented CD8(+) T-cell responses against tumors with tumor vaccines. Here, we report the protective efficacy of vaccine-elicited antitumor immune responses with an aggressive HBc-expressing B16-HBc melanoma, which expressed HBc as a self and model antigen, tumor model. We demonstrated that the significantly better memory responses or marked inhibition on tumor growth could be achieved after coadministration of cytokine adjuvants RANTES and Flt3L in a DNA prime-protein boost regimen. Furthermore, the augmentation of DNA prime-protein boost regimens by cytokines gene was due to the improvement the immunopotency of DNA vaccine and subsequently the augmented Ag-specific and IFN-gamma mediating CD8(+) T-cell responses after protein boosting. Hence, this study demonstrates for the first time that combinatorial use of chemotactic and potent DC-specific growth factor molecules provides a useful strategy for enhancing antitumor responses.

[Immunostimulatory and antitumor effect of Flt3L and CCL5 on DNA vaccine in DNA prime/protein boost strategy].

AIM: To investigate the immune enhancment and antitumor effect of the recombinant plasmids pFlt3L and pCCL5 in DNA prime/protein boost regimens. METHODS: The mice were coimmunized with HBcAg DNA vaccine and the two cytokines DNA constructs by intramuscular injection for three times at an interval of 2 weeks. Then the mice were boosted with HBc particle proteins or DNA vaccines, respectively. The immune efficacy was evaluated by tumor growth curve. To further investigate the mechanism of inhibiting tumor growth, lymphocytes proliferation response and the number of IFN-gamma-producing cells in splenocytes were measured by MTT or flow cytometry, respectively. The levels of IL-2 and IL-4 in supernatant of spleno-lymphocyte cultures were measured by ELISA. The CTL activity of spleno-lymphocyte was detected with LDH release assay. RESULTS: Compared with negative control, DDP/Adj group significantly inhibit tumor growth; splenocytes proliferation response and the numbers of IFN-gamma-producing cells in DDD/Adj group and DDP/Adj group were significantly higher (P<0.05 or P<0.01). The levels of IL-2 in supernatant of spleno-lymphocyte cultures in DDD/Adj group and DDP/Adj group were also markedly higher than that of negative control (P<0.05); but the levels of IL-4 were no differences in all groups (P>0.05). The CTL activities in group of DDS/Adj and DDD/Adj were stronger than that of other groups (P<0.01 or P<0.05). While, the CTL killing activity in DDS/Adj group was over that of DDD/Adj (P<0.01 or P<0.05). CONCLUSION: The significant Th1 response and specific CTL against B16-HBc tumor cells are elicited by the combination of Flt3L and CCL5 in the DNA prime/protein boost strategy.

[Establishment of B16-HLA-MAGE-3 melanoma cell line coexpressing HLA-A 0201/K(b) and MAGE-3 and its role in tumor vaccine evaluation].

AIM: To establish a tumor model in HLA-A2.1 transgenic mice to examine the efficacy of MAGE-3 vaccine, a cell line coexpressing HLA-A 0201/K(b) and MAGE-3 is established. METHODS: B16-HLA-MAGE-3 melanoma was obtained by means of cotransfection of HLA-A 0201/K(b) and MAGE-3 to B16 melanoma. RT-PCR, FCM analysis and Western blot were used to detect the mRNA or protein of HLA-A 0201/K(b) or MAGE-3 expression in B16-HLA-MAGE-3. The ability of MAGE-3 antigen to be processed and presented in the B16-HLA-MAGE-3 cell line were observed by CTL activity detection and tumor challenge test. RESULTS: Transcription and protein expression of HLA-A 0201/H-2k(b) and MAGE-3 were demonstrated in B16-HLA-MAGE-3 cells. CTL activity of splenocytes in immunized mice against B16-HLA-MAGE-3 was detected and the growth of B16-HLA-MAGE-3 in immunized mice was also inhibited. CONCLUSION: MAGE-3 antigen is able to be processed and presented efficiently by B16-HLA-MAGE-3 melanoma cells and this cell can be employed to test HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice.

Expression of hepatitis B virus proteins in transgenic mice alters lipid metabolism and induces oxidative stress in the liver.

BACKGROUND/AIMS: Hepatitis B virus transgenic mice (HBV-Tg mice) have been widely used as animal models in the study of pathogenesis and control of hepatitis B. It is important for the evaluation of such animal models to define the physiological differences between HBV-Tg and wild-type mice. The aim of this research was to investigate whether the integrated system biology approach that combines proteomics and metabonomics describes the physiological changes and provides new insights into the pathogenesis of the early stages of HBV infection. METHODS: In this study the protein and metabolite profiles of the liver were established based on two-dimensional electrophoresis and HPLC/MS analysis. RESULTS: Several protein molecules, whose expression was altered in HBV-Tg mouse liver, were identified including protective enzymes against oxidative stress and regulatory proteins related to lipid metabolism. Metabonomics confirmed the potential derangement of lipid metabolism by discovering the intermediate and the final products of lipid metabolism that were markedly changed in transgenic mice. CONCLUSIONS: This study demonstrated that HBV antigens could impair host cell lipid metabolism and induce modest oxidative stress in vivo.