Objective To investigate the effect of α-1,3-galactosyltransferase (α-1,3GT)silencing through RNA interference on the immunological and the physiological functions of porcine endothelial cells.Methods The primary porcine aortic endothelial cells were isolated and immortalized through transfection of SV40LT.The lentivirus carrying the α-1,3GT shRNA was established and transfected into the cells.The expression of α-1,3GT and α-1,3Gal after transfection was detected.The transfected cells were co-cultured with human serum to establish cell model of xenotransplantation.The effects of α-1,3GT) silencing through RNA interference on the immunological and the physiological functions of porcine endothelial cells were observed by using MTT,flow cytometry,immunohistochemistry and immunofluorescence.Results The physiological function of endothelial cells showed no significant difference before and after immortalization,but the expression of α-1,3GT and Gal was decreased greatly after RNA interference.After co-culture,transfected group cells showed higher proliferation rate but lower apoptosis rate,and their binding capability to human IgM,IgG C3 and C5b-9 was decreased as compared with mismatch and control groups.Conclusion Down-regulation of the α-1,3GT could reduce the binding of porcine endothelial cells to human immunoglobulin and complement.The endothelial cells with a-1,3GT silencing by RNA interference can be used for the study on immunology of xenotransplantation.

Objective To investigate the expression of Notch1 in hepatocellular carcinoma (HCC) tissues and to evaluate its relationship with clinicopathological features and prognosis of HCC patients.Methods A total of 110 samples of HCC tissues and non-cancerous adjacent liver tissues were collected from Xijing Hospital of the Fourth Military Medical University from June 2005 to June 2008.The expressions of Notch1 and the relationship between Notch1 expression and the clinicopathological features and prognosis of HCC patients were analyzed using the chi-square test and Fisher exact probability.The survival curve was drawn by Kaplan-Meier method,and the survival was analyzed by using the log-rank test.Results Different expressions of Notch1 were detected in HCC tissues.Negative expressions of Notch1 were detected in 30 samples (27.27％),weak positive expressions of Notch1 in 32 samples (29.09％),positive expressions of Notch1 in 21 samples (19.09％),and strong positive expression of Notch1 in 27 samples (24.55％).There were some weak positive stainings of Notch1 in non-cancerous adjacent liver tissues,and all the Notch1 expressions in the non-cancerous adjacent liver tissues were at a lower level.Positive expression of Notch1 was correlated with the tumor grade,satellite lesions,portal vein tumor thrombus,metastasis and AJCC stages (x2 =6.490,8.959,9.068,23.432,22.123,P ＜ 0.05).The 3-year survival time of patients with high Notch1 expressions was 8 months,which was significantly lower than 20 months of patients with low Notch1 expressions (Log-rank value =35.096,P ＜ 0.05).The results of univariate analysis showed that positive expressions of Notch1,tumor differentiation,number of tumors,satellite lesions,portal vein tumor thrombosis,lymph node metastasis,AJCC stage were correlated with the survival of hepatic cancer patients (RR =3.364,3.402,2.164,8.548,9.470,16.717,6.011,P ＜ 0.05).The results of multivariate analysis showed that positive expression of Notch1,tumor differentiation,satellite lesions,portal vein tumor thrombus,AJCC stage were independent predictors for the survival of HCC patients (RR =1.883,2.787,3.460,5.254,2.952,P ＜ 0.05).Conclusions The expression of Notch1 in noncancerous adjacent liver tissues is weak,and the expression of Notch1 in liver tissues is significantly higher than that in noncancerous adjacent liver tissues.Notch1 might be invloved in the development of HCC.The expression of Notch1 has a close relationship with the clinicopathological features and prognosis of HCC,and it may be an independent prognostic predictor of HCC.

Background: Pig organs are commonly used in xenotransplantation, and alpha-1,3-galactose has been shown to be the main cause of hyperacute rejection. The development of transgenic pigs that lack alpha-1,3-galactosyltransferase (GGTA1) has overcome this problem to a certain extent, but transgenic pigs are difficult to maintain, making their usefulness in basic research limited. For this reason, we propose to establish a cell model to study hyperacute rejection.Methods: Immortalized primary porcine aortic endothelial cells were transfected with a short hairpin RNA targeted to GGTA1. Cell proliferation, apoptosis, complement C3 activation, and the binding of human immunoglobulins and components of the complement system, including IgM, IgG, C3, and C5b-9, were examined.Results: After RNA interference, GGTA1 was found to be reduced at both the transcript and protein level as assessed by quantitative polymerase chain reaction and flow cytometry, respectively. When cultured in the presence of human serum, the proliferation rate of the transfected cells was higher than that of untransfected cells, and the apoptosis rate was lower. Additionally, activation of C3 and the binding of human immunoglobulins IgM and IgG and complement component C3 and C5b-9 to the transfected cells were lower than in the immortalized group but higher than in untransfected cells.Conclusions: RNA interference of GGTA1 in cultured porcine endothelial cells reduces the reaction of immunoglobulin and complement system with the cells. Therefore, this in vitro cell model could be useful for further study of xenotransplantation. (C) 2013 Elsevier Inc. All rights reserved.

Liver transplantation is the best treatment for end-stage liver disease.Because of the severe shortage of donor sources,most of the patients died while waiting for liver grafts.Artificial liver support system can improve the liver function in a short time,and help patients to pass the waiting periods.Artificial liver support system takes place of composition,detoxification and metabolism function of liver,stabilizes the physiological and biochemical index of liver,relieves the burden of liver and helps patients to prepare for the liver transplantation.With the wide application of artificial liver support system,new types of the artificial liver support systems gradually conquered the defects of the old types,but they still have their own defects.This review concludes the merits and demerits of artificial liver systems,its clinical application and the problems so as to help the treatment of end-stage liver disease.

Background Dendritic cells (DCs) release bioactive exosomes that play an important role in immune regulation. Because they express low levels of class I major histocompatibility complex (MHC) and co-stimulatory molecules, exosomes derived from donor immature DCs (imDex) prolong allograft survival by inhibiting T-cell activation. However, this effect is limited and does not induce immunological tolerance when imDex are administered alone. Thus, we tested the effect of combined treatment with donor imDex and low-dose rapamycin on inducing tolerance in a mouse cardiac transplantation model. Methods ImDex were obtained from the culture supernatant of immature DCs derived from donor mouse (C57BL/6) bone marrow and were injected with suboptimal doses of rapamycin into recipient mouse (BALB/c) before and after transplantation. The capacity of this treatment to induce immune tolerance was analyzed in vitro and in vivo using the mouse cardiac transplantation model. Results Donor imDex expressed moderate levels of MHC class II and low levels of MHC class I and co-stimulatory molecules, but neither imDex nor subtherapeutic rapamycin dose alone induced cardiac allograft tolerance. Combined treatment with imDex and rapamycin, however, led to donor specific cardiac allograft tolerance. This effect was accompanied by decreased anti-donor antigen cellular response and an increased percentage of spleen CD4+CD25+ T cells in recipients. Furthermore, this donor specific tolerance could be further transferred to naïve allograft recipients through injection of splenocytes, but not serum, from tolerant recipients. Conclusion Combined with immunosuppressive treatment, donor imDex can prolong cardiac allograft survival and induce donor specific allograft tolerance.

Thymosin alpha 1 (Tα1) has immunomodulatory and anti-tumor effects in patients and has been commercialized in worldwide. An innovative technique is therefore impending to achieve high-yield expression and purification of Tα1 to meet the increasing requirements for clinical applications. Tα1 can enhance T cells, dendritic cells and antibody responses, and also augment an anti-tumor immune response. In the current study, we developed a novel technique to produce Tα1 concatemer and investigated its capability in anti-tumor immunotherapy. We expressed the recombinant 2×Tα1 concatemer protein (Tα1② protein) in Escherichia coli. The purity of Tα1② was higher than 95% as assessed by HPLC analysis. In vitro, Tα1② could stimulate the proliferation of mouse splenic lymphocyte, and increase the apoptosis of tumor cell lines. In vivo, Tα1② significantly inhibited the tumor growth in B16 tumor-bearing mice. Compared with Tα1, the Tα1② is of more effective bioactivity than Tα1. The purified Tα1② is a promising substitute for synthetic Tα1 because of its potent anti-tumor effects. We concluded that the expression system for Tα1 concatemer was constructed successfully, which could serves as a highly efficient tool for the production of large quantities of the highly active protein.

Transcription factor forkhead box protein 3 (FOXP3) is a specific marker of naturally occurring regulatory T cells (Tregs). Recently, various reports have suggested that FOXP3 may represent a tumor escape mechanism in cancer cells apart from its roles in Tregs. In the present study, the clinical and biological characteristics of FOXP3 were evaluated in human gastric cancer. The expression and localization of FOXP3 in gastric cancer cell lines was analyzed to evaluate its cellular biological features. Sections of human gastric cancer specimens were stained using immunohistochemistry (IHC) to assess the relationship between FOXP3 expression and tumor differentiation, in order to identify its clinical characteristics in gastric cancer. Expression of FOXP3 mRNA and protein was found in four gastric cancer cell lines (AGS, SGC-7901, MKN-28 and MKN-45). IHC of the gastric cancer sections revealed that more than 56% of gastric cancers displayed nuclear or cytoplasmic FOXP3 staining. Furthermore, a linear relationship between the differentiation of the gastric cancer tissues and FOXP3 expression intensity was shown. IHC and confocal analysis showed that the expression of FOXP3 was mainly present in the nucleus of tumor cells in the tissues and cell lines. Thus, FOXP3 nuclear staining may be associated with the risk of poor tumor differentiation. Apart from the lymphocytes, no FOXP3 staining was noted in the normal gastric tissues and para-tumor tissues. The high frequency of FOXP3 expression in gastric cancer tissue is a significant finding in the investigation of tumor differentiation and immune escape. This mechanism provides a further understanding of gastric cancer and a novel therapeutic strategy is presented.

Objective To construct a recombinant bacillus Calmette-Guérin(BCG) vaccines based on different tandem repeats of MUC1 and GM-CSF, rBCG-MVNTR1/4/8-CSF, and to observe the ability of three recombinant BCG vaccines in the inhibition of breast cancer. Methods After MUC1 variable-number tandem repeats (MVNTR1/4/8) were cloned in a stepwise manner, the E. coli-Mycobacteria shuttle expression vector pDE22-MVNTR1/4/8-CSF were constructed by fusing MVNTR1/4/8 and GM-CSF, and then used to transform competent BCG by electroporation after identification by restriction endonuclease digestion analysis and DNA sequencing. A novel breast cancer vaccines, rBCG-MVNTR1/4/8-CSF was constructed. The expression of fused MVNTR1/4/8-CSF protiens was analyzed by SDS-PAGE and Western blot. The ability of rBCG vaccines inhibiting the growth of breast cancer was observed in hu-PBL-SCID mice. The specific T cell responses in mice were assessed by immunohistochemistry. Results The expression of recombinant MVNTR1/4/8-CSF fusion proteins were detected by SDS-PAGE and Western Blot in rBCG-MVNTR1/4/8-CSF vaccines, respectively. Tumor incidence in mice prophylactic immunized with rBCG-MVNTR4-CSF (37.5%) or rBCG-MVNTR8-CSF (25%) significantly decreased compared with PBS and BCG-pDE22 control ( 100% ) at 42 days after tumor implantation ( P ＜ 0. 05 ). MCF-7 tumor growth inhibition in rBCG- MVNTR4/8-CSF-immunized mice was more significant than that in controls ( P ＜0. 01 ).The inhibition effect of three rBCG vaccines on breast rumor growth appeared to rise with increase of numbers of the tandem repeats of MUC1. Survival rate was 75% of mice in the rBCG-MVNTR4-CSF-treated group and 87. 5% of mice in the rBCG-MVNTR8-CSF-treated groups at 70 days after tumor implantation; however,survival rate was only 12. 5% in control group( P ＜0. 05). The CD4-positive and CD8-positive lymphocytes were detected only in rBCG-MVNTR4/8-CSF-immunized mice. Conclusions rBCG-MVNTR4/8-CSF immunization inhibits breast cancer growth in mice.

Importance of the field: The challenge in breast cancer vaccine development is to find the best combination of antigen, adjuvant and delivery system to produce a strong and long-lasting immune response. Mucin 1 (MUC1) is a potential candidate target for breast cancer immunotherapy. Bacillus Calmette-Guerin (BCG) is used widely in human vaccines. Furthermore, it can potentially offer unique advantages for developing a safe and effective multi-vaccine vehicle. Due to these properties, the development of MUC1 based recombinant BCG (rBCG) vaccines for breast cancer immunotherapy has gained great momentum in recent years.Areas covered in this review: Our aim is to discuss the recent progress in MUC1-based breast cancer immunotherapy and to highlight the advantages of MUC1-based rBCG vaccines as the new breast cancer vaccines.What the reader will gain: Several promising MUC1-based rBCG vaccines have been shown to induce MUC1-specific antitumor immune responses in preclinical studies. This review updates and evaluates this very important and rapidly developing field, and provides a critical perspective and information source for its potential clinical applications.Take home message: MUC1-based rBCG vaccines have been shown to elicit an effective anti-tumor immune response in vivo demonstrating its potential utility in breast cancer treatment.

We had prepared earlier a prokaryotic-expressed tumor necrosis factor-alpha (TNF-&agr;) mutant that exhibited a higher antitumor activity and a lower systemic toxicity compared with that of wild-type TNF-&agr; in both syngeneic murine tumor models and human tumor xenografts models. For its clinical use as an antitumor agent, we evaluated repeated-dose toxicity, anaphylaxis, genetic toxicity, pharmacokinetic, and metabolism in different animals according to the criteria of the biological investigational new drug application. It was found to be safe at a dose of 4×106 IU/kg/day for 60 days after administration in rhesus monkeys, but the TNF-&agr; antibody level and liver toxicity needed to be monitored. No systemic anaphylaxis or genetic toxicity was found and the pharmacokinetic characteristics of the recombinant mutated human TNF-&agr; (rmhTNF-&agr;) were suited for clinical use. More than 96.3% of rmhTNF-&agr; could be reclaimed from the urine and feces in 24 h after administration, which indicated the main excretion route. The results proved that the characteristics of this rmhTNF-&agr; satisfied clinical trial requirements. The related positive clinical trial results will be reported in future. This study of novel rmhTNF-&agr; is of considerable importance, not only given the proven usefulness of TNF-&agr; local application therapies under isolated limp perfusion and isolated hepatic perfusion conditions for selected indications, but also implicated for systemic application of TNF-&agr;.

AIM:To study the biological and clinical characteristics of transcription factor forkhead box protein 3 (FOXP3) in hepatocellular carcinoma (HCC).METHODS:We analyzed the expression and localization of FOXP3 in HCC tissues and cell lines to evaluate its biological features. The relationship between FOXP3 staining and clinical risk factors of HCC was assessed to identify the clinical characteristics of FOXP3 in HCC.RESULTS:The mRNA and protein expression of FOXP3 were found in some hepatoma cell lines. Immunohistochemical (IHC) analysis of HCC sections revealed that 48% of HCC displayed FOXP3 staining, but we did not find any FOXP3 staining in normal liver tissues and para-tumor tissues. IHC and Confocal analysis showed that the expressions of FOXP3 were mainly present in the nucleus and cytoplasm of tumor cells in tissues or cell lines. In HCC, the distribution of FOXP3 was similar to that of the cirrhosis, but not to the hepatitis B virus. Those findings implicate that FOXP3 staining seems to be associated with the high risk of HCC.CONCLUSION:The clinical characteristics of FOXP3 in HCC warrants further studies to explore its functions and roles in the cirrhosis and development of HCC.

Purpose Mucin-1 (MUC1) is a breast tumor-associated antigen. However, clinical trials with MUC1 showed that, with respect to its expression levels, MUC1 is a relatively poor immunogen in human beings. Evidence showed that MUC1-specific immunodominant B and T cell epitopes are derived from the variable-number tandem repeat (VNTR) region. Therefore, immunotherapy that targets multiple VNTRs may induce anti-MUC1 immune responses. GM-CSF has been shown to increase the percentage and activity of antigen-presenting cells. In this study, we constructed two recombinant Bacillus Calmette-Guérin (BCG) vaccines that combine the expression of multiple tandem repeats of MUC1 and CSF. The effect of two novel breast cancer vaccines (rBCG-MVNTR4-CSF and rBCG-MVNTR8-CSF) on the growth of breast tumor on hu-PBL-SCID mice was evaluated. Methods We coupled VNTRs (4 and 8) of MUC1 with GM-CSF (MVNTR4-CSF and MVNTR8-CSF). The MVNTR4-CSF and MVNTR8-CSF were inserted into the pDE22 plasmid and transformed into competent BCG by electroporation. The effect of both BCG vaccines on the growth of breast tumor on hu-PBL-SCID mice was evaluated. Results The growth of MUC1-positive breast tumors from hu-PBL-SCID mice immunized with two vaccines was significantly inhibited. Conclusions rBCG-MVNTR4-CSF and rBCG-MVNTR8-CSF vaccines may be good candidates for breast tumor immunotherapy.

Thirty-eight isolates of Escherichia coli (E.coli) were isolated from chickens either ill or dead suspected with E.coli. These chickens come from chicken breeding farms in the districts of Baoding, Qinhuangdao and Beijing of China. The results of the antibiotics sensitivity test in the thirty-eight isolates to twenty-four kinds of antibiotics show that all isolates have presented antibiotic resistance disparately as well as multiresistance. All isolates are sensitive to Cephalosporins antibiotics and Aminoglycosides antibiotics such as Amikacin, Cefazolin, Cefradine, Cefoperazone and Ceftriaxone. Thirty-eight isolates are extremely resistant to Penicillins antibiotics, Lincomycins antibiotics, Tetracyclines antibiotics and Macrolides antibiotics such as Midecamycine, Lincomycin, Carbenicillin, Tetracycline, Amoxicillin. Antibiotics sensitivity test provides a guidance for antibiotic application and scientific research on poultry farms.

AIM:To clone,express and purify recombinant Thymosin alpha 1 ② protein (Tα1②) in E.coli. METHODS:A 189 bp recombinant gene of 2×Tα1 tandem repeats was synthesized and cloned into pET-22b(+) vector. The expression plasmid was then transformed into E.coli. BL21(DE3). The expression of the recombinant protein Tα1② was induced with IPTG and detected by SDS-PAGE and Western blot. The biological activity of Tα1② was identified by MTT assay. RESULTS:A novel protein with expected molecular mass was expressed. The expressed product could be recognized by a monoclonal antibody against the human Tα1. The purified Tα1② stimulated the proliferation of mice splenic lymphocytes. CONCLUSION:The recombinant Tα1② is successfully cloned,expressed and purified. The purified Tα1②protein has the same function as synthesized Tα1 but higher bioactivity.

Adjuvants are necessary to elicit high titers of antibodies in vaccine-immunization procedures. We previously developed a mouse tumor necrosis factor-alpha (TNF-alpha) autovaccine (mTNF-PADRE) capable of inducing anti-TNF-alpha antibodies. In this study, we investigated the therapeutic effect of adjuvant-free administration of the autovaccine on collagen-type-II-induced rheumatoid arthritis (CIA) in mice. Our results showed that the vaccine could ameliorate the symptoms of CIA in mice. In addition, this study suggests that it is possible to control the antibody levels in mice immunized with mTNF-PADRE without adjuvant.

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. CD167 is a tyrosine kinase receptor for native type I collagen, its physiologic functions include matrix homeostasis and cell growth, adhesion, branching, and migration, but the specific role of CD167 in T cells has not yet been characterized. In this study, we found that CD167 expression on T cells was up-regulated after activation. Cooperation of CD167 engagement with suboptimal TCR/CD3 signals induced T-cell proliferation, enhanced expression of activation markers such as CD25 and CD69, elevated intracellular calcium mobilization and tyrosine phosphorylation, and introduced a bias toward a TH1/Tc1 immune response. Cooperation of CD167 engagement also enhanced mixed lymphocyte responses to alloantigens. Moreover, CD167 rapidly localized to the aggregated lipid rafts upon T-cell activation, this provided a molecular base for the signaling machinery of CD167. Together these findings, we demonstrate for the first time that CD167 could serve as a novel costimulatory receptor for T-cell activation.

Many approaches targeting MUC1 for breast tumor immunotherapy have been attempted. However, preclinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in human. B7 molecules that bind CD28 provide an antigen-nonspecific signal, which, along with an antigen-specific signal, is crucial for T-cell activation. In the present study, we constructed a novel Bacillus Calmette-Guérin-based breast cancer vaccine that coexpressed four VNTRs (variable-number tandem repeats) of MUC1 and CD80 (rBCG-MVNTR4-CD80). The aim of our study was to enhance anti-MUC1 tumor immunity by vaccination of hu-PBL-SCID mice with the recombinant BCG vaccine. The inhibition effect on tumors from the mice immunized with rBCG-MVNTR4-CD80 significantly increased, compared with rBCG-MVNTR4, BCG-pDE22, and phosphate-buffered saline immunized mice (p < 0.05, p < 0.05, p < 0.05). ELISpot assays showed that there was a significant increase in interferon-gamma production in the splenocytes from the mice immunized with rBCG-MVNTR4-CD80. In addition, CD4 and CD8-positive lymphocytes in tumors from rBCG-MVNTR4-CD80-immunized animals were detected. These data showed that rBCG-MVNTR4-CD80 immunization elicited tumor-specific immune response, which closely related with the B7 molecule (CD80), indicating that the vaccine may be a good candidate for MUC1-positive breast cancer immunotherapy.

In this study, we constructed several novel breast cancer vaccines, Bacillus Calmette-Guerin (BCG)-MUC1 variable-number tandem repeats (VNTR) 1/4/8-CSF, that consist of BCG and express 1, 4, and 8 of the tandem repeats of MUC1 and human granulocyte-macrophage colony-stimulating factor (GM-CSF). The ability of the three recombinant BCG vaccines to inhibit breast cancer growth was observed in human (hu)-peripheral blood lymphocyte (PBL)-severe combined immunodeficient (SCID) mice. Prophylactic protective responses were successfully induced and the tumor incidence in mice immunized with recombinant BCG (rBCG)-MVNTR4-CSF (37.5%) or rBCG-MVNTR8-CSF (25%) was significantly decreased compared with control (100%) at 42 days after tumor implantation (P<0.05 vs. control group). We also found that CD4-positive and CD8-positive lymphocytes were detected only in tumors grown in rBCG-MVNTR4/8-CSF-immunized animals, and strong IFN-gamma responses were induced by immunization with rBCG-MVNTR4-CSF and rBCG-MVNTR8-CSF vaccines. This study suggests a potential role of coexpressed GM-CSF and tandem repeats of MUC1 in eliciting tumor-specific immune response. Our results indicate that rBCG-MVNTR4-CSF and rBCG-MVNTR8-CSF immunization preventively inhibited breast cancer growth in hu-PBL-SCID mice and the both rBCG vaccines may be good candidates for breast cancer immunotherapy. European Journal of Cancer Prevention 18:416-423 (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Tumor growth and metastasis depend on vessel formation, and inhibition of angiogenesis of tumor by production of anti-angiogenic drugs should be a promising approach for cancer therapy. Tumstatin is an angiogenesis inhibitor. The anti-angiogenic activity of tumstatin is localized to the 54–132 amino acids. The gene fragment encoding amino acids 45–132 of tumstatin (tum-5) was subcloned into pcDNA3.1 (pcDNA-tum5). Tum-5 protein could be expressed and secreted in CHO cells after transfection. The conditioned medium (containing tum-5 protein) from the transfectant has an anti-angiogenic effect on HUVEC cells in vitro. The anti-tumor effect of pcDNA-tum5 on mice bearing S180 tumors was evaluated. The results showed that pcDNA-tum-5 has a significant inhibition activity in the growth of the tumors. This study suggests that the gene delivery of tum-5 may be an effective strategy for cancer therapy.