Therapeutic effects of human monoclonal PSMA antibody-mediated TRIM24 siRNA delivery in PSMA-positive castration-resistant prostate cancer.

Background and Aims: Prostate specific membrane antigen (PSMA) is specifically expressed on prostate epithelial cells and markedly overexpressed in almost all prostate cancers. TRIM24 is also up-regulated from localized prostate cancer to metastatic castration-resistant prostate cancer (CRPC). Because of the high relevance of TRIM24 for cancer development and the universal expression of PSMA in CPRC, we investigated the efficacy of human monoclonal PSMA antibody (PSMAb)-based platform for the targeted TRIM24 siRNA delivery and its therapeutic efficacy in CRPC in vivo and in vitro. Methods: The therapeutic complexes were constructed by conjugating PSMAb and sulfo-SMCC-protamine, and encapsulating TRIM24 siRNA. Flow cytometry, immunofluorescence, and fluorescence imaging were performed to detect the receptor-binding, internalization, and targeted delivery of PSMAb-sulfo-SMCC-protamine (PSP)-FAM-siRNA complex (PSPS) in vitro and in vivo. CCK-8, plate-colony formation, apoptosis, cell cycle, and Transwell assays were performed to evaluate the therapeutic potential of the PSP-TRIM24 siRNA complex in vitro, whereas the in vivo therapeutic efficacy was monitored by small animal imaging, radiography, and micro CT. Results: We confirmed that PSP could efficiently protect siRNA from enzymatic digestion, enable targeted delivery of siRNA, and internalize and release siRNA into PSMA-positive (PSMA+) prostate cancer cells in vitro and in vivo. Silencing TRIM24 expression by the PSP-TRIM24 siRNA complex could dramatically suppress proliferation, colony-formation, and invasion of PSMA+ CRPC cells in vitro, and inhibit tumor growth of PSMA+ CRPC xenografts and bone loss in PSMA+ CRPC bone metastasis model without obvious toxicity at therapeutic doses in vivo. Conclusion: PSMAb mediated TRIM24 siRNA delivery platform could significantly inhibit cell proliferation, colony-formation, and invasion in PSMA+ CRPC in vitro and suppressed tumor growth and bone loss in PSMA+ CRPC xenograft and bone metastasis model.

Tumor-suppressive miR-145 co-repressed by TCF4-ß-catenin and PRC2 complexes forms double-negative regulation loops with its negative regulators in colorectal cancer.

The frequently dysregulated Wnt/ß-catenin signaling in different malignancies, by activation of its own or orchestration with other co-factors, regulates various oncogenic or tumor-suppressive genes. Among these genes, miRNAs, which are negative posttranscriptional regulators, are also embedded in the Wnt signaling network. Different from the Wnt-induced oncogenic miRNAs, the specific mechanism underlying the Wnt-repressed tumor-suppressive miRNAs is much less understood. In our study, firstly by analyzing a ChIP-seq dataset against TCF4, the core transcription factor for initiation of Wnt signaling in colorectal cancer (CRC) cells, we screened out several tumor-suppressive miRNAs potentially regulated by Wnt signaling. Then through siRNA-mediated knock-down tests and protein and chromatin immunoprecipitations, we found the TCF4-ß-catenin complex can recruit the histone trimethylation complex PRC2 as a co-repressor while binding to the TCF4-binding element (TBE) in the promoter regions of miR-145, miR-132 and miR-212. Thus, upon Wnt signaling activation, the PRC2-mediated trimethylation of histone H3 at lysine 27 increases at these promoter regions, leading to decreased miRNA levels. Furthermore, we found that by targeting TCF4 and SUZ12, the key components of the negative regulation complexes, the tumor-suppressive miR-145 co-repressed by Wnt signaling and histone trimethylation, forms double-negative regulation loops with its negative regulators in CRC cells. And the inverse associations between miR-145 and its targets/negative regulators have also been demonstrated in nude mice and clinical samples. Collectively, we elucidated the detailed molecular mechanism of how dysregulated Wnt/ß-catenin signaling and tumor-suppressive miRNAs reciprocally regulate each other in CRC cells.

Isolation and determination of four potential antimicrobial components from Pseudomonas aeruginosa extracts.

Background:Pseudomonas aeruginosa can cause disease and also can be isolated from the skin of healthy people. Additionally, it exhibits certain antimicrobial effects against other microorganisms.Methods: We collected 60 strains of P. aeruginosa and screened their antimicrobial effects against Staphylococcus aureus (ATCC 25923) using the filter paper-disk method, the cross-streaking method and the co-culture method and then evaluated the antimicrobial activity of the chloroform-isolated S. aureus extracts against methicillin-resistant S. aureus (MRSA, Gram-positive cocci), vancomycin intermediate-resistant S. aureus (VISA, Gram-positive cocci), Corynebacterium spp. (CS, Gram-positive bacilli), Acinetobacter baumannii (AB, Gram-negative bacilli), Moraxella catarrhalis (MC, Gram-negative diplococcus), Candida albicans (CA, fungi), Candida tropicalis (CT, fungi), Candida glabrata (CG, fungi) and Candida parapsilosis (CP, fungi). Results: The PA06 and PA46 strains have strong antimicrobial effects. High-performance liquid chromatography (HPLC) analysis revealed that the major components of PA06 and PA46 that exhibit antimicrobial activity are functionally similar to phenazine-1-carboxylic acid (PCA) and pyocyanin. Preparative HPLC was performed to separate and isolate the 4 major potential antimicrobial components: PA06ER10, PA06ER16, PA06ER23 and PA06ER31. Further, the molecular masses of PA06ER10 (260.1), PA06ER16 (274.1), PA06ER23 (286.1) and PA06ER31 (318.2) were determined by electrospray ionization (ESI) mass spectrometry. Conclusion:P. aeruginosa can produce small molecules with potential antimicrobial activities against MRSA, VISA, CS, MC, CA, CT, CG and CP but not against AB.

CD4+T cell specific B7-H1 selectively inhibits proliferation of naïve T cells and Th17 differentiation in experimental autoimmune encephalomyelitis.

It is widely acknowledged that interleukin 17-producing T helper (Th17) cells are critically participant in the pathogenesis of multiple sclerosis. In the current study, we identified that the expression of CD4+T cells specific co-inhibitory molecule B7-homologue 1(B7-H1) in spleenocytes and mononuclear cells isolated from brains and spinal cord were positive correlated with Th1 and Th17 cells generation and disease severity in experimental autoimmune encephalomyelitis (EAE). Furthermore, B7-H1 transgenic mice developed milder EAE symptoms and fewer Th17 cells than B7-H1 wild type mice. We also found the proliferation of naïve CD4+CD62+T cells isolated from B7-H1 transgenic mice was inhibited. And naïve T cells isolated from B7-H1 transgenic mice produced fewer Th17 cells than WT mice in Th17-polarizing conditions, but the Th1, Th2, and inducible Treg differentiation were the similar in naïve T cells isolated from B7-H1 transgenic mice and WT mice. In conclusion, our study show CD4+T cells specific B7-H1 is a slective inhibitor in proliferation of naïve T cells, Th17 differentiation and pathogenesis of multiple sclerosis.

Anti-tumor effects of a recombinant anti-prostate specific membrane antigen immunotoxin against prostate cancer cells.

BACKGROUND: To evaluate anti-prostate cancer effects of a chimeric tumor-targeted killer protein. METHODS: We established a novel fusion gene, immunocasp-3, composed of NH2-terminal leader sequence fused with an anti-prostate-specific membrane antigen (PSMA) antibody (J591), the furin cleavage sequences of diphtheria toxin (Fdt), and the reverse coding sequences of the large and small subunits of caspase-3 (revcaspase-3). The expressing level of the immunocasp-3 gene was evaluated by using the reverse transcription-PCR (RT-PCR) and western blot analysis. Cell viability assay and cytotoxicity assay were used to evaluate its anti-tumor effects in vitro. Apoptosis was confirmed by electron microscopy and Annexin V-FITC staining. The antitumor effects of immunocasp-3 were assessed in nude mice xenograft models containing PSMA-overexpressing LNCaP cells. RESULTS: This study shows that the immunocasp-3 proteins selectively recognized and induced apoptotic death in PSMA-overexpressing LNCaP cells in vitro, where apoptotic cells were present in 15.3% of the cells transfected with the immunocasp-3 expression vector at 48 h after the transfection, in contrast to 5.5% in the control cells. Moreover, LNCaP cells were significantly killed under the condition of the co-culture of the immunocasp-3-secreting Jurkat cells and more than 50% of the LNCaP cells died when the two cell lines were co-cultured within 5 days. In addition, The expression of immunocasp-3 also significantly suppressed tumor growth and greatly prolonged the animal survival rate in vivo. CONCLUSION: A novel fusion gene, immunocasp-3, may represent a viable approach to treating PSMA-positive prostate cancer.

Epigenetically regulated miR-145 suppresses colon cancer invasion and metastasis by targeting LASP1.

MiR-145 is a tumor-suppressive microRNA that participates in the malignant progression of colorectal cancer (CRC). Although miR-145 has been reported to inhibit proliferation and to induce apoptosis of CRC cells, the reports about its role in invasion and metastasis are controversial. The regulation of miR-145 its own expression also requires further elucidation. In this study, we firstly found that miR-145 is markedly downregulated in the metastatic tumors of CRC patients. Then through gain- and loss-of function studies, we demonstrated that miR-145 suppresses the invasion and metastasis of CRC cells. We also provided experimental evidences which include direct binding assays and verifications on tissue specimens to confirm that LIM and SH3 protein 1 (LASP1) is a direct target of miR-145. Furthermore, we identified the core promoter regions of miR-145 and observed the cooperation between histone methylation and transcription factors through binding to these core promoter regions to regulate the expression of miR-145 in CRC cells. Our study provides an insight into the regulatory network in CRC cells, thus offering new targets for treating CRC patients.

Predictive value of HLA-G and HLA-E in the prognosis of colorectal cancer patients.

HLA-G and HLA-E are non-classical HLA Ib molecules. Recently, increasingly more reports have shown that HLA-G is highly expressed in different malignancies. In this article, we detected the expression levels of HLA-G and HLA-E in primary colorectal cancer patients. Our results showed that 70.6% and 65.7% of the colorectal cancer tissues had positive HLA-G or HLA-E expression, respectively, and that 46.1% positively expressed both molecules. We also analyzed the correlations between the expression levels of HLA-G, HLA-E or both combined and the clinical outcomes of the patients. Kaplan-Meier analysis results showed that the expression levels of HLA-G or HLA-E alone and the combined expression of both molecules were all statistically correlated with the overall survival of colorectal cancer patients. Cox multivariate analysis showed that only HLA-G expression can serve as independent factor for OS. Our results also showed that the expression of HLA-E was significantly correlated with tumor metastasis.

c-Myc-mediated epigenetic silencing of MicroRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma.

UNLABELLED: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01). CONCLUSIONS: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC.

[Construction, expression, and identification of the gene of human anti-prostate specific membrane antigen single-chain antibody].

OBJECTIVE: To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins. METHODS: The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA. RESULTS: The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen. CONCLUSION: Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.

MiR-568 inhibits the activation and function of CD4⁺ T cells and Treg cells by targeting NFAT5.

CD4(+) T cells play critical roles in orchestrating adaptive immune responses. Their activation and proliferation are critical steps that occur before they execute their biological functions. Despite the important role of this process, the underlying molecular events are not fully understood. MicroRNAs (miRNAs) have been shown to play important roles in lymphocyte development and function. However, the miRNAs that regulate T-cell differentiation, activation and proliferation are still largely unknown. In our previous study, using a miRNA array, we found that several miRNAs (including miR-202, 33b, 181c, 568 and 576) are differentially expressed between resting and activated CD4(+) T cells. In this study, we focused on the function of miR-568 during CD4(+) T-cell activation. We showed that the expression level of miR-568 decreased during the activation of T cells, including Jurkat cells and human peripheral blood CD4(+) T cells. When Jurkat or human peripheral blood CD4(+) T cells were transfected with miR-568 mimics, cell activation was significantly inhibited, as shown by the inhibited expression of activation markers such as CD25, CD69 and CD154; decreased IL-2 production; and inhibited cell proliferation. Using software predictions and confirmatory experiments, we demonstrated that nuclear factor of activated T cells 5 (NFAT5) is a target of miR-568. Treg cells are an important CD4(+) T-cell subpopulation, so we also evaluated the function of miR-568 in Treg-cell activation and differentiation. We showed that the miR-568 level decreased, while the NFAT5 protein level increased during CD4(+)CD25(+) Treg-cell activation, and the transfection of miR-568 mimics inhibited the NFAT5 expression, inhibited the production of both TGF-ß and IL-10 and also inhibited the proliferation of Treg cells. Our further study showed that over-expression of miR-568 can inhibit Treg-cell differentiation and can inhibit the suppressive effect of these cells on effector cells. In addition, inhibition of NFAT5 by siRNA-mediated knockdown can inhibit the activation and differentiation of Treg cells. These findings reveal that miR-568 can inhibit the activation and function of both CD4(+) T cells and Treg cells by targeting NFAT5. Since miR-568 plays an important role in both CD4(+) T cells and Treg cells, these findings may provide leads for the development of novel treatments for human inflammatory and autoimmune diseases.

B7-H1 expression is associated with poor prognosis in colorectal carcinoma and regulates the proliferation and invasion of HCT116 colorectal cancer cells.

BACKGROUND AND OBJECTIVE: The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells. METHODS: Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells. RESULTS: Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition. CONCLUSIONS: Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.

Pseudogene OCT4-pg4 functions as a natural micro RNA sponge to regulate OCT4 expression by competing for miR-145 in hepatocellular carcinoma.

The POU transcription factor OCT4 is a pleiotropic regulator of gene expression in embryonic stem cells. Recent studies demonstrated that OCT4 is aberrantly expressed in multiple types of human cancer; however, the underlying molecular mechanism remains largely unknown. In this study, we report that OCT4-pg4, a pseudogene of OCT4, is abnormally activated in hepatocellular carcinoma (HCC). The expression level of OCT4-pg4 is positively correlated with that of OCT4, and both gene transcripts can be directly targeted by a tumor-suppressive micro RNA miR-145. We find that the non-coding RNA OCT4-pg4 is biologically active, as it can upregulate OCT4 protein level in HCC. Mechanistic analysis revealed that OCT4-pg4 functions as a natural micro RNA sponge to protect OCT4 transcript from being inhibited by miR-145. In addition, our study also showed that OCT4-pg4 can promote growth and tumorigenicity of HCC cells, thus exerting an oncogenic role in hepatocarcinogenesis. Furthermore, survival analysis suggests that high OCT4-pg4 level is significantly correlated with poor prognosis of HCC patients. Taken together, our finding adds a new layer of post-transcriptional regulation of OCT4 and sheds new light on the treatment of human HCC.

Ectopically expressed perforin-1 is proapoptotic in tumor cell lines by increasing caspase-3 activity and the nuclear translocation of cytochrome C.

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.

Human activated CD4(+) T lymphocytes increase IL-2 expression by downregulating microRNA-181c.

MicroRNAs, a large family of small regulatory RNAs, are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner, thereby controlling diverse aspects of cell function, including immune reaction. In this study, we screened and identified a group of differentially expressed miRNAs in naive and activated CD4(+) T cells. Among the miRNAs studied, miR-181c was proven to have the potential to regulate CD4(+) T cell activation. miR-181c was downregulated in the process of CD4(+) T cell activation, and transfection of miR-181c mimics partially repressed the activation of both Jurkat cells and human peripheral blood mononuclear cells (PBMC) CD4(+) T cells. We further showed that miR-181c can bind to the IL-2 3' UTR and repress its expression by inhibiting translation. Moreover, miR-181c mimics reduced activated CD4(+) T cell proliferation. Taken together, our results show that miR-181c serves as a negative regulator that modulates the activation of CD4(+) T cells.

[Expression of a chimeric gene containing poly-arginine as the protein transduction domain and its killing activity against HER2 positive gastric cancer SGC-7901 cells].

AIM: To construct a eukaryotic expression vector for chimeric gene containing poly-arginine as the protein transduction domain(PTD) and transiently transfect this vector into HER2 positive SGC-7901 cells and HER2 negative HeLa cells to examine its effect on cell growth. METHODS: PCR amplication was used to obtain the gene of active form caspase-3 fused with nonaarginine, and then fusion gene was cloned into eukaryotic expression vector containing e23sFv DNA fragment. After this chimeric gene transfected into SGC-7901 cells and HeLa cells by Lipofectamine 2000™; reagent, indirect immunofluorescence and cell counting were used to examine the expression in these two cells and the effect on cell growth. RESULTS: The eukaryotic expression vector, named pCMV-e23sFv-R9;-casp3, encoding e23sFv/caspase-3 containing nonaarginine as the PTD was successfully constructed. e23sFv- R9;-casp3 protein was expressed in a secretary manner in both SGC-7901 cells and HeLa cells. Transfected SGC-7901 cells were found obvious growth inhibitory, morphology change and condensed nucleus, whereas neither growth inhibitory nor apparent morphology change was detected in transfected HeLa cells. CONCLUSION: Of the secretary expressed chimeric protein, the antibody moiety against HER2 can mediate targeted recognition, the nonaarginine translocating peptide can promote activation and translocation of the effector molecule, and the active caspase-3 can effectively induce cell killing.

A survivin double point mutant has potent inhibitory effect on the growth of hepatocellular cancer cells.

Survivin is an attractive target in cancer therapy. Previous studies have demonstrated that survivin dominant-negative mutants T34A and C84A were able to induce apoptosis in cancer cells. Given that they had different mechanisms in inducing apoptosis, our study was undertaken to determine whether a survivin double point mutant (TC34,84AA) could achieve more potent inhibitory effect on the growth of hepatocellular cancer cells. Adenoviruses expressing survivin mutants were constructed and transduced into hepatocellular cancer cells. The inhibitory effect of the survivin mutants on cancer cell growth was measured. Transduction of cancer cells with all three survivin mutants resulted in significant apoptosis. Compared with survivin mutants T34A or C84A alone, the cancer killing effect of survivin TC34,84AA was much stronger. In addition, the survivin mutants were more sensitive than wild type survivin to the degradation in the ubiquitin-proteasome pathway. Our results suggest that adenovirus-delivered dominant-negative survivin TC34,84AA promotes apoptosis-mediated hepatocellular carcinoma suppression, and could potentially be a promising candidate for cancer therapies.

Recombinant immunoproapoptotic proteins with furin site can translocate and kill HER2-positive cancer cells.

We previously reported the selective killing of HER2-positive tumor cells by a class of immunoproapoptotic proteins containing single-chain antibody, translocation domain of Pseudomonas exotoxin A (domain II; PEA II), and constitutively active human apoptotic molecules. In this study, a novel class of antitumor immunoproapoptotic proteins was explored to mediate tumor-specific apoptosis both in vitro and in vivo. Three furin cleavage sequences, including a synthetic polyarginine tract, and two furin cleavable sequences from PEA and diphtheria toxin were respectively used to replace PEA II in the previously constructed immunoproapoptotic protein. When produced and secreted by the genetically modified Jurkat cells, the novel targeted proapoptotic proteins selectively bound to HER2, which is often overexpressed on tumor cell surface. Followed by receptor-mediated endocytosis and furin cleavage in the endosome, the recombinant proteins could translocate into the cytosol, leading to irreversible cell death. Moreover, delivery of these proteins by either i.m. plasmid injection or i.v. injection of plasmid-expressing Jurkat cells led to tumor regression and prolonged animal survival in a nude mouse xenograft tumor model, indicating in vivo antitumor activity of the recombinant proteins. We conclude that the new class of immunoproapoptotic proteins show comparable activity with PEA II-containing counterpart and provide an attractive therapeutic alternative as they contain much less exogenous fragments.

Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery.

UNLABELLED: RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sCkappa-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sCkappa-tP, a constant region of the kappa chain (Ckappa). S-tP and sCkappa-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sCkappa-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. CONCLUSION: Our results describe a potential method for the targeted delivery of siRNA or siRNA-producing plasmids against HBV, using anti-HBsAg fusion proteins.

TGF-beta insensitive dendritic cells: an efficient vaccine for murine prostate cancer.

Dendritic cells (DCs) are highly potent initiators of the immune response, but DC effector functions are often inhibited by immunosuppressants such as transforming growth factor beta (TGF-beta). The present study was conducted to develop a treatment strategy for prostate cancer using a TGF-beta-insensitive DC vaccine. Tumor lysate-pulsed DCs were rendered TGF-beta insensitive by dominant-negative TGF-beta type II receptor (TbetaRIIDN), leading to the blockade of TGF-beta signals to members of the Smad family, which are the principal cytoplasmic intermediates involved in the transduction of signals from TGF-beta receptors to the nucleus. Expression of TbetaRIIDN did not affect the phenotype of transduced DCs. Phosphorylated Smad-2 was undetectable and expression of surface co-stimulatory molecules (CD80/CD86) were upregulated in TbetaRIIDN DCs after antigen and TGF-beta1 stimulation. Vaccination of C57BL/6 tumor-bearing mice with the TbetaRIIDN DC vaccine induced potent tumor-specific cytotoxic T lymphocyte responses against TRAMP-C2 tumors, increased serum IFN-gamma and IL-12 level, inhibited tumor growth and increased mouse survival. Furthermore, complete tumor regression occurred in two vaccinated mice. These results demonstrate that blocking TGF-beta signals in DC enhances the efficacy of DC-based vaccines.

Development of a phage displayed disulfide-stabilized Fv fragment vaccine against Vibrio anguillarum.

Anti-idiotype monoclonal antibody 1E10 can mimic the protective epitope of Vibrio anguillarum and be used as vaccine to prevent fish infection of V. anguillarum. In this study, the variable heavy (V(H)) domain and variable light (V(L)) domain of mAb1E10 were cloned by RT-PCR and were linked to each other by a disulfide bond engineered at position 44 of V(H) and position 105 of V(L) that lie between structurally conserved framework positions. Mutated V(H) 44 and V(L) 105 were inserted into phagemid pCANTAB5E. When co-transfected by recombinant pCANTAB5E and helper phage M13KO7, the host Escherichia coli cells secreted disulfide-stabilized Fv fragment (dsFv) which displayed on the surface of filamentous phage. The binding specificity of the phage-displayed dsFv was proved by ELISA method. Protection experiment showed that Japanese flounders can develop high titer of antibody against the dsFv and survival ratio of vaccinated group was significantly different from control groups. Thus, this phage-displayed dsFv may be used as vaccine against V. anguillarum in fishery.