Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo.

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.

The combination of ATA classification and FNA results can improve the diagnostic efficiency of malignant thyroid nodules.

PURPOSE: To determine the diagnostic efficiency of the ATA classification and ultrasound-guided fine-needle aspiration (FNA) results in identifying the risk factors of malignancy, we analyzed the thyroid nodules of patients who underwent thyroidectomy and compared preoperative ATA classifications with FNA results. METHODS: We retrospectively analyzed 274 nodules of 196 patients who underwent ultrasonography, FNA and thyroidectomy. Histopathological findings of thyroid nodules were considered as the Au standard in the analysis of the diagnostic efficiency of the ATA classification and FNA results. Univariate analysis and binary multivariate logistic regression analysis were applied to identify the ultrasound features associated with malignancy. RESULTS: The overall malignancy rate of 274 nodules was 41.6%. The areas under the ROC curves (AUCs) for the ATA classification and FNA results were 0.88 and 0.878, respectively (P < 0.001). The sensitivity and specificity of the ATA classification were 86 and 86.9%, whereas those of FNA results were 68.5 and 91.4%, respectively. The specificity (98.7%) and sensitivity (94.3%) increased after the combined use of the ATA classification and FNA results. Taller-than-wide shape, microcalcifications, hypoechogenicity and irregular margins were independent risk factors for malignancy. Microcalcifications had the highest OR (7.58), and taller-than-wide shape had the highest specificity in BSRTC I, II, III and IV cytology. CONCLUSION: The diagnostic efficiency of the ATA classification and FNA results in identifying malignant nodules was high, and the use of both criteria improved the diagnostic accuracy. Taller-than-wide shape, microcalcifications, hypoechogenicity and irregular margins were independent risk factors for malignancy.

Up-regulation of CIT promotes the growth of colon cancer cells.

Colon cancer is one of the major causes of cancer mortality worldwide. However, the underlying mechanism and therapeutic targets of colon cancer have not yet been fully elucidated. In the present study, we demonstrate that citron rho-interacting, serine/threonine kinase 21 (CIT) promotes the growth of human colon cancer cells. CIT is overexpressed in human colon cancer tissues and cell lines. High expression of CIT predicts poor survival for patients with colon cancer. In colon cancer cells, CIT knockdown represses cellular proliferation and colony formation. Our in vivo xenograft experiments showed that CIT knockdown reduces the growth rate of colon cancer cells and the final tumor weight. We found that CIT knockdown induces cell cycle arrest and apoptosis in colon cancer cells. Further microarray and bioinformatics analyses indicated that CIT regulates the p53 signaling pathway, which may account for the effects of CIT on colon cancer cells. Taken together, our findings provide evidence that CIT may promote the development of colon cancer, at least in part, through the p53 signaling pathway. Therefore, CIT may be a potential therapeutic target for colon cancer treatment.

A synthetic multi-epitope antigen enhances hepatitis C virus-specific B- and T-cell responses.

Combining results from previous studies, a multi-epitope antigen PCXZ against the hepatitis C virus was synthesized in this study. The antigenic specificity of PCXZ was determined by recognizing antibodies in serum samples from hepatitis C virus patients, but not from healthy subjects or subjects who had the hepatitis B virus. The characteristics of PCXZ immunogenicity were evaluated in BALB/c mice. Strong antibody responses were generated in mice immunized with either naked PCXZ or PCXZ in Freund's adjuvant. As for the T-cell responses, Freund's adjuvant significantly increased interferon-γ secretion and enhanced the lytic activity of cytotoxic T lymphocytes. The epitope Pa, one component of PCXZ, made the most significant contribution to specific CTL lysis; this epitope was also a B-cell epitope and was able to induce high IgG titers. In summary, PCXZ was found to be highly immunogenic, and elicited both humoral and cellular immune responses in mice.

Tumor antigen delivered by Salmonella III secretion protein fused with heat shock protein 70 induces protection and eradication against murine melanoma.

Attenuated Salmonella typhimurium possess the ability to stimulate innate immune responses and preferentially allocate within the solid tumor. These two main characteristics make attenuated Salmonella one of the most attractive vehicles for development of vaccine and also targeted cancer therapies. However, location of Salmonella prevents the process of antigen presentation. Salmonella Type III secretion system can be utilized to circumvent this problem because this system secretes the protein it encoded outside the cells. Heat shock protein 70 (Hsp70) is referred to as an "immunochaperone" for its capacity to elicit tumor-specific adaptive immune responses in the form of Hsp70-TAA (tumor associated antigen) complex. Hsp70 facilitates the cross-presentation of exogenous antigens through its receptor on antigen-presenting cells and therefore activates an antigen-specific cytotoxic T lymphocyte (CTL) response, which can directly contribute to potent anti-tumor immunity. Here, we designed a novel therapeutic vaccine utilizing the type III secretion system and Hsp70 to deliver and present the tumor-specific antigen. This live recombinant bacteria vaccine, when administrated orally, successfully broke the immune tolerance, induced a specific CTL response against tumor cells, and therefore revealed protective and therapeutic effects against generation and growth of B16F10 melanoma in C57BL/6J mice.

Enzymatic synthesis of 2'-deoxyadenosine and 6-methylpurine-2'-deoxyriboside by Escherichia coli DH5alpha overexpressing nucleoside phosphorylases from Escherichia coli BL21.

Genes encoding purine nucleoside phosphorylase (deo D), uridine phosphorylase (udp) and thimidine phosphorylase (deo A) from Escherichia coli BL21 were cloned and overexpressed in E. coli DH5alpha. The recombinant strains were employed to synthesize 2'-deoxyadenosine (dAR) and 6-methylpurine-2'-deoxyriboside (MePdR). Experimental parameters such as strains, temperature, pH, reagent concentration and cell mass were optimized. Under the optimal situation, 96% adenine was converted to dAR and 95% 6-methylpurine (MeP) was converted to MePdR in an hour, using 0.2 per thousand (dry wt./v) cell paste as biocatalyst.

The treatment and prevention of mouse melanoma with an oral DNA vaccine carried by attenuated Salmonella typhimurium.

Therapeutic vaccines of cancer are attractive for their capacity of breaking the immune tolerance and invoking long-term immune response targeting cancer cells without autoimmunity. An efficient antigen delivery system is the key issue of developing an effective cancer vaccine. Attenuated Salmonella typhimurium as the carrier of cancer vaccine are able to transfer DNA from the prokaryote to the eukaryote and preferentially replicate within the tumor tissue. Heat shock protein 70 delivers the tumor-associated antigens to antigen presenting cells through its polypeptide-binding domain and breaks immune tolerance of the cancer cells. Here we described a novel low-copy-number DNA vaccine based on the Hsp70-TAA complex and carried by the attenuated S. typhimurium strain SL3261. Oral administration of this vaccine elicited specific CTL-mediated lysis of the melanoma tumor cells and marked activation of the T-cells. The therapeutic vaccine effectively protected 57.1% C57BL/6J mice from lethal challenge with B16F10 melanoma tumor cells in prophylactic settings and eraicated 62.5% tumor growth in therapeutic settings. This approach may provide a new strategy for the prevention and treatment of cancer.

The intrabody targeting of hTERT attenuates the immortality of cancer cells.

hTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells.

Oral administration of attenuated S. typhimurium carrying shRNA-expressing vectors as a cancer therapeutic.

RNAi has been successfully applied in genomic research, and it also holds considerable promise as a therapeutic approach to suppress disease-causing gene expression. Here, we show that attenuated S. typhimurium were capable of delivering shRNA-expressing vectors to mammalian cells and inducing RNAi in vitro and in vivo. Upon oral administration, S. typhimurium carrying shRNA-expressing vectors targeting bcl2 induced significant gene silencing in murine melanoma cells that led to a remarkably delayed tumor growth and prolonged survival in the mouse model. These results suggest that bacteria mediated RNAi may be a new potent approach to the treatment of cancers.