Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt­TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

TGF-ß1 protein expression in non-small cell lung cancers is correlated with prognosis.

To investigate the expression intensity and prognostic significance of TGF-ß1 protein in non-small cell lung cancer (NSCLC), immunohistochemistry was carried out in 194 cases of NSCLC and 24 cases of normal lung tissues by SP methods. The PU (positive unit) value was used to assess the TGF-ß1 protein expression in systematically selected fields under the microscope with Leica Q500MC image analysis. We found that the TGF-ß1 PU value was nearly two-fold higher in NSCLC than in normal lung tissues (p=0.000), being associated with TNM stages (p=0.000) and lymph node metastases (p=0.000), but not to patient age, gender, smoking history, tumor differentiation, histological subtype and tumor location (P>0.05). Univariate analysis indicated that patients with high TGF-ß1 protein expression and lymph node metastases demonstrated a poor prognosis (both p=0.000, ). Multivariate analysis showed that TGF-ß1 protein expression (RR = 2.565, p=0.002) and lymph node metastases (RR=1.874, p= 0.030) were also independent prognostic factors. Thus, TGF-ß1 protein expression may be correlated to oncogenesis and serve as an independent prognostic biomarker for NSCLC.

Value of T cell receptor gamma alternate reading frame protein and keratin 5 in endometrial carcinoma.

BACKGROUND: Tumors with different gene expression develop and progress in different ways. To deepen our understanding of the progression in endometrial cancer, and provide a useful tool for accurate diagnosis and prognosis assessment, we identified the new molecular prognostic markers in endometrial carcinoma and analyzed the relationship of them with clinical and pathological features of endometrial carcinoma. METHODS: Ninety-four cases of endometrial endometrioid adenocarcinoma with complete data from the Peking University People's Hospital from 2000 to 2008 and 40 cases of normal endometrium were enrolled. Among these, 30 endometrial endometrioid adenocarcinoma samples of different International Federation of Gynecology and Obstetrics (FIGO) stage were selected for further Agilent genome-wide microarray analysis. Significance analysis of microarrays (SAM) was used to identify genes that are significantly associated with tumor progress. Immunohistochemistry was utilized to identify the genes of interest in endometrial carcinoma and normal endometrium. The relationship between the genes and the age, clinical stage, histological grade, myometrium invaded depth, lymph node metastasis status, and the expression of ER, PR, P53, and PTEN were analyzed by χ(2) test. RESULTS: Analysis between FIGO 1988 stage I and stage III identified a 362-gene "progress signature"; 171 down-regulated and 191 up-regulated genes. Among the alterative genes, TARP (T cell receptor gamma alternate reading frame protein) and KRT5 (keratin 5) decreased 3.57 fold and 5.8 fold in FIGO stage III patients. The expression of TARP in endometrial carcinoma increased compared to normal endometrium, while that of KRT5 decreased (P < 0.05). The expression of TARP and KRT5 decreased when stage, histological grading, myometrium invaded depth increased (P < 0.05). In the cases with lymph node metastasis, the expression of TARP decreased, while the expression of KRT5 did not differ (both P < 0.05) both. The expression of P53 had a negative relationship with the expression of KRT5 (P < 0.05), but not with the expression of TARP (P > 0.05). There was no correlation between the expression of TARP and KRT5 and the expression of ER, PR, PTEN (all P > 0.05). There was no significant difference in TARP and KRT5 expression in patients aged 50 or younger and patients older than 50 (P > 0.05). CONCLUSION: The expression of TARP and KRT5 was correlated with the progress of endometrial cancer and their role needs further study.

Overexpression of the insulin receptor isoform A promotes endometrial carcinoma cell growth.

Epidemiological studies have demonstrated that type 2 diabetes mellitus (T2DM) and hyperinsulinemia are associated closely with endometrial carcinoma risk, although the molecular mechanism remains unclear. Insulin receptor isoformA expression is upregulated in many cancer cells and tissues, which suggests that IR-A-mediated signaling pathways may have important implications for cancer pathogenesis. We measured the expression of insulin receptor isoforms (IR-A and IR-B in the normal endometrium tissues, the endometrial carcinoma tissues and the endometrial carcinoma cell lines. We found that the total insulin receptor (IR) and IR-A expression mRNA levels and the ratio of IR-A to total IR in endometrial carcinoma specimens were significantly higher than them in control endometrial tissue specimens(P<0.05). Further analysis indicated that the tendency was more prominently in patients with T2DM. IR-A mRNA was differentially expressed in four endometrial carcinoma cell lines (Ishikawa, KLE, RL95-2 and HEC-1-A. RL95-2 cells have a low endogenous IR-A expression, and these were used to construct a stable cell line overexpressing IR-A. We found that IR-A overexpression significantly increased cell proliferation, the proportion of cells in S phase, activation of the Akt pathway and tumorigenicity of xenografts in nude mice. In contrast, there was no significant difference in the the percentage of apoptotic cells between cells overexpressing IR-A and control cells. Moreover, levels of phosphorylated ERK1/2 protein were significantly decreased in cells overexpressing IR-A relative to controls. These findings reveal the pivotal role of IR-A in endometrial cancer carcinogenesis, and suggest that the association of elevated IR-A levels with cell proliferation and tumorigenicity may be causally linked to its effect on the proportion of cells in S phase and the activation of the Akt pathway.

Effects of insulin, insulin-like growth factor-I and -II on proliferation and intracellular signaling in endometrial carcinoma cells with different expression levels of insulin receptor isoform A.

BACKGROUND: Hyperinsulinemia, insulin-like growth factor (IGF)-I and -II (IGF-II) are associated with increased risk of endometrial carcinoma. Insulin receptor isoform A (IR-A) is more frequently expressed in endometrial carcinoma than in normal endometrial tissues. To better understand their roles in endometrial carcinoma, we investigated the effects of insulin, IGF-I, and IGF-II in endometrial carcinomas cells with different IR-A expression levels. METHODS: To explore the role of IR-A in mediating the activity of IGF-I, IGF-II, and insulin, we investigate the cellular proliferation of endometrial carcinoma cell lines RL95-2 and RL95-2-IR-A by MTS assays. Then we examined the protein kinase Akt phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in both cell lines by Western blotting. The effect of IGF-II and AG1024 on cell cycle progression and apoptosis was assessed by flowcytometry. To examine whether the effects of IGFs were mediated by IR-A, we blocked IGF-I receptor (IGF-IR) in both cell lines using AG1024, an IGF-IR-specific inhibitor. RESULTS: IGF-I and IGF-II significantly enhanced proliferation of both cell lines (P < 0.05). By contrast, insulin significantly increased proliferation of RL95-2-IR-A cells only (P < 0.05). IGF-I and IGF-II significantly increased pAkt levels in RL95-2 cells and pERK1/2 levels in RL95-2-IR-A cells (all, P < 0.05). Insulin increased pERK1/2 levels in RL95-2-IR-A cells only (P < 0.05). LY294002 and PD98059 inhibited the specific signaling activities and cellular proliferation. After AG1024 pretreatment, neither IGF-I nor IGF-II affected pAkt levels in RL95-2 cells. IGF-II, but not IGF-I, increased pERK1/2 levels in RL95-2-IR-A cells. After AG1024 pretreatment, the proliferation rate and DNA content corresponding to the S phase increased and apoptosis decreased significantly in IGF-II-treated RL95-2-IR-A cells only (P < 0.05). CONCLUSIONS: The proliferation effect of insulin is mediated by IR-A. When IR-A dominates in a cell line, IGF-II activated cell proliferation mainly through the ERK1/2 pathway. On the other hand, IGF-II activated cell proliferation mainly through the Akt pathway. IR-A can at least partly mediate the proliferative and anti-apoptotic effects of IGF-II through the ERK1/2 pathway.

[Association of HAb18G with clinicopathologic features and prognosis in non-small cell carcinoma of lung].

OBJECTIVE: To study the association between HAb18G expression, tumor parameters, metastatic potential and prognosis in non-small cell lung carcinoma (NSCLC). METHODS: Immunohistochemical study for HAb18G protein using SP methods was carried out in 144 cases of NSCLC. Nineteen cases of benign lung lesions and 41 cases of normal lung tissue were used as controls. The intensity (positive unit/PU) of HAb18G expression was assessed quantitatively by image analysis software. The results were correlated with tumor parameters, metastatic potential and follow-up data. RESULTS: The intensity of HAb18G protein expression was significantly higher in NSCLC than that in controls (P = 0.000). In squamous cell carcinomas and adenocarcinomas, the expression of HAb18G protein in well-differentiated tumors was lower than that in moderately to poorly differentiated tumors (P = 0.001). Tumors of TNM stage IV had stronger expression than tumors of lower stages (P = 0.000). HAb18G PU was greater in tumors with lymph node metastasis than those without nodal metastasis (P = 0.045). The PU value of tumors with maximal diameter greater than 5 cm was higher than that of the smaller tumors (P = 0.000). It was also higher in male than in female patients (P = 0.046). There was no association between HAb18G protein expression and age of patients, history of smoking, tumor types and gross morphology (P > 0.05). The five-year survival rate in cases with low HAb18G protein expression was higher than that in cases with high expression (P = 0.006). Univariate analysis indicated that patients with high HAb18G protein expression carried a poor prognosis (P = 0.007). Multivariate analysis showed that expression of HAb18G protein was an independent prognostic factor in patients with NSCLC (P = 0.032, relative risk 3.962). CONCLUSIONS: HAb18G protein expression is associated with tumor progression and prognosis. It may represent a useful biomarker for prognostic evaluation.

[Progress of research on DNA vaccines against parasitosis].

One of the effective prevention and treatment strategies to parasitosis is to develop safe and effective vaccines. The DNA vaccine is a new kind of vaccine developed in last 10 years. In recent years, many advances in DNA vaccines against parasitosis have been made. This article reviews the advances in the mechanism, construction, optimization, adjuvants and delivery ways of DNA vaccines and the advances in the study of DNA vaccines against some parasitosis including malaria, schistosomiasis, cysticercosis and toxoplasmosis in recent years.

[Quantitative analysis of Tiam1 expression in lung cancer and its clinical significance].

OBJECTIVE: To investigate the relationship between the expression of T lymphoma invasion and metastasis inducing factor 1 (Tiam1) and the progression, metastasis, TNM stage, and histological types of lung carcinoma. METHODS: Immunohistochemistry was performed to detect the expression of Tiam1 in 116 lung carcinoma specimens. The expression intensity (measured in positive unit, PU) of Tiam1 in these tissues was assessed quantitatively using Imagepro Plus image analysis software. RESULTS: The PU of Tiam1 was significantly greater in primary lung carcinomas with lymph node metastases than in those without metastases (t=-2.089, P=0.039). Lung cancers of TNM stage II-IV had stronger expression than those of stage I (t=-2.272, P=0.025). The PU of Tiam1 differed significantly between different histological types of lung cancer, and squamouscell cell carcinoma had a lower PU than adenocarcinoma, large cell carcinoma and small cell carcinoma (P<0.05). The intensity of Tiam1 expression was not associated with the patients' gender, age, general types, smoking history, pneumoconiosis or differentiation of lung carcinoma. CONCLUSION: These results strongly suggest that Tiam1 is an invasion and metastasis inducing factor of lung carcinoma. The overexpression of Tiam1 is closely associated with lymph node metastases, TNM stage and histological types of lung carcinoma.

[Construction of full-length cDNA library of erythrocytic stage Plasmodium vivax].

Blood samples were collected from vivax malaria patients without antimalarial drug therapy. After filtration through Plasmodipur filter to remove white blood cells, Plasmodium vivax-infected RBCs were enriched by Percoll. Total P. vivax in red blood cells was isolated. A full-length cDNA library of erythrocytic stage P. vivax was constructed by the SMART cDNA library construction kit. The volume and recombinant rate of the library were evaluated. The inserted fragments were identified by PCR amplification. The titer of cDNA library was 1.14 x 10(6). The length of inserted fragment ranged from 900 to 2 500 bp, and the recombination efficiency accounted for 97.2%.

[Cloning and efficient prokaryotic expression of soluble stage-specific antigen cC1 from Cysticercus cellulosae].

OBJECTIVE: To clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli. METHODS: The cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting. RESULTS: The fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography. CONCLUSION: The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.