Evaluating And Testing Of A Potential Dna Vaccine Against Vibrio Cholerae

Although it has been more than 100 years since the first attempt to produce a cholera vaccine was made, an effective cholera vaccine has yet to be developed. In this study, the level of protection produced by a potential DNA vaccine (PVax/ctxB) was tested against the ctxB toxin of Vibrio cholerae on Balb/c mice. First, the intramuscular vaccination method was validated using pCMV plasmid that encodes HbsAg, which was detected 5 days after the injection into the tibial muscle. Next, 4 groups of mice were intramuscularly injected with either the pVax/ctxB vaccine construct or pVaxl as the negative control. The first and second groups received 2 injections spaced 3 weeks apart, while the other two groups were given 3 injections spaced 3 weeks apart. This was then followed by challenging the mice with 105 or 107 cfu/ml/mouse from clinical isolates of V. cholerae after 3 weeks of the last injection. Antibody levels for both IgG and serum IgA were monitored using ELISA, and showed high production of IgG after the first booster injection with no significant change of IgA levels

Application of Proteomics Approaches in the Identification of New Markers and Therapeutic Targets for Breast Cancer

Breast cancer is the most common cancer in most parts of the world and is a leading cause of death among women. Even though the incidence of the disease is increasing each year, early detection and improved treatments have increased the survival rates. Currently, only a few markers are used for either early diagnosis, treatment response or for survival of breast cancer. In this study, two-dimensional gel electrophoresis (2DGE) was used in the quest for new potential biomarkers for the disease. Breast cancer cell lines and normal breast cell line were used to optimize the conditions to produce the respective proteome maps. Fresh frozen samples representing tumor and adjacent normal tissues were then collected from patients who underwent breast surgery at HUKM, HKL and Hospital Putrajaya. A total of sixty samples representing tumor and adjacent normal tissues were collected from June 2005 to December 2006 and were screened using 2DGE. Subsequently, 24 samples representing the different stages of infiltrating ductal carcinoma were used for further analysis using 17 cm IPG strips with 2 pH ranges 3-10 and 4-7, and the gels were analyzed using PDQuest 7.3 software. Several protein spots of interest were then excised and analyzed using MALDI-TOF spectrometer. Tumor rejection antigen (gp96), heat shock protein 90α, nucleosome assembly protein 1-like 1 and opioid-binding cell adhesion molecule precursor were identified and found to be upregulated in breast cancer cell lines when compared to the normal breast cell line. Calreticulin, tumor rejection antigen (gp96), heat shock protein 60 and cytokine induced apoptosis inhibitor 1 were found to be up-regulated by 2 folds or more in tumor tissues when compared to the adjacent normal tissues. On the other hand, actin γ 2 and protein tyrosine phosphatase were found to be down-regulated in tumor tissues. Since Calreticulin, a calcium binding protein was more intense at different stages of the disease with its expression confirmed by Western blotting, it was chosen for further investigations. Quantitative RT-PCR with GAPDH as a house keeping gene was used to monitor the level of gene expression and to correlate the mRNA levels with calreticulin levels. In the samples that represent later stages of the disease, mRNA levels were found to be highly expressed in tumor tissues when compared to the adjacent normal tissues where in average more than 18 folds increase was observed. The mRNA level was also found to be decreased in stage IV sample where 12 folds increase was observed, indicating a possible role of calreticulin in the progression of the disease. In conclusion, the proteomics approaches were utilized in this study and was found to be valuable in the search for potential new biomarkers for breast cancer

Calreticulin mediates an invasive breast cancer phenotype through the transcriptional dysregulation of p53 and MAPK pathways

Background The introduction of effective novel biomarkers of invasion and metastasis is integral for the advancement of breast cancer management. The present study focused on the identification and evaluation of calreticulin (CRT) as a potential biomarker for breast cancer invasion. Methods Two-dimensional gel protein electrophoresis and MALDI-TOF were utilized in the analysis of fresh-frozen invasive intra-ductal carcinoma specimens. Calreticulin-associated expression was analyzed using immunohistochemistry of FFPE non-malignant/malignant breast specimens. A CRT-knockdown model of MCF7 cell line was developed using siRNA and the CRT genotype/phenotype correlations based on migration and trans-well invasion assays were determined. Finally, microarray-based global gene expression profiling was conducted to elucidate the possible calreticulin pro-invasive regulatory pathways. Results Two-dimensional gel protein electrophoresis and MALDI-TOF analysis showed upregulation of calreticulin expression in tumor tissues as compared to the normal adjacent tissues. Meta-analysis of the immunohistochemical results confirmed significantly higher expression of calreticulin (p < 0.05) in the stromal compartments of malignant tissues as compared to non-malignant tissues. Migration and transwell invasion assays showed significant loss in the migratory and invasive potential of CRT-knockdown cells (p < 0.05). Global gene expression profiling successfully identified various putative gene networks such as p53 and MAPK pathways that are involved in calreticulin breast cancer signaling. Conclusion Besides confirming calreticulin overexpression in invasive breast cancer tissues, this study reveals a calreticulin-dependent pro-invasive potential and suggests possible contributing pathways. Defining the mechanistic role of invasion and characterizing the possible calreticulin-dependent molecular targets will be the focus of future work

Changes in Lactate Production, Lactate Dehydrogenase Genes Expression and DNA Methylation in Response to Tamoxifen Resistance Development in MCF-7 Cell Line

Lactate dehydrogenase (LDH) is a key enzyme in the last step of glycolysis, playing a role in the pyruvate-to-lactate reaction. It is associated with the prognosis and metastasis of many cancers, including breast cancer. In this study, we investigated the changes in LDH gene expression and lactate concentrations in the culture media during tamoxifen resistance development in the MCF-7 cell line, and examined LDHB promoter methylation levels. An upregulation of 2.9 times of LDHB gene expression was observed around the IC50 concentration of tamoxifen in treated cells, while fluctuation in LDHA gene expression levels was found. Furthermore, morphological changes in the cell shape accompanied the changes in gene expression. Bisulfate treatment followed by sequencing of the LDHB promoter was performed to track any change in methylation levels; hypomethylation of CpG areas was found, suggesting that gene expression upregulation could be due to methylation level changes. Changes in LDHA and LDHB gene expression were correlated with the increase in lactate concentration in the culture media of treated MCF-7 cells