METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.
RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.
CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.
Method: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins.
Result: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed.
Conclusion: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.
Methods: The study was conducted in the Department of Medical Microbiology and Parasitology, University Putra Malaysia in 2014-2017. Saline extract protein from the infective larvae of S. ratti was used to immunize BALB/c mice and subsequent fusion of the B-cells with myeloma cells (SP2/0) using 50% PEG. The hybridomas were cultured in HAT medium and cloned by limiting dilutions. Positive hybrids were screened by indirect ELISA. The ascites fluid from the antibody-secreting hybridoma was purified and the MAb was characterized by western-blots and evaluated in sandwich ELISA for reactivity against the homologous and heterologous antigens.
Results: An IgG1 that recognizes a 30 and 34 kDa protein bands was obtained. The MAb was recognized by all S. ratti-related antigens and cross-reacted with only Toxocara canis antigens in both assays. The minimum antigen detection limit was found to be 5 ng/ml. All antibody-positive rat and dog sera evaluated have shown antigen-positive reactions in Sandwich-ELISA.
Conclusion: The MAb produced, was able to detect antigens in strongyloidiasis and toxocariasis in animal models and may also be useful for the serological detection of active strongyloidiasis and visceral toxocariasis in human sera.