Materials and Methods: SF1 was produced by optimized methodology for bioassay-guided fractionation. Fourier transform infrared (FTIR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) were carried out to characterize the SF1. SF1 was screened for cytotoxicity activity toward HeLa, SiHa, and normal cells (NIH) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The anticancer mechanism of SF1 was evaluated toward SiHa cells, which showed highest cytotoxicity toward SF1 treatment. The mechanism includes cell cycle progression and protein expression, which was detected using specific antibody-conjugated fluorescent dye, p53-FITC, by flow cytometry.
Results: Major constituents of SF1 were alkaloids with amines as functional group. SF1 showed highest cytotoxic activity against SiHa (half-maximal inhibitory concentration [IC50] < 10 µg/mL) compared to HeLa cells. Cytoselectivity of SF1 was observed with no IC50 detected on normal NIH cells. On flow cytometry analysis, SF1 was able to induce apoptosis on SiHa cells by arresting cell cycle at G1/S and upregulation of p53 protein.
Conclusion: SF1 showed anticancer activity by inducing apoptosis through arrested G1/S cell cycle checkpoint-mediated mitochondrial pathway.
METHODS: EMA detection was performed by flow cytometry in monocytes and monoblasts. EMA expression was compared with other known markers of monocytic-macrophage lineage (CD11c, CD14 and intracellular CD68). Samples of purified monocytes were obtained from 20 healthy volunteers. Twenty-two cases of monocytic AML (M4 and M5) were studied and controls were selected from 20 cases of acute lymphoblastic leukaemia (ALL) and 18 cases of non-monocytic AML (M0, M1, M2, M3, and M7).
RESULTS: EMA was shown to be expressed strongly on the surface of all purified monocytes. EMA expression was observed on blast cells in 18/22 (81.8%) cases of AML M4 and M5, but not in that of non-monocytic AML or ALL. In this study EMA monoclonal antibody has demonstrated a strong association (P<0.001) with all the other known markers of monocytic-macrophage lineage in acute leukaemia subtypes. EMA had also shown 100% specificity and 81.8% sensitivity in the diagnosis of AML M4 and M5.
CONCLUSIONS: The monoclonal antibody EMA (clone E29) is a useful marker in the classification of acute myeloid leukaemia and can be used as a supplementary analysis for the diagnosis of acute leukemia with monocytic involvement.
METHODS: We analysed the frequency, phenotype and functionality of peripheral blood MAIT cells, as well as γδ T cells, invariant natural killer T (iNKT) cells and natural killer (NK) cells with flow cytometry in a cross-sectional paediatric cohort (aged 2-15) consisting of 51 children with newly diagnosed type 1 diabetes, 27 autoantibody-positive (AAb+) at-risk children, and 113 healthy control children of similar age and HLA class II background. The frequency of MAIT cells was also assessed in a separate cross-sectional adult cohort (aged 19-39) of 33 adults with established type 1 diabetes and 37 healthy individuals of similar age.
RESULTS: Children with newly diagnosed type 1 diabetes displayed a proportional increase of CD8-CD27- MAIT cells compared with healthy control children (median 4.6% vs 3.1% of MAIT cells, respectively, p = 0.004), which was associated with reduced expression of C-C chemokine receptor (CCR)5 (median 90.0% vs 94.3% of MAIT cells, p = 0.02) and β7 integrin (median 73.5% vs 81.7% of MAIT cells, p = 0.004), as well as decreased production of IFN-γ (median 57.1% vs 69.3% of MAIT cells, p = 0.04) by the MAIT cells. The frequency of MAIT cells was also decreased in AAb+ children who later progressed to type 1 diabetes compared with healthy control children (median 0.44% vs 0.96% of CD3+ T cells, p = 0.04), as well as in adult patients with a short duration of type 1 diabetes (less than 6 years after diagnosis) compared with control individuals (median 0.87% vs 2.19% of CD3+ T cells, p = 0.007). No alterations in γδ T cell, iNKT cell or NK cell frequencies were observed in children with type 1 diabetes or in AAb+ children, with the exception of an increased frequency of IL-17A+ γδ T cells in children with newly diagnosed diabetes compared with healthy control children (median 1.58% vs 1.09% of γδ T cells, p = 0.002).
CONCLUSIONS/INTERPRETATION: Changes in the frequency and phenotype of circulating MAIT cells were detectable before, at the onset and after diagnosis of type 1 diabetes in cross-sectional cohorts. Our results suggest a possible temporal association between peripheral blood MAIT cell alterations and the clinical onset of type 1 diabetes. Graphical abstract.
METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.
RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses.
CONCLUSIONS: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.
METHODS: In this study, transfection of VP3 and silencing of CD147 genes was achieved through the treatment of tumors with pVIVO1-GFP/VP3 (VP3), psiRNA-CD147/2 (shCD147/2), and their combination of CT26 colon cancer cell-induced in mice. The effectiveness of tumor-treatment was ascertained by electrophoresis, TUNEL assay, and flow cytometry analysis. While histopathological and biochemical analysis were used as toxic side effect identification.
RESULTS: The tumor growth delay index (TGDI) after treatment with VP3, shCD147/2, and their combination treatments increased by 1.3-, 1.2-, 2.0- and 2.3-fold respectively, over untreated control. The VP3-shCD147/2 combination treatment was more efficacious then either VP3 or shCD147/2 alone in the retardation of mouse CT26 colorectal cell tumor allograft.
CONCLUSION: The antitumor effect of the combination treatment is the result of synergistic effects of VP3 and shCD147/2 on the tumor cells resulting in apoptosis. Thus, the study shows that combination of VP3 and shCD147/2 treatment can be developed into a potential approach for anticolorectal cancer treatment regimen.