OBJECTIVE: This study aimed to elucidate the polarization of M1 and M2 macrophage from CAD patients as well as to investigate the expression of MerTK in these macrophage phenotypes.
METHODS: A total of 14 (n) CAD patients were recruited and subsequently grouped into "no apparent CAD", "non-obstructive CAD" and "obstructive CAD" according to the degree of stenosis. Thirty ml of venous blood was withdrawn to obtain monocyte from the patients. The M1 macrophage was generated by treating the monocyte with GMCSF, LPS and IFN-γ while MCSF, IL-4 and IL-13 were employed to differentiate monocyte into M2 macrophage. After 7 days of polarization, analysis of cell surface differentiation markers (CD86+/CD80+ for M1 and CD206+/CD200R+ for M2) and measurement of MerTK expression were performed using flow cytometry.
RESULTS: Both M1 and M2 macrophage expressed similar level of CD86, CD80 and CD206 in all groups of CAD patients. MerTK expression in no apparent CAD patients was significantly higher in M2 macrophage compared to M1 macrophage [12.58 ± 4.40 vs. 6.58 ± 1.37, p = 0.040].
CONCLUSION: Differential polarization of macrophage into M1 and M2 was highly dynamic and can be varied due to the microenvironment stimuli in atherosclerotic plaque. Besides, higher expression of MerTK in patients with the least coronary obstructive suggest its vital involvement in efferocytosis.
METHODS: Anti-retroviral therapy (ART) naïve HIV-seropositive individuals (progressors, n=16) and long-term non-progressors (LTNPs, n=10) were recruited for this study. We employed multi-color flow cytometry on frozen peripheral blood mononuclear cells (PBMCs) to determine iNKT subset frequencies, the levels of co-inhibitory 2B4 expression, and intracellular IFN-γ production. CD1d tetramer was used to characterize iNKT cells.
RESULTS: We report significantly lower level of 2B4 expression on bulk LTNPs iNKT cells as well as on their CD4 subsets compared to HIV progressors. Furthermore, the iNKT cells from LTNPs produced higher amount of IFN-γ than HIV progressors as detected by intracellular cytokine staining. Interestingly, the frequency of 2B4iNKT cells of progressors but not LTNPs significantly correlates with CD4 T cell count, HIV viral load and IFNγ production by iNKT cells.
CONCLUSION: Our results suggest that in addition to suppressed HIV replication, diminished 2B4 expression and associated co-inhibitory signaling, and substantial production of IFN-γ could contribute to preserved iNKT cell phenotype in LTNPs.
MATERIALS AND METHODS: MCF-7 cells were plated at a density of 15105 cells/well in 6-well plates. After 24h, cells were treated with a series of concentrations of rapamycin while only adding DMEM medium with PEG for the control regiment and grown at 37oC, 5% CO2 and 95% air for 72h. Trypan blue was used to determine the cell viability and proliferation. Untreated and rapamycin-treated MCF-7 cells were also examined for morphological changes with an inverted-phase contrast microscope. Alteration in cell morphology was ascertained, along with a stage in the cell cycle and proliferation. In addition, cytotoxicity testing was performed using normal mouse breast mammary pads.
RESULTS: Our results clearly showed that rapamycin exhibited inhibitory activity on MCF-7 cell lines. The IC50 value of rapamycin on the MCF-7 cells was determined as 0.4μg/ml (p<0.05). Direct observation by inverted microscopy demonstrated that the MCF-7 cells treated with rapamycin showed characteristic features of apoptosis including cell shrinkage, vascularization and autophagy. Cells underwent early apoptosis up to 24% after 72h. Analysis of the cell cycle showed an increase in the G0G1 phase cell population and a corresponding decrease in the S and G2M phase populations, from 81.5% to 91.3% and 17.3% to 7.9%, respectively.
CONCLUSIONS: This study demonstrated that rapamycin may potentially act as an anti-cancer agent via the inhibition of growth with some morphological changes of the MCF-7 cancer cells, arrest cell cycle progression at G0/G1 phase and induction of apoptosis in late stage of apoptosis. Further studies are needed to further characterize the mode of action of rapamycin as an anti-cancer agent.
METHODOLOGY/PRINCIPAL FINDINGS: Salivaricin 9 was found to be auto-regulated when an induction assay was applied and this finding was used to develop a successful salivaricin 9 production system in liquid medium. A combination of XAD-16 and cation exchange chromatography was used to purify the secondary metabolite which was shown to have a molecular weight of approximately 3000 Da by SDS-PAGE. MALDI-TOF MS analysis indicated the presence of salivaricin 9, a 2560 Da lantibiotic. Salivaricin 9 is a bactericidal molecule targeting the cytoplasmic membrane of sensitive cells. The membrane permeabilization assay showed that salivaricin 9 penetrated the cytoplasmic membrane and induced pore formation which resulted in cell death. The morphological changes of test bacterial strains incubated with salivaricin 9 were visualized using Scanning Electron Microscopy which confirmed a pore forming mechanism of inhibition. Salivaricin 9 retained biological stability when exposed to high temperature (90-100°C) and stayed bioactive at pH ranging 2 to 10. When treated with proteinase K or peptidase, salivaricin 9 lost all antimicrobial activity, while it remained active when treated with lyticase, catalase and certain detergents.
CONCLUSION: The mechanism of antimicrobial action of a newly discovered lantibiotic salivaricin 9 was elucidated in this study. Salivaricin 9 penetrated the cytoplasmic membrane of its targeted cells and induced pore formation. This project has given new insights on lantibiotic peptides produced by S. salivarius isolated from the oral cavities of Malaysian subjects.