Characterization of peripheral natural killer cells in primary Sjögren's syndrome: impaired NK cell activity and low NK cell number

The aim of this study was to compare the number of peripheral blood natural killer (NK) cells, NK cell activity, expression of NK cell activating receptors, and serum cytokine levels in patients with primary Sj?gren's syndrome (SS) vs normal controls. The authors found that NK cell number, NK cell killing activity, and the expression of activating receptors CD2 and NKG2D were significantly decreased, and the expression of NKp46, as well as the percentage of apoptotic NK cells, were significantly increased in primary SS patients compared with healthy controls. NK cell killing activity on a per-cell basis was similar in primary SS patients and healthy controls. Moreover, the levels of IL-18 and TNF-alpha, cytokines that have been shown to promote NK cell death, were significantly increased in sera from patients with primary SS compared with controls. These data suggest that reduced NK cell numbers, probably a result of apoptotic death, may contribute to impaired NK cell activity in patients with primary SS.     

Fine environmental particulate engenders alterations in human lung epithelial A549 cells

Particulate matter (PM), a component of urban air pollution that derives primarily from the combustion of fossil fuels, is responsible for a number of health effects in humans. Recent studies have demonstrated that the fine particles (PM(2.5)) present in high numbers in PM samples can be more harmful than larger particles, since they are more efficiently retained in the peripheral lung. In the present study, we have investigated the biological effects of PM(2.5) on human lung epithelial cell line A549. Morphological analysis performed by immunofluorescence and electron microscopy showed that fine particles interact with the cell surface, where they induce evident alterations and, subsequently, are internalized in the cytoplasm. Cytoskeletal components, in particular microfilaments and microtubules, cause modifications upon challenge with PM(2.5). Of interest, an early cell response to the fine particulate is an increase of reactive oxygen species content, which can account for the observed cytoskeletal changes and the production of proinflammatory cytokines in A549 cells. In particular, exposure to PM(2.5) promoted a dose- and time-dependent release of TNF-alpha and IL-6 in the cell medium.     

Application of Genetically Encoded Photoconvertible Protein SAASoti for the Study of Enzyme Activity in a Single Live Cell by Fluorescence Correlation Microscopy

Fluorescent Correlation Spectroscopy (FCS) allows us to determine interactions of labeled proteins or changes in the oligomeric state. The FCS method needs a low amount of fluorescent dye, near nanomolar concentrations. To control the amount of fluorescent dye, we used new photoconvertible FP SAASoti. This work is devoted to the proof of principle of using photoconvertible proteins to measure caspase enzymatic activity in a single live cell. The advantage of this approach is that partial photoconversion of the FP makes FCS measurements possible when studying enzymatic reactions. To investigate the process, in vivo we used HeLa cell line expressing the engineered FRET sensor, SAASoti-23-KFP. This FRET sensor has a cleavable (DEVD) sequence in the linker between two FPs for the detection of one of the key enzymes of apoptosis, caspase-3. Caspase-3 activity was detected by registering the increase in the fluorescent lifetimes of the sensor, whereas the diffusion coefficient of SAASoti decreased. This can be explained by an increase in the total cell viscosity during apoptosis. We can suppose that in the moment of detectible caspase-3 activity, cell structure already has crucial changes in viscosity.     

Identification of CD123+ myeloid dendritic cells as an early-stage immature subset with strong tumoristatic potential

CD123 has been identified as a specific surface marker for plasmacytoid dendritic cells (PDCs). However, CD123 has recently been shown to be expressed on freshly isolated or in vitro generated myeloid dendritic cells (MDCs). In this article, we investigated whether the expression of CD123 on monocyte-derived MDCs was related to their function, especially to tumor-inhibiting potential. MDCs were induced from cord blood CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days, and then CD123(+) cells were isolated by positive immunomagnetic cell selection. We observed that CD123(+) cells lost monocyte CD14 expression, acquired immature myeloid dendritic cell phenotype and morphology. They exerted more significant endocytosis and less antigen-presenting function than CD123(-)MDCs which are often referred to as typical MDCs. Meanwhile, CD123(+) MDCs exhibited more significant tumor-inhibiting activity toward hematological tumor cell lines of U937 and Jurkat even at a low effector:target ratio. CD123(+) MDCs expressed higher level of cytoplasmic TNF-alpha-related apoptosis-inducing ligand (TRAIL), but no detectable surface TRAIL and very little soluble TRAIL. Pretreatment with recombinant human TRAIL receptor 2:Fc fusion protein significantly reduced the tumor-inhibiting effect of CD123(+) MDCs, but not of CD123(-) MDCs. Overall, our data demonstrated that CD123(+) MDCs were an early-stage immature DC subset, with a significant tumor-inhibiting activity partially via involvement of enhanced cytoplasmic TRAIL.     

Thymoquinone-induced suicidal erythrocyte death

Thymoquinone is a nutrient with anticarcinogenic activity by stimulating suicidal death of tumor cells. Similar to nucleated cells, erythrocytes may experience suicidal death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and by cell shrinkage. Triggers and signaling of eryptosis include increase in cytosolic Ca²⁺activity, ceramide formation, and stimulation of protein kinase C. The present experiments explored, whether thymoquinone influences eryptosis. According to annexin V-binding, thymoquinone (?3 μM) increased the percentage of phosphatidylserine-exposing erythrocytes. According to forward scatter in FACS analysis, thymoquinone (?10 μM) led to cell shrinkage. The effect of thymoquinone was not paralleled by appreciable ceramide formation (immunofluorescent antibody) or hemolysis (hemoglobin release). It was not significantly blunted in the nominal absence of extracellular Ca²⁺ but was inhibited by staurosporine (500 nM). In conclusion, thymoquinone triggers suicidal erythrocyte death, an effect paralleling the apoptotic effect on nucleated cells.