Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting ...increased production of reative oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS‐dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1‐fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1‐fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl‐2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl‐1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine‐cathepsins but not caspases, and the differential effects of inhibitors of cysteine‐cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe‐induced apoptosis in neutrophils, ROS‐dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.
Early metastasis is responsible for frequent relapse and high mortality of hepatocellular carcinoma (HCC), but its underlying mechanisms remain unclear. Epithelial–mesenchymal transition (EMT) has ...been considered a key event in metastasis. Based on histological examination of serial HCC sections and three‐dimensional reconstruction, we found a novel and prevalent vascular pattern, vessels that encapsulated tumor clusters (VETC) and formed cobweb‐like networks. The presence of VETC (VETC+) predicted higher metastasis and recurrence rates of HCC. Using clinical samples and mouse xenograft models, we further showed that VETC was composed of functional vessels with blood perfusion and induced by tumor cells at the early stage of HCC. Subsequent investigations revealed that HCC cell–derived angiopoietin‐2 was a prerequisite for VETC formation and that the VETC pattern was a critical factor promoting HCC metastasis as knockdown of angiopoietin‐2 abolished this vascular pattern and consequently attenuated in vivo tumor metastasis. Interestingly, abrogation of EMT by knockdown of Snail or Slug significantly diminished in vivo metastasis of VETC– xenografts but did not affect that of VETC+ ones, although silencing of Snail or Slug substantially reduced the in vitro migration of both VETC+ and VETC– HCC cells. In contrast to human VETC– cases, EMT signatures were rarely observed in VETC+ cases with metastatic potential. Further analysis revealed that VETC provided an efficient metastasis mode by facilitating the release of whole tumor clusters into the bloodstream. Conclusion: Our findings identify a novel metastasis mechanism that relies on vascular pattern but is independent of EMT, which may provide new targets for antimetastasis therapy and offer a basis for selecting patients who may benefit from certain molecularly targeted drugs. (Hepatology 2015;62:452–465
Overcoming local immunosuppression is critical for immunotherapy to produce robust anti-tumor responses. Myeloid-derived suppressor cells (MDSCs) are key regulators of immunosuppressive networks and ...promote tumor progression. However, it remains unclear whether and how tumor-infiltrating MDSCs are shaped in response to anti-PD-1 treatment and what their impact on therapeutic efficacy is in colorectal cancer (CRC). In this study, the levels of infiltrating MDSCs were significantly higher in the non-responding organoids and were selectively reduced in the responding group, with MDSCs showing increased apoptosis and attenuated functional activity after anti-PD-1 treatment. A negative correlation between T-cell activation and MDSC function was also observed in fresh human CRC tissues. Mechanistic studies revealed that autocrine IFN-α/β upregulated TRAIL expression on activated T cells to elicit MDSC apoptosis via the TRAIL-DR5 interaction and acted synergistically with TNF-α to inhibit MDSC function of suppressing the T-cell response through the JNK-NMDAR-ARG-1 pathway. Moreover, blockade of IFN-α/β and TNF-α abolished the therapeutic efficacy of anti-PD-1 treatment by preserving the frequency and suppressive activity of infiltrating MDSCs in a CRC mouse model. This result suggested that reprogramming MDSCs by IFN-α/β and TNF-α from activated T cells was necessary for successful anti-PD-1 treatment and might serve as a novel strategy to improve the response and efficacy of anticancer therapy.
B cells are prominent components of human solid tumours, but activation status and functions of these cells in human cancers remain elusive. Here we establish that over 50% B cells in hepatocellular ...carcinoma (HCC) exhibit an FcγRII
activated phenotype, and high infiltration of these cells positively correlates with cancer progression. Environmental semimature dendritic cells, but not macrophages, can operate in a CD95L-dependent pathway to generate FcγRII
activated B cells. Early activation of monocytes in cancer environments is critical for the generation of semimature dendritic cells and subsequent FcγRII
activated B cells. More importantly, the activated FcγRII
B cells from HCC tumours, but not the resting FcγRII
B cells, without external stimulation suppress autologous tumour-specific cytotoxic T-cell immunity via IL-10 signals. Collectively, generation of FcγRII
activated B cells may represent a mechanism by which the immune activation is linked to immune tolerance in the tumour milieu.
Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the ...early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (P
) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV P
contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of P
to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action.
Objective
To compare the accuracy, correlation and agreement between the bispectral index (BIS) and BISpro during propofol anaesthesia.
Methods
The BIS, BISpro, heart rate, target-concentration of ...propofol and Observer’s Assessment of Alertness and Sedation (OAA/S) score were recorded every 30 s in female patients scheduled for hysteroscopic surgery. Propofol anaesthesia was induced by an initial target-controlled concentration (1.0 μg/ml) followed by a stepwise increase (0.5 μg/ml) until the patient was unresponsive. Spearman’s correlation coefficient and prediction probability were calculated for the association between sedation levels and the above parameters. The ability of investigated parameters to distinguish between OAA/S scores was analysed. Bland–Altman analysis was used to compare the agreement between BIS and BISpro. The BIS and BISpro cut-off values for lost response were also determined.
Results
Out of 30 patients in total, a high correlation was found between BIS and BISpro, and both correlated well with OAA/S score. Only BIS was able to distinguish all investigated OAA/S states accurately, but the ability to predict OAA/S score 5 to loss of response was comparable between BIS and BISpro. The calculated cut-off values were 68 for BIS and 70 for BISpro.
Conclusion
BISpro and BIS are reliable monitors of general anaesthesia during sedation.
Trial registration number: Chinese Clinical Trial Registry (URL: www.chictr.org.cn): ChiCTR1900024037 (retrospectively registered).
Numerous plant viruses that cause significant agricultural problems are persistently transmitted by insect vectors. We wanted to see if apoptosis was involved in viral infection process in the ...vector. We found that a plant reovirus (rice gall dwarf virus, RGDV) induced typical apoptotic response during viral replication in the leafhopper vector and cultured vector cells, as demonstrated by mitochondrial degeneration and membrane potential decrease. Fibrillar structures formed by nonstructural protein Pns11 of RGDV targeted the outer membrane of mitochondria, likely by interaction with an apoptosis-related mitochondrial protein in virus-infected leafhopper cells or nonvector insect cells. Such association of virus-induced fibrillar structures with mitochondria clearly led to mitochondrial degeneration and membrane potential decrease, suggesting that RGDV Pns11 was the inducer of apoptotic response in insect vectors. A caspase inhibitor treatment and knockdown of caspase gene expression using RNA interference each reduced apoptosis and viral accumulation, while the knockdown of gene expression for the inhibitor of apoptosis protein improved apoptosis and viral accumulation. Thus, RGDV exploited caspase-dependent apoptotic response to promote viral infection in insect vectors. For the first time, we directly confirmed that a nonstructural protein encoded by a persistent plant virus can induce the typical apoptotic response to benefit viral transmission by insect vectors.
Rice stripe virus (RSV), a tenuivirus, is transmitted by small brown planthopper (SBPH) in a persistent-propagative manner. In this study, sequential infection of RSV in the internal organs of SBPH ...after ingestion of virus indicated that RSV initially infected the midgut epithelium, and then progressed to the visceral muscle tissues, through which RSV spread to the entire alimentary canal. Finally, RSV spread into the salivary glands and reproductive system. During viral infection, the nonstructural protein NS4 of RSV formed cytoplasmic inclusions in various tissues of viruliferous SBPH. We demonstrated that the ribonucleoprotein particles of RSV were closely associated with NS4-specific inclusions in the body of viruliferous SBPH through a direct interaction between NS4 and nucleoprotein of RSV. Moreover, the knockdown of NS4 expression due to RNA interference induced by dsRNA from NS4 gene significantly prevented the spread of RSV in the bodies of SBPHs without a significant effect on viral replication in continuous cell culture derived from SBPH. All these results suggest that the nonstructural protein NS4 of RSV plays a critical role in viral spread by the vector insects.
RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that ...regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in
. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.
Objective
Non-steroidal anti-inflammatory drugs have been shown to effectively decrease postoperative pain and reduce opioid requirements. Flurbiprofen axetil is an injectable non-selective ...cyclooxygenase inhibitor that has a high affinity for inflammatory tissues to achieve targeted drug therapy and prolonged duration of action. This meta-analysis examined the use of preoperative flurbiprofen axetil and its impact on postoperative analgesia.
Methods
An electronic literature search of the Library of PubMed, Cochrane CENTRAL, and EMBASE databases was conducted in Feb 2016. Searches were limited to randomized controlled trials. The primary outcome was pain scores. The secondary outcomes included cumulative postoperative opioid consumption and opioid-related adverse effects.
Results
A total of nine RCT studies involving 457 patients were included in this study. Compared to patients without perioperative flurbiprofen axetil, patients treated with preoperative flurbiprofen axetil had lower pain scores at 2 h (SMD −1.00; 95% CI −1.57 to −0.43,
P
= 0.0006), 6 h (SMD −1.22; 95% CI −2.01 to −0.43;
P
= 0.002), 12 h (SMD −1.19; 95% CI −2.10 to −0.28;
P
= 0.01), and 24 h (SMD −0.79; 95% CI −1.31 to −0.27;
P
= 0.003) following surgery. Preoperative flurbiprofen axetil had no significant effect on postoperative opioid consumption (SMD −13.11; 95% CI −34.56 to 8.33;
P
= 0.23). There was no significant difference between the groups with regard to adverse effects. Compared to patients with postoperative flurbiprofen axetil, however, preoperative flurbiprofen axetil resulted in decreased pain score only at 2 h after operation.
Conclusions
Preoperative use of flurbiprofen axetil will result in significantly lower postoperative pain scores, but no difference in nausea, vomiting, and opioid consumption compared to those who did not receive flurbiprofen axetil. However, more homogeneous and well-designed clinical studies are necessary to determine whether preoperative flurbiprofen axetil administration has more efficacy than that given at the end of surgery.
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