Genetic Polymorphisms in NLRP3 Inflammasome-Associated Genes in Patients with B-Cell Non-Hodgkin's Lymphoma.

PURPOSE: The role of NLRP3 inflammasome in the progression of many diseases has been increasingly recognized. However, the function of this molecular assembly in the development and progression of B-cell non-Hodgkin's lymphoma remains unclear. PATIENTS AND METHODS: In this study, we investigated the polymorphisms in the NLRP3 inflammasome associated genes in 281 patients with B-cell non-Hodgkin's lymphoma and 385 age- and gender-matched healthy controls. RESULTS: We found that IL-18 (rs1946518) and NFκB-94 ins/del (rs28362491) contributed to susceptibility to B-cell non-Hodgkin's lymphoma. Specifically, the allele "G" in IL-18 (rs1946518) and allele "ins" in NFκB-94 ins/del (rs28362491) were significantly associated with the risk of disease. The AA genotype of CARD8 (rs2043211) and the higher level of serum lactate dehydrogenase (LDH) led to statistically poorer B-cell non-Hodgkin's lymphoma survival. Less frequent genotype TT of CARD8 (rs2043211) was observed in patients with higher LDH level, clinical stages III-IV of disease, and IPI 3-5, although the relationship did not reach statistical significance. However, IPI is an independent prognostic factor for B-cell non-Hodgkin's lymphoma. CONCLUSION: IL-18 (rs1946518) and NFκB-94 ins/del (rs28362491) gene polymorphisms appear to be the factors influencing the risk of B-cell non-Hodgkin's lymphoma. CARD8 (rs2043211) polymorphisms are important factors for the survival of patients with this disease.

RIP1K Contributes to Neuronal and Astrocytic Cell Death in Ischemic Stroke via Activating Autophagic-lysosomal Pathway.

Although the receptor-interacting protein 1 kinase (RIP1K)-regulated necroptosis can be evoked by cerebral ischemia, the effects of RIP1K in mediating neuronal and astrocytic cell death and the underlying mechanisms remain poorly understood. This study evaluates the contribution of RIP1K to ischemic stroke-induced neuronal and astrocytic cell death, and the activation of autophagic-lysosomal pathway. Using an in vitro oxygen and glucose deprivation (OGD) in primary cultured neurons or astrocytes and a permanent middle cerebral artery occlusion (pMCAO) model in rats or mice, we observed the role of RIP1K in the ischemic neuronal and astrocytic cell death and the underlying mechanisms by pharmacological or genetic inhibition of RIP1K. pMCAO or OGD condition led to an increase in RIP1K, RIP3K and RIP1K-RIP3K complex. RIP1K knockdown or necrostatin-1 (Nec-1, a specific inhibitor of RIP1K) treatment reduced infarct volume, improved neurological deficits, increased microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) levels, and attenuated neuronal or astrocytic necrotic cell death in the ischemic cortex. RIP1K knockdown decreased RIP1K-RIP3K complex formation, light chain 3 II (LC3II) and active cathepsin B levels and lysosomal membrane permeability (LMP). Furthermore, a combination of Nec-1 and an inhibitor of autophagy or cathepsin B produced an enhancement of protective effect on neuronal or astrocytic cell death. RIP1K-mediated necroptosis may play important roles in ischemia-induced neuronal and astrocytic cell death through the activation of autophagic-lysosomal pathway.

Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes.

Our previous study and others have demonstrated that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death. However, the mechanisms of ischemia-induced autophagy remain largely unknown. In this study, we established a rat's model of permanent middle cerebral artery occlusion (pMCAO) and an in vitro oxygen and glucose deprivation (OGD) model. Autophagy was inhibited by either pharmacological treatment with 3-methyladenine (3-MA) and wortmannin (Wort) or genetic treatment with knockdown of Atg5 in primary cultured astrocytes and knockout of Atg5 in mouse embryonic fibroblast (MEF) cells, respectively. We found that pharmacological or genetic inhibition of autophagy reversed pMCAO or OGD-induced increase in LC3-II, active cathepsin B and L, tBid, active caspase-3 and cytoplastic cytochrome c (Cyt-c), and suppressed the injury-induced reduction in mitochondrial Cyt-c in ischemic cortex, in injured astrocytes and MEF cells. Immunofluorescence analysis showed that 3-MA or Wort treatment reversed OGD-induced release of cathepsin B and L from the lysosome to the cytoplasm and activation of caspase-3 in the astrocytes. Furthermore, treatment of 3-MA or Wort decreased OGD-induced increase in lysosomal membrane permeability and enhanced OGD-induced upregulation of lysosomal heat shock protein 70.1B (Hsp70.1B) in astrocytes. Inhibition of autophagy by 3-MA or Wort reduced infarction volume in rats and protected OGD-induced astrocytic cell injury. A non-selective caspase inhibitor z-VAD-fmk or a specific caspase-3 inhibitor Q-DEVD-OPh also rescued OGD-induced astrocytic cell injury. In conclusion, our presenting data suggest that inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membranes, possibly due to upregulation of the lysosomal Hsp70.1B in ischemic astrocytes.

Induction of autophagy and apoptosis by miR-148a through the sonic hedgehog signaling pathway in hepatic stellate cells.

Autophagy is an evolutionarily conserved biological process that is activated in response to stress. Increasing evidence indicate that dysregulated miRNAs significantly contribute to autophagy and are thus implicated in various pathological conditions, including hepatic fibrosis. MiR-148a, a member of the miR-148/152 family, has been found to be downregulated in hepatic fibrosis and human hepatocellular carcinoma. However, the role of miR-148a in the development of hepatic fibrosis remains largely unknown. In this study, we describe the epigenetic regulation of miR-148a and its impact on autophagy in hepatic stellate cells (HSCs), exploring new targets of miR-148a. We found that miR-148a expression was significantly increased under starvation-induced conditions in LX-2 and T-6 cells. In addition, dual-luciferase reporter assays showed that miR-148a suppressed target gene expression by directly interacting with the 3'-untranslated regions (3'-UTRs) of growth arrest-specific gene 1 (Gas1) transcripts. Intriguingly, Gas1, which encodes a Hedgehog surface binding receptor and facilitates the Hedgehog (Hh) signaling pathway, inhibited autophagosome synthesis. Furthermore, we demonstrated a novel function for miR-148a as a potent inducer of autophagy in HSCs. Overexpressing of miR-148a increased autophagic activity, which inhibited proliferation and promoted apoptosis in HSCs. In conclusion, these data support a novel role for miR-148a as a key regulator of autophagy through the Hh signaling pathway, making miR-148a a potential candidate for the development of novel therapeutic strategies.

Transforming growth factor­ß1 reduces apoptosis via autophagy activation in hepatic stellate cells.

Autophagy is a metabolic process that is important in fibrogenesis, in which cellular components are degraded by lysosomal machinery. Transforming growth factor ß1 (TGF­ß1) is a potent fibrogenic cytokine involved in liver fibrosis; however, it remains elusive whether autophagy is regulated by TGF­ß1 in this process. In the present study, the function of TGF­ß1­mediated autophagy in the proliferation and apoptosis of hepatic stellate cells (HSCs) was investigated. A rat HSC cell line (HSC­T6) was incubated with or without TGF­ß1 followed by bafilomycin A1, and microtubule-associated proteins 1A/1B light chain 3 (LC3) small interfering (si)RNA was used to inhibit autophagy in order to assess the association between TGF­ß1 and autophagy. HSC­T6 cell transient transfection was accomplished with a pLVX­AcGFP­N1­rLC3B­encoding plasmid. An MTS assay and flow cytometry were utilized to detect proliferation and apoptosis of HSC­T6 cells. Quantitative polymerase chain reaction, immunofluorescence and western blot analysis were used to detect the presence of activation markers. Proliferation was increased and apoptosis was reduced in HSC­T6 cells treated with TGF­ß1 compared with cells subjected to serum deprivation. However, when HSC­T6 cells were treated with bafilomycin A1 and LC3 siRNA, increased apoptosis and reduced proliferation were observed. In addition, protein and mRNA expression levels of the autophagy marker LC3 were significantly increased. GFP­LC3 punctate markings were more prolific following TGF­ß1 treatment of HSC­T6 cells, indicating that TGF­ß1 may rescue HSC­T6 cells from serum deprivation and reduce apoptosis via autophagy induction. The present study elucidated the possible functions of TGF­ß1­mediated autophagy in the pathological process of liver fibrosis.

[Population pharmacokinetics of vancomycin and prediction of pharmacodynamics in the Chinese people].

Population pharmacokinetics of vancomycin (VAN) in the Chinese patients was described by using nonlinear mixed-effects modeling (NONMEM). 619 VAN serum concentrations data from 260 patients including 177 males and 83 females were collected separately from two centers. A one-compartment model was used to describe this sparse data. No significant difference was observed between two center datasets by introducing SID covariate. The final model was as CL= (θ (base0+ θ(max) x(1 -e(-θ(Age)(Age/72) and V = θ x θ (Age)(Age/72). The creatinine clearance (CL(Cr)) and Age were identified as the most significant covariate in the final model. Typical values of clearance (CL) and volume of distribution (V) in the final model were 2.91 L x h(-1) and 54.76 L, respectively. Internal model validation by Bootstrap and NPDE were performed to evaluate the robustness and prediction of the final model. The median and 95% confidence intervals for the final model parameters were based on 1000 Bootstraps. External model evaluation was conducted using an independent dataset that consisted of 34 patients to predict model performance. Pharmacodynamic assessment for VAN by AUC (0-24 h) to MIC ratios of over 400 was considered to be the best to predict treatment outcomes for patients. AUC (0-24 h) was calculated by clearance based on the above population model. The results indicate that the conventional dosing regimen probably being suboptimal concentrations in aged patients. The approach via population pharmacokinetic of VAN combined with the relationship of MIC, Age, CL(Cr) and AUC(0-24 h)/MIC can predict the rational dose for attaining efficacy.

[Influence of educational status, burn area and coping behaviors on the complication of psychological disorders in severely burned patients].

OBJECTIVE: To discuss how the educational status, burn area and coping behaviors influence the psychological disorders in severely burned patients. METHODS: Sixty-four severely burned patients hospitalized in Guangzhou Red Cross Hospital, Guangdong Provincial Work Injury Rehabilitation Center, and Guangdong General Hospital were enrolled with cluster random sampling method. Data of their demography and situation of burns were collected. Then their coping behavior, psychological disorders including anxiety, depression and post-traumatic stress disorder (PTSD) plus its core symptoms of flashback, avoidance, and hypervigilance were assessed by medical coping modes questionnaire, self-rating anxiety scale (SAS), self-rating depression scale (SDS), PTSD checklist-civilian version (PCL-C) respectively. Correlation was analyzed between demography, burn area, coping behavior and psychological disorders. The predictive powers of educational status, burn area and coping behaviors on the psychological disorders were analyzed. The qualitative variables were assigned values. Data were processed with t test, Spearman rank correlation analysis, and multiple linear regression analysis. RESULTS: (1) The patients scored (19.0 ± 3.4) points in confrontation coping behavior, which showed no statistically significant difference from the domestic norm score (19.5 ± 3.8) points (t = -1.13, P > 0.05). The patients scored (16.6 ± 2.4) and (11.0 ± 2.2) points in avoidance and resignation coping behaviors, which were significantly higher than the domestic norm score (14.4 ± 3.0), (8.8 ± 3.2) points (with t values respectively 7.06 and 7.76, P values both below 0.01). The patients' standard score of SAS, SDS, PCL-C were (50 ± 11), (54 ± 11), and (38 ± 12) points. Respectively 89.1% (57/64), 60.9% (39/64), 46.9% (30/64) of the patients showed anxiety, depression, and PTSD symptoms. (2) Four independent variables: age, gender, marital status, and time after burns, were correlated with the psychological disorders, but the correlativity was not statistically significant (with rs values from -0.089 to 0.245, P values all above 0.05). Educational status was significantly negatively correlated with anxiety, depression, PTSD and its core symptoms of flashback, avoidance (with rs values from -0.361 to -0.253, P values all below 0.05). Educational status was negatively correlated with hypervigilance, but the correlativity was not statistically significant (rs = -0.187, P > 0.05). Burn area was significantly positively correlated with the psychological disorders (with rs values from 0.306 to 0.478, P values all below 0.05). Confrontation coping behavior was positively correlated with the psychological disorders, but the correlativity was not statistically significant (with rs values from 0.121 to 0.550, P values all above 0.05). Avoidance coping behavior was correlated with the psychological disorders, but the correlativity was not statistically significant (with rs values from -0.144 to 0.193, P values all above 0.05). Resignation coping behavior was significantly positively correlated with the psychological disorder (with rs values from 0.377 to 0.596, P values all below 0.01). (3) Educational status had predictive power on the anxiety, PTSD and flash back symptoms of patients (with t values from -2.19 to -2.02, P values all below 0.05), but not on depression, avoidance and hypervigilance (with t values from -1.95 to -0.99, P values all above 0.05). Burn area had no predictive power on the psychological disorders (with t values from 0.55 to 1.78, P values all above 0.05). Resignation coping behavior had predictive power on the psychological disorders (with t values from 3.10 to 6.46, P values below 0.01). Confrontation and avoidance coping behaviors had no predictive power on the psychological disorders (with t values from 0.46 to 2.32 and -0.89 and 1.75 respectively, P values all above 0.05). CONCLUSIONS: The severely burned patients with lower educational status, larger burn area, and the more frequently adapted resignation coping behavior are more likely to suffer from anxiety, depression, and PTSD.

Relationship between NRAGE and the radioresistance of esophageal carcinoma cell line TE13R120.

BACKGROUND AND OBJECTIVE: The mRNA levels of 59 genes, detected by cDNA microarray, were up-regulated in the radioresistant human esophageal cacinoma cell line TE13R120 as compared with its parental cell line TE13 before and after radiation, and the expression of NRAGE gene showed a gradually up-regulating tendency. This study aimed to further detect the differences of NRAGE gene and protein expression and apoptosis between TE13R120 and TE13 cells, and to investigate the relationship between the NRAGE and the radioresistance of TE13R120 cells and its mechanism. METHODS: The two cell lines were irradiated by 6°Co γ-ray at different conditions. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry were used to detect the expression of NRAGE. Flow cytometry (FCM) was used to detect the cell apoptosis before and after irradiation. RESULTS: The mRNA level of NRAGE was higher in TE13R120 cells than in TE13 cells before and after irradiation (before radiation: 0.25 ± 0.03 vs. 0.49 ± 0.03; 4 Gy 4 h: 0.31 ± 0.03 vs. 0.53 ± 0.02; 4 Gy 16 h: 0.32 ± 0.04 vs. 0.59 ± 0.04; 4 Gy 24 h: 0.36 ± 0.05 vs. 0.72 ± 0.04; 2 Gy 12 h: 0.32 ± 0.02 vs. 0.64 ± 0.04; 6 Gy 12 h: 0.36 ± 0.02 vs. 0.79 ± 0.05; 10 Gy 12 h: 0.46 ± 0.04 vs. 0.85 ± 0.01; P < 0.01), and the mRNA level of NRAGE was increased gradually with the increase of radiation dose and time in the two cell lines (P < 0.05 and P < 0.01). Western blot results showed no difference of NRAGE protein level in cytoplasm between TE13R120 cells and TE13 cells before and after irradiation, but its level in nuclei was higher in TE13R120 cells than in TE13 cells at different radiation time and dosages. Immunocytochemistry showed similar results as Western blot. FCM showed no significant difference in apoptosis rate between TE13R120 and TE13 cells before and after radiation. CONCLUSION: NRAGE may play an important role in the radiation responses of the two cell lines, and may participate in the formation of radioresistance of TE13R120 cells by changing its subcellular localization, but its relationship with cell apoptosis has not been confirmed.