[Co-exposure of carbon black and cadmium induces autophagy and inflammation in human bronchial epithelial cells via PERK pathway].

Objective: To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells. Methods: In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 µg/ml Cd, 100 µg/ml CBNPs, 100 µg/ml CBNPs+2 µg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results: There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined (P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group (P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression (P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group (P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group (P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene (P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 (P<0.05), but the interaction between the two chemicals had no statistical significance (P>0.05) . Conclusion: CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

[Effect of serum interleukin-21 on B cell secretory capacity and apoptosis in patients with systemic lupus erythematosus].

Objective: To investigate the secretory capacity and apoptosis of interleukin (IL)-21 induced normal B cells by co-culture with serum from patients with systemic lupus erythematosus (SLE). Methods: Serum from twenty new-onset SLE patients and 20 healthy donors were collected. CD(19)(+) B cells from the normal controls were co-cultured with serum from SLE patients in the presence or absence of IL-21-R-FC(4 µg/ml). Supernatant IgG and IgM concentration were measured by immunoturbidimetric assay on day 5. Supernatant anti-dsDNA level was determined by ELISA. The percentage of apoptotic cells was detected by flow cytometer. Results: IgG, IgM and anti-dsDNA levels in normal B cells with SLE serum were significantly higher than those in the serum of SLE patients alone [(5.84±1.79)g/L vs (4.25±1.48)g/L, P=0.000; (0.46±0.21)g/L vs (0.43±0.21)g/L, P=0.003; (127.76±70.24)IU/ml vs (115.15±63.88) IU/ml, P=0.014 respectively]. However, no significant differences were found in the group of normal B cells with non-homologous serum from normal controls (P>0.05). Supernatant IgG, IgM and anti-dsDNA levels in normal B cells with SLE serum significantly decreased while IL-21R-fusion protein was added [(5.26±1.62)g/L vs (5.84±1.79)g/L, P=0.006; (0.42±0.20)g/L vs (0.46±0.21)g/L, P=0.002; (118.00±69.62)IU/ml vs (127.76±70.24)IU/ml, P=0.012 respectively]. The apoptotic rate of B cells with SLE serum was significantly higher than that with normal serum [(47.88±12.65)% vs (38.86±10.32)%, P=0.004]. But adding IL-21R-fusion conversed the apoptotic rates [(42.08±12.52)% vs (47.88±12.65)%, P=0.001]. Conclusions: SLE serum could induce normal B cells to form immunoglobulin secreting cells and producing autoantibodies, or apoptosis in pathological conditions. IL-21 might be considered as a potential therapeutic target of SLE.

Identification of LCK mutation in a family with atypical epidermodysplasia verruciformis with T-cell defects and virus-induced squamous cell carcinoma.

BACKGROUND: Inherited epidermodysplasia verruciformis (EV) is a rare skin disorder characterized by susceptibility to specific types of human papilloma virus (HPV) and is strongly associated with skin carcinomas. Inactivating mutations in EVER1/EVER2 account for most cases of EV. However, more phenotypes related to but distinct from EV have been reported with an immunodeficiency state but without EVER1/EVER2 mutation, and the genetic basis for these atypical EV cases is poorly understood. OBJECTIVES: To identify the causative gene responsible for three siblings affected by atypical EV but without EVER1/EVER2 mutation. METHODS: Whole-exome sequencing followed by Sanger sequencing was performed to identify the gene responsible for the patients with atypical EV enrolled in our study. RESULTS: A homozygous splicing mutation was detected in LCK (c.188-2A>G). This mutation resulted in an exon 3 deletion T lymphocyte-specific protein tyrosine kinase isoform, which further led to frameshift mutation and subsequent mRNA decay. CONCLUSIONS: We demonstrate a novel mutation in LCK in a family affected by atypical EV with T-cell defects, HPV infection and virus-induced malignancy, providing new clues in the understanding of host defences against HPV and better genetic counselling of patients with the EV phenotype.

Association of the -2510A/G chemokine (C-C motif) ligand 2 polymorphism with knee osteoarthritis in a Korean population.

OBJECTIVE: To investigate the possible association between polymorphisms [the -2510A/G promoter polymorphism (rs1024611) and the Cys35Cys coding polymorphism (rs4586) in exon 2] of the chemokine (C-C motif) ligand 2 (CCL2) gene and knee osteoarthritis (OA) in a Korean population. METHODS: DNA was obtained from 153 Korean primary knee OA patients and 270 healthy controls. CCL2 genomic variants (-2510A/G and Cys35Cys polymorphisms) were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In additional, the effect of -2510A/G on CCL2 transcription was examined, using a luciferase reporter gene construct transfected into HMC-1 cells. RESULTS: The -2510A/G promoter polymorphism was associated with OA [genotype frequency, p = 0.041; allele frequency, p = 0.017, odds ratio (OR) = 1.45, 95% confidence interval (CI) = 1.07-1.96]. Significant association was observed between the G carrier of the -2510A/G promoter polymorphism and primary knee OA patients (p = 0.021, OR = 2.25, 95% CI = 1.12-4.52). The G carrier of the -2510A/G promoter polymorphism was also associated with both clinically subtyped OA patients (OA patients with functionally poor index and radiographically severe OA patients). However, no significant difference was found in the Cys35Cys polymorphism. Haplotype frequency analysis revealed a significant difference (chi(2) = 8.98, p = 0.030). The CCL2 serum level of subjects with the G carrier (290.0+/-87.5 pg/mL) of the -2510A/G promoter polymorphism was statistically higher than that of subjects with the non-G carrier (161.5+/-48.3 pg/mL). The luciferase activity was significantly greater from interleukin (IL)-1beta-induced cells transfected with constructs containing G at position -2510. CONCLUSIONS: The G carrier of the -2510A/G promoter polymorphism was found to be associated with primary knee OA, and could be a susceptibility factor in the development of primary knee OA in the Korean population.

Topiramate stimulates glucose transport through AMP-activated protein kinase-mediated pathway in L6 skeletal muscle cells.

The use of topiramate (TPM) in the treatment of binge-eating disorder, bulimia nervosa, and antipsychotic-induced weight gain has recently increased, however, the exact molecular basis for its effects on body weight reduction and improved glucose homeostasis, is yet to be elucidated. Here we investigated the effect and signaling pathway of TPM on glucose uptake in L6 rat skeletal muscle cells, which account for >70% of glucose disposal in the body. Intriguingly, we found that TPM (10 microM) stimulated the rate of glucose uptake up to twofold increase. And TPM-stimulated glucose transport was inhibited with the overexpression of dominant-negative form of AMP-activated protein kinase (AMPK), an important mediator in glucose transport, implicating that AMPK-mediated pathway is involved. The TPM-stimulated glucose transport was blocked by SB203580, a specific inhibitor of AMPK downstream mediator, p38 mitogen-activated protein kinase (MAPK) protein. LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, which is another crucial mediator in independent glucose transport pathway, did not inhibit TPM-stimulated glucose transport. We also found that TPM increased the phosphorylation level of AMPK and p38 MAPK, whereas no effect on the activity of PI 3-kinase of TPM, when assessed by PI 3-kinase assay, was observed. These results together suggest that TPM stimulates glucose transport, not via PI 3-kinase mediated, but via AMPK-mediated pathway in skeletal muscle cells, thereby contributing to the body weight regulation and glucose homeostasis.