Hydroxychloroquine Potentiates Apoptosis Induced by PPARα Antagonist in 786-O Clear Cell Renal Cell Carcinoma Cells Associated with Inhibiting Autophagy.

Clear cell renal cell carcinoma (ccRCC) is the major pathological pattern of renal cell carcinoma. The ccRCC cells exhibit a certain degree of inherent drug resistance due to some genetic mutations. In recent years, peroxisome proliferator-activated receptor-α (PPARα) antagonists have been reported as a targeted therapeutic drug capable of inducing apoptosis and cell cycle arrest in the ccRCC cell line. Autophagy, which can be induced by stress in eukaryotic cells, plays a complex role in the proliferation, survival, and death of tumor cells. In our study, we found that the expression of PPARα was low in highly differentiated ccRCC tissues and 786-O cell line but high in poorly differentiated ccRCC tissues. The level of PPARα expression in ccRCC tissues is correlated to the grade of differentiation, but not to the sex or age of ccRCC patients. The findings also revealed that the PPARα antagonist GW6471 can lower cell viability and induce autophagy in the 786-O ccRCC cell line. This autophagy can be inhibited by hydroxychloroquine. When treated with a combination of hydroxychloroquine and GW6471, the viability of the 786-O cells was decreased further when compared to the treatment with GW6471 or hydroxychloroquine alone, and apoptosis was promoted. Meanwhile, when human kidney 2 cells were cotreated with hydroxychloroquine and GW6471, cell viability was only slightly influenced. Hence, our finding indicates that the combination of GW6471 and hydroxychloroquine may constitute a novel and potentially effective treatment for ccRCC. Furthermore, this approach is likely to be safe owing to its minimal effects on normal renal tissues.

Contrasting ecosystem responses to climatic events and human activity revealed by a sedimentary record from Lake Yilong, southwestern China.

Global climate change and human activities have significantly impacted lake ecosystems at an accelerating rate in recent decades, but the differences between the responses of lake ecosystems to these two stressors remain unclear. Thus an improved understanding of the long-term influences of climatic and anthropogenic disturbances is necessary for the management of lake ecosystems. In order to address these issues, a sedimentary record was obtained from Lake Yilong in Yunnan Province in southwestern China, where the climate and natural environment are dominated by the Indian Summer Monsoon and there is a long history of human occupation and intensive human activity. The chronology is based on AMS 14C dates from 13 samples of plant macrofossils and charcoal, which show that the record spans the last ~12,000 yr. Geochemical indices were used to reconstruct hydro-climatic variations and lake ecosystem responses. The results indicate that a cold and humid climate prevailed from the late Pleistocene to the beginning of the Holocene, which was interrupted by an abrupt decrease in precipitation during 9.7-8.7 ka (1 ka = 1000 cal yr BP, corresponding to the 9.3 ka event). A persistent drying trend occurred during the middle and late Holocene, and there was an increase in the intensity of human activity during the past 1500 years. A comparison of the effects of a natural climatic event and human disturbance reveals contrasting lake ecosystem responses. The lake ecosystem was resilient to the 9.3 ka event and subsequently recovered; however, long-term human activity in the watershed, including deforestation and cultivation, reduced the stability of the lake ecosystem and positive feedback effects were strengthened, leading to the deviation of the system far from its previous stable state. It is concluded that, compared to climate change, human activities have had a much more serious impact on lake ecosystem.

B cell-activating factor regulates the survival of B lymphocytes infected with human cytomegalovirus.

BACKGROUND: Previous studies have suggested that B lymphocytes can be polyclonally activated by human cytomegalovirus (HCMV), and individuals infected by HCMV exhibit characteristic features of an autoimmunity disease. B cell-activating factor (BAFF) plays important roles in the survival and differentiation of B cells; however, few studies have examined the potential role of BAFF on B cells infected by HCMV. METHODS: HCMV virus strain (HCMV AD-169) was concentrated by normal methods and used to infect microbead-purified tonsil CD19+ B cells. Cells and supernatants were collected at the 1st, 3rd, 5th, and 7th day of co-culture, respectively. Cellular phenotypes, including expression of BAFF and its cognate receptors (BAFF-R, TACI, and BCMA) were detected by flow cytometry (FCM); cells apoptosis rates were also examined by FCM; and IgG titers in supernatants was detected by ELISA. In parallel, neutralizing anti-BAFF-R antibody was applied to observe the effect of BAFF/BAFF-R signaling on apoptosis and the IgG secretion ability of B cells stimulated by HCMV. RESULTS: LogTCID50 of 3rd and 4th generation of HCMV was -3.54 and -3.28, respectively. FCM results showed that the purity of CD19+ B cells was >98%. BAFF-R was highly expressed and upregulated on HCMV-infected B cells (93.5%-99.3%), compared with B cells prior to HCMV infection and uninfected group; while BAFF-R expression gradually decreased with time and to the lowest level at 5th day (81%) in the control medium-only group. In contrast, expression of TACI and BCMA gradually increased during culture in both HCMV-infected and medium-only control B cells. Furthermore, the apoptosis rate of HCMV-infected and medium-only control B cells did not vary significantly during culture, but IgG secretion ability of HCMV-infected B cells significantly increased over time while no changes were observed with the medium-only control. Importantly, the apoptosis rate of B cells significantly increased when BAFF/BAFF-R signal was blocked prior to HCMV infection (P<0.05), although no significant changes of IgG levels were observed (P>0.05). CONCLUSIONS: BAFF-R was consistently expressed on B cells infected by HCMV. Enhancement of BAFF/BAFF-R signaling decreased the apoptosis rate and extended the survival of B cells.

[miR-338-5p modulates B cell biological functions by targeting NF-κB1].

Objective To explore the effects of miR-338-5p on the nuclear factor κB1 (NF-κB1) expression and the IgG-producing ability of B cells. Methods Dual-luciferase reporter assay was used to test the target gene of miR-338-5p. The purified CD20+ B cells were transfected with miR-338-5p agomiR, miR-338-5p antagomiR, NF-κB1 siRNA (siNF-κB1) and their corresponding negative control reagents, and then cultured with anti-IgM antibody and/or recombinant human B cell activating factor (rhBAFF). Real-time RCR and Western boltting were applied to determine the mRNA and protein levels of NF-κB1. IgG level in the supernatant was detected by ELISA. Results Compared with the control group, the hRluc/hLuc relative luciferase activity was significantly elevated in miR-338-5p mimic and NF-κB1-3'-UTR reporter co-transfected group. In the co-culture system with anti-IgM antibody and rhBAFF, the NF-κB1 mRNA, p105, p50 and IgG levels in B cells transfected with miR-338-5p agomiR were significantly increased, while the NF-κB1 mRNA and IgG levels in B cells transfected with miR-338-5p antagomiR were significantly decreased. The effect of siNF-κB1 on B cells was opposite to that of miR-338-5p agomiR. Correlation analysis suggested that NF-κB1 mRNA level was significantly positively correlated with IgG concentration. Conclusion miR-338-5p regulates the biological functions of B cells by positively regulating NF-κB1 expression and indirectly regulating BAFF signal.

Comparison of Tolerogenic Dendritic Cells Induced by Liver X Receptor Agonist from Bone Marrow-derived Cells and Natural Tolerogenic Dendritic Cells.

BACKGROUND: Mouse dendritic cells (DCs) possess the tolerogenic potentiality after being induced by liver X receptor (LXR) agonist; while the characteristics and mechanisms of the induced DCs are still little known. METHODS: Mouse bone marrow cells were pulsed with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) with or without LXR agonist-T0901317 for 7 days, or induced with lipopolysaccharide for 3 days. Cellular biomarkers, inducing ability of T cell proliferation and regulatory T cells (Treg) formation, and cytokines in culture supernatants were tested. OVA-specific CD4⁺ T cells purified from BALB/c DO11.10 RAG⁻/⁻ mice were purified in Treg induction assay. NF-κB inhibitors and PI3K inhibitors were applied during cell culture. Natural tDCs (N-tDCs) isolated from mouse spleens were used as controls. RESULTS: Compared with the N-tDCs, the morphology of T-tDCs (induced by T0901317 for 7 days) was similar to that of normal DCs but had slightly lower or moderate expression levels of CD80, CD86, and MHCII. The N-tDCs could significantly inhibit T cell proliferation, while T-tDCs lost their stimulatory effect on T cell viability. The co-treatment of T0901317 with NF-κB inhibitors or PI3K inhibitors propagated the inhibitory effect and resulted in significantly reduced T cell viability. The N-tDCs could induce the formation of Tregs, which was enhanced in the presence of TGF-ß; however, T-tDCs did not have this capability. T-tDCs secreted much higher levels of IL-10 than did the N-tDCs. Neither the T-tDCs nor the N-tDCs produced indoleamine 2,3-dioxygenase. CONCLUSIONS: Compared with N-tDCs, T-tDCs have inhibitory effects. But they should function via different modes, involving the NF-κB and PI3K signal pathways.

B-cell-activating factor code and human cytomegalovirus infection in renal transplant recipients.

The objective of the present study was to explore the correlation between the BAFF signal and HCMV-TLR activation in RTx recipients complicated by HCMV. Peripheral blood (anticoagulated by EDTA-Na2 ) and urine of 113 RTx recipients were collected; healthy volunteers were controlled. Urine HCMV-DNA was detected by real-time PCR. Recipients were classified into a positive group (>10,000 copies/mL urine) and a negative group (<10,000 copies/mL urine). ELISA results showed that sBAFF, sera anti-HCMV pp65 immunoglobulin (Ig)G antibody, and total IgG all significantly increased in recipients with positive HCMV-DNA (>10,000 copies/mL urine) (P < 0.05) compared with negative recipients (<10,000 copies/mL urine). In the positive group, HCMV-DNA copies and total IgG positively correlated with sBAFF (r = 0.988 and 0.625, respectively) (P < 0.05). Luminex assay results suggested that the incidence of anti-HLA I and II and MICA antibody obviously increased in positive recipients. The expression level of BAFF and BAFF-R increased in positive recipients. A total of 88 particular genes-involved in TLR signaling pathways, NF-κB signaling pathways, and cytokine-cytokine receptor signaling pathways-were detected in real-time PCR chip assay. A total of 46 genes were differentially expressed greater than two-fold, and the expression characteristic of BAFF-R was concordant with FACS results. Our findings are that activation of HCMV would induce or enhance the activation of BAFF code in RTx recipients, which may independently or cooperatively participate in renal allograft injury and decrease the long-term outcome of renal allografts.