A novel mucosal bivalent vaccine of EV-A71/EV-D68 adjuvanted with polysaccharides from Ganoderma lucidum protects mice against EV-A71 and EV-D68 lethal challenge.

BACKGROUND: Human enteroviruses A71 (EV-A71) and D68 (EV-D68) are the suspected causative agents of hand-foot-and-mouth disease, aseptic meningitis, encephalitis, acute flaccid myelitis, and acute flaccid paralysis in children. Until now, no cure nor mucosal vaccine existed for EV-A71 and EV-D68. Novel mucosal bivalent vaccines are highly important for preventing EV-A71 and EV-D68 infections. METHODS: In this study, formalin-inactivated EV-A71 and EV-D68 were used as antigens, while PS-G, a polysaccharide from Ganoderma lucidum, was used as an adjuvant. Natural polysaccharides have the characteristics of intrinsic immunomodulation, biocompatibility, low toxicity, and safety. Mice were immunized intranasally with PBS, EV-A71, EV-D68, or EV-A71 + EV-D68, with or without PS-G as an adjuvant. RESULTS: The EV-A71 + EV-D68 bivalent vaccine generated considerable EV-A71- and EV-D68-specific IgG and IgA titres in the sera, nasal washes, saliva, bronchoalveolar lavage fluid, and feces. These antibodies neutralized EV-D68 and EV-A71 infectivity. They also cross-neutralized infections by different EV-D68 and EV-A71 sub-genotypes. Furthermore, compared with the PBS group, EV-A71 + EV-D68 + PS-G-vaccinated mice exhibited an increased number of EV-D68- and EV-A71-specific IgA- and IgG-producing cells. In addition, T-cell proliferative responses, and IFN-γ and IL-17 secretion in the spleen were substantially induced when PS-G was used as an adjuvant with EV-A71 + EV-D68. Finally, in vivo challenge experiments demonstrated that the immune sera induced by EV-A71 + EV-D68 + PS-G conferred protection in neonate mice against lethal EV-A71 and EV-D68 challenges as indicated by the increased survival rate and decreased clinical score and viral RNA tissue expression. Taken together, all EV-A71/EV-D68 + PS-G-immunized mice developed potent specific humoral, mucosal, and cellular immune responses to EV-D68 and EV-A71 and were protected against them. CONCLUSIONS: These findings demonstrated that PS-G can be used as a potential adjuvant for EV-A71 and EV-D68 bivalent mucosal vaccines. Our results provide useful information for the further preclinical and clinical development of a mucosal bivalent enterovirus vaccine against both EV-A71 and EV-D68 infections.

Metabolic Reprogramming in Response to Alterations of Mitochondrial DNA and Mitochondrial Dysfunction in Gastric Adenocarcinoma.

We used gastric cancer cell line AGS and clinical samples to investigate the roles of mitochondrial DNA (mtDNA) alterations and mitochondrial respiratory dysfunction in gastric adenocarcinoma (GAC). A total of 131 clinical samples, including 17 normal gastric mucosa (N-GM) from overweight patients who had received sleeve gastrectomy and 57 paired non-cancerous gastric mucosae (NC-GM) and GAC from GAC patients who had undergone partial/subtotal/total gastrectomy, were recruited to examine the copy number and D310 sequences of mtDNA. The gastric cancer cell line AGS was used with knockdown (KD) mitochondrial transcription factor A (TFAM) to achieve mitochondrial dysfunction through a decrease of mtDNA copy number. Parental (PT), null-target (NT), and TFAM-KD-(A/B/C) represented the parental, control, and TFAM knocked-down AGS cells, respectively. These cells were used to compare the parameters reflecting mitochondrial biogenesis, glycolysis, and cell migration activity. The median mtDNA copy numbers of 17 N-GM, 57 NC-GM, and 57 GAC were 0.058, 0.055, and 0.045, respectively. The trend of decrease was significant (p = 0.030). In addition, GAC had a lower mean mtDNA copy number of 0.055 as compared with the paired NC-GM of 0.078 (p < 0.001). The mean mtDNA copy number ratio (mtDNA copy number of GAC/mtDNA copy number of paired NC-GM) was 0.891. A total of 35 (61.4%) GAC samples had an mtDNA copy number ratio ≤0.804 (p = 0.017) and 27 (47.4%) harbored a D310 mutation (p = 0.047), and these patients had shorter survival time and poorer prognosis. After effective knockdown of TFAM, TFAM-KD-B/C cells expressed higher levels of hexokinase II (HK-II) and v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT, but lower levels of phosphorylated pyruvate dehydrogenase (p-PDH) than did the NT/PT AGS cells. Except for a higher level of p-PDH, the expression levels of these proteins remained unchanged in TFAM-KD-A, which had a mild knockdown of TFAM. Compared to those of NT, TFAM-KD-C had not only a lower mtDNA copy number (p = 0.050), but also lower oxygen consumption rates (OCR), including basal respiration (OCRBR), ATP-coupled respiration (OCRATP), reserve capacity (OCRRC), and proton leak (OCRPL)(all with p = 0.050). In contrast, TFAM-KD-C expressed a higher extracellular acidification rate (ECAR)/OCRBR ratio (p = 0.050) and a faster wound healing migration at 6, 12, and 18 h, respectively (all with p = 0.050). Beyond a threshold, the decrease in mtDNA copy number, the mtDNA D310 mutation, and mitochondrial dysfunction were involved in the carcinogenesis and progression of GACs. Activation of PDH might be considered as compensation for the mitochondrial dysfunction in response to glucose metabolic reprogramming or to adjust mitochondrial plasticity in GAC.

The Role of Plasma Cell-Free Mitochondrial DNA and Nuclear DNA in Systemic Lupus Erythematosus.

BACKGROUND: The roles of plasma cell-free (pcf) mitochondrial DNA (mtDNApcf) and nuclear DNA (nDNApcf) in the pathogenesis of systemic lupus erythematosus (SLE) remain unclear. We analyzed the relative copies of mtDNApcf and nDNApcf and investigated their association with the levels of plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG), plasma malondialdehyde (MDA) and mRNA of leukocyte C-type lectin domain family 5 member A (CLEC5A) in SLE patients. METHODS: A total of 80 SLE patients and 43 healthy controls (HCs) were enrolled. Their plasma samples were subjected to the measurements of mtDNApcf copies, nDNApcf copies, 8-OHdG and MDA, respectively. Their leukocytes were analyzed for CLEC5A mRNA expression. RESULTS: SLE patients had higher nDNApcf copies (2.84 ± 1.99 vs. 2.00 ± 0.88, p = 0.002), lower mtDNApcf copies (4.81 ± 6.33 vs. 9.83 ± 14.20, p = 0.032), higher plasma 8-OHdG (0.227 ± 0.085 vs. 0.199 ± 0.041 ng/mL, p = 0.016), lower plasma MDA (3.02 ± 2.20 vs. 4.37 ± 2.16 µM, p = 0.001) and similar leukocyte CLEC5A mRNA expression levels (1.21 ± 1.17 vs. 1.26 ± 1.05, p = 0.870), as compared with those of HCs. Among the HCs, SLE patients with SLE Disease Activity Index (SLEDAI) ≤8, and SLE patients with SLEDAI >8, their respective mtDNApcf copies decreased stepwisely (9.83 ± 14.20 vs. 6.28 ± 7.91 vs. 3.19 ± 3.35, p = 0.054). The nDNApcf copies of HCs, SLE patients without nephritis, and SLE patients with nephritis were increased stepwisely (2.00 ± 0.88 vs. 2.63 ± 1.74 vs. 3.16 ± 2.34, p = 0.043). Among SLE patients, higher nDNApcf copies were associated with higher levels of plasma 8-OHdG (p < 0.001) but lower plasma MDA (p = 0.019). Among HCs but not SLE patients, higher nDNApcf copies (p = 0.013) or lower mtDNApcf copies (p < 0.001) were related to higher levels of leukocyte CLEC5A mRNA expression. CONCLUSIONS: Higher nDNApcf, lower mtDNApcf, increased ROS-elicited oxidative DNA damage and dysregulated leukocyte CLEC5A expression might be implicated in the pathogenesis of SLE.

Involvement of increased p53 expression in the decrease of mitochondrial DNA copy number and increase of SUVmax of FDG-PET scan in esophageal squamous cell carcinoma.

We appraised Warburg effect through analysis of mitochondrial DNA (mtDNA) copy number and maximum standard uptake value (SUVmax) of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) scan and their alterations in esophageal squamous cell carcinoma (ESCC). Later T-status and longer longitudinal tumor length were associated with lower mtDNAESCC copy number (p < .05) but higher SUVmax-ESCC (p < .05), respectively. Lower mtDNAESCC copy number correlated with higher SUVmax-ESCC, reciprocally (p < .05). ESCCs expressing mutant p53 protein had lower mtDNAESCC copy number (p = .056) but higher SUVmax-ESCC (p = .046). We conclude that mutant p53 protein may be involved in the Warburg effect of ESCC.

Alterations of oxygen consumption and extracellular acidification rates by glutamine in PBMCs of SLE patients.

We evaluated plasma glutamine levels and basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB) of peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients and healthy controls (HCs). Lower plasma glutamine levels correlated with higher SLE disease activity indexes (p=0.025). Incubated in DMEM containing 100mg/dL glucose, SLE-PBMCs displayed lower mOCRB (p=0.018) but similar ECARB (p=0.467) to those of HC-PBMCs, and their mOCRB got elevated (p<0.001) without altering ECARB (p=0.239) by supplementation with 2 or 4mM glutamine. We conclude that impaired mitochondrial respiration of SLE-PBMCs could be improved by glutamine under euglycemic condition.

Role of mitochondrial function in the invasiveness of human colon cancer cells.

We investigated the role of mitochondrial function in the invasiveness of human colorectal cancer (CRC) cell lines, using paired primary SW480 and metastatic SW620 cells, and appraised the clinical relevance of the alteration of mtDNA copy number in 33 pairs of CRC specimens after surgical resection. Suppression of mitochondrial function was achieved by the exposure of cells to oligomycin A (OA) or by knockdown of mitochondrial transcriptional factor A (TFAM) to evaluate their effects on energy metabolism, reactive oxygen species, protein expression levels of epithelial-mesenchymal transition (EMT) markers and invasive activity of CRC cells. We found that SW620 cells expressed higher levels of TFAM and mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) and cytochrome c oxidase subunit II (COX-II) and nuclear DNA-encoded NADH ubiquinone oxidoreductase subunit A9 (NDUFA9), iron-sulfur protein subunit B of succinate dehydrogenase (SDHB), ubiquinol­cytochrome c reductase core protein I/II (UQCRC1/2) and cytochrome c oxidase subunit IV (COX-IV) when compared with the SW480 cells. The mtDNA copy number, ADP-triggered oxygen consumption rate (OCR) and respiratory control ratio (RCR) of succinate-supported respiration in the SW620 cells were higher than those noted in the SW480 cells. The intracellular levels of H2O2 and O2-• in the SW620 cells were lower than levels noted in the SW480 cells. Moreover, SW620 cells displayed lower protein levels of hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI) and lactate dehydrogenase (LDH), and lower lactate production rate, and expressed higher levels of EMT markers N-cadherin, vimentin and Snail, and showed higher Transwell migration and invasion activities as compared with the SW480 cells. After OA treatment, SW620 cells exhibited a decrease in OCR and RCR of succinate-supported respiration, an increase in lactate production rate and intracellular levels of H2O2 and O2-•. Moreover, the level of vimentin and Transwell migration activity of the SW620 cells were decreased. After TFAM knockdown, the protein levels of TFAM, ND6 and COX-II, and mtDNA copy number, OCR and RCR of succinate-supported respiration in the SW620-KD#4 and SW620-KD#5 cells were all lower than those noted in the SW620­Control cells. By contrast, the protein level of HK-II, lactate production rate, the intracellular levels of H2O2 and O2-• in the SW620-KD#4 and SW620-KD#5 cells were all higher than those noted in the SW620-Control cells. Subsequently, both SW620-KD#4 and SW620-KD#5 cells had lower Transwell invasion activity than did the SW620-Control cells. Furthermore, we found that deeper invasion (P=0.025) and longer tumor length (P=0.069) were associated with higher mtDNA copy ratios in the 33 pairs of CRC specimens obtained from surgical resection. Taken together, we conclude that higher mtDNA copy number and mitochondrial function may confer an invasive advantage to CRCs.

Oxidative DNA and mitochondrial DNA change in patients with SLE.

We evaluated plasma IL-10, IFN-alpha, IL-23, IFN-gamma, IP-10, MCP-1, 8-OHdG, leukocyte mtDNA, serum anti-dsDNA antibodies and disease activity index (SLEDAI) in SLE patients. 93 patients (35 nephritis, 4 under dialysis, 5 under rituximab) and 50 healthy controls were recruited. Compared with healthy controls, SLE patients had higher IL-10, IFN-alpha, IL-23, IFN-gamma, IP-10 and MCP-1 (p<0.05). High IFN-alpha (p=0.031) and IP-10 (p=0.026) correlated with high SLEDAI; high IFN-alpha (p<0.001), IL-23 (p=0.023) and IP-10 (p<0.001) correlated with high anti-dsDNA. High IL-10 (p=0.014), IL-23 (p<0.001), IFN-gamma (p<0.001) and MCP-1 (p=0.002) correlated with high 8-OHdG and high IL-23 (p<0.001), INF-gamma (p<0.001), IP-10 (p=0.023) and MCP-1 (p=0.002) correlated with low leukocyte mtDNA. mtDNA 4977 deletion correlated with high mtDNA (p=0.011) and low IL-10 (p=0.009). MCP-1 (p=0.043) decreased after rituximab therapy. 54 SLE patients without nephritis, 35 with nephritis but without dialysis, and 4 with nephritis under dialysis exhibited stepwise increases in IL-23 (p=0.009) and MCP-1 (p=0.015). These data suggest that oxidative DNA and mtDNA alterations and coordinate changes in cytokines/chemokines are implicated in progression of SLE and rituximab in amelioration of SLE.

Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0.007); and drug resistance to doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a drug-resistant phenotype in vitro.