Intravenous Injection of Na Ions Aggravates Ang II-Induced Hypertension-Related Vascular Endothelial Injury by Increasing Transmembrane Osmotic Pressure.

Introduction: Extracellular protein nanoparticles (PNs) and ions perform synergistical functions in the control of transmembrane osmotic pressure (OP) under isotonic conditions. Intravenous injection may disrupt the ion balance and alter PN levels in blood plasma, changing transmembrane OP and damaging vascular endothelial cells. Methods: Na ions were injected into AngII-induced HUVECs to simulate cell injury in vitro, and tail vein infusion of Na ions into hypertensive rats was performed to assess vascular damage. Optical measurements using an intermediate filament (IF) tension probe were conducted to detect indicators related to transmembrane OP. Immunofluorescence, Western blotting and small interfering RNA (siRNA) transfection were employed to investigate inflammasomes and the relationship between Abl2 and inflammation. Results: Electrolyte injections with sodium ions (but not glucose and hydroxyethyl starch) induced the production of ASC and NLRP3 inflammasomes in Ang II-induced HUVECs; this in turn resulted in the disorder of calcium signals, and changes in transmembrane OP and cell permeability. Moreover, injection of Na ions into Ang II-induced HUVECs activated the mechanosensitive protein Abl2, involved in inflammation-induced transmembrane OP changes. A drug combination was identified that could induce OP recovery and block hyperpermeability induced by cytoplasmic inflammatory corpuscles in vivo and in vitro. Conclusion: Changes in extracellular PNs and ions following chemical stimuli (Ang II) participate in the regulation of transmembrane OP. Furthermore, injection of Na ions causes vascular endothelial injury in Ang II-induced cells in vitro and hypertension rats in vivo, suggesting it is not safe for hypertensive patients, and we propose a new drug combination as a solution.

Association of mean platelet volume/lymphocyte ratio with inflammation in non-dialysis patients with chronic kidney disease stages 1-4: A retrospective study.

Objective: The mean platelet volume/ratio (MPVLR) is recognized as a novel marker of inflammation. We evaluated whether the MPVLR is associated with inflammation in non-dialysis patients with chronic kidney disease (CKD) stages 1-4. Methods: A total of 402 non-dialysis patients with CKD stages 1-4 were included. The indicators of hematological, renal function (urea, serum creatinine [Scr], estimated glomerular filtration rate [eGFR], and urine albumin to creatinine ratio [ACR]) and the markers of inflammation (high-sensitivity C-reactive protein [hsCRP] and fibrinogen [FIB]) were recorded. The MPVLR values at different CKD stages were analyzed. Next, based on the absence (hsCRP level < 5 mg/L) and presence (hsCRP level > 5 mg/L) of inflammation, the patients were categorized, and the differences in indices between the two groups were observed. The patients were divided into two groups based on the median MPVLR value (6.39) at admission. The laboratory indices of patients with CKD were compared. Simultaneously, a correlation analysis was performed to identify the association between the MPVLR and each parameter. A binary logistic regression analysis was performed to test whether the MPVLR was associated independently with the presence of inflammation in non-dialysis patients with CKD. The receiver operating characteristic (ROC) curve was used to analyzed diagnostic performance of the MPVLR in evaluating the inflammation of non-dialysis patients with CKD stages 1-4. Results: The MPVLR was higher in patients with CKD stages 3-4 than in those with CKD stages 1 and 2. Significant differences in urea, Scr, eGFR, ACR, lymphocyte (LYM), red blood cell (RBC), hemoglobin (HGB), RBC distribution width (RDW-CV), MPVLR, and FIB values were observed between the groups with and without inflammation. The patients with a higher MPVLR had higher urea, Scr, ACR, WBC, neutrophils (NEU), RDW-CV, platelet distribution width (PDW), mean platelet volume (MPV), and hsCRP values and lower eGFR, LYM, RBC, HGB, and platelet (PLT) values. The MPVLR showed a positive correlation with age, urea, Scr, WBC, NEU, RDW-CV, PDW, MPV, and hsCRP values and a negative correlation with the eGFR, LYM, RBC, HGB, and PLT values. A logistic analysis revealed that the MPVLR was associated independently with the presence of inflammation in non-dialysis patients with CKD, after adjustment for the confounding factors (odds ratio = 1.020; P = 0.024). Furthermore, MPVLR exhibited a modest diagnostic performance for the assessment of inflammation in non-dialysis patients with CKD stages 1-4, with an area under the curve (AUC) of 0.706, and the sensitivity, specificity being 46.2% and 83.2%, respectively. Conclusions: The MPVLR was associated independently with the presence of inflammation in non-dialysis patients with CKD and may be useful for monitoring inflammation.

Metabolic profiling reveals new serum signatures to discriminate lupus nephritis from systemic lupus erythematosus.

Background: Lupus nephritis (LN) occurs in 50% of patients with systemic lupus erythematosus (SLE), causing considerable morbidity and even mortality. Previous studies had shown the potential of metabolic profiling in the diagnosis of SLE or LN. However, few metabonomics studies have attempted to distinguish SLE from LN based on metabolic changes. The current study was designed to find new candidate serum signatures that could differentiate LN from SLE patients using a non-targeted metabonomics method based on ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Method: Metabolic profiling of sera obtained from 21 healthy controls, 52 SLE patients and 43 LN patients. We used SPSS 25.0 for statistical analysis. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and metabolic pathway analysis were used to analyze the metabolic data. Results: Upon comparison of SLE and LN groups, 28 differential metabolites were detected, the majority of which were lipids and amino acids. Glycerolphospholipid metabolism, pentose and glucuronate interconversions and porphyrin and chlorophyll metabolism were obviously enriched in LN patients versus those with SLE. Among the 28 characteristic metabolites, five key serum metabolites including SM d34:2, DG (18:3(9Z,12Z,15Z)/20:5(5Z,8Z,11Z,14Z,17Z)/0:0), nervonic acid, Cer-NS d27:4, and PC (18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z) performed higher diagnostic performance in discriminating LN from SLE (all AUC > 0.75). Moreover, combined analysis of neuritic acid, C1q, and CysC (AUC = 0.916) produced the best combined diagnosis. Conclusion: This study identified five serum metabolites that are potential indicators for the differential diagnosis of SLE and LN. Glycerolphospholipid metabolism may play an important role in the development of SLE to LN. The metabolites we screened can provide more references for the diagnosis of LN and more support for the pathophysiological study of SLE progressed to LN.

Significant metabolic alterations in non-small cell lung cancer patients by epidermal growth factor receptor-targeted therapy and PD-1/PD-L1 immunotherapy.

Background: Cancer-related deaths are primarily attributable to lung cancer, of which non-small cell lung cancer (NSCLC) is the most common type. Molecular targeting therapy and antitumor immunotherapy have both made great strides in the treatment of NSCLC, but their underlying mechanisms remain unclear, especially from a metabolic perspective. Methods: Herein, we used a nontargeted metabolomics approach based on liquid chromatography-mass spectrometry to analyze the metabolic response of NSCLC patients to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) or PD-1/PD-L1 inhibitors. Multiple analyses, including principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA) and pathway analysis, were used for metabolic data analysis. Additionally, differential metabolites were analysed and identified by publically available and integrated databases. Results: After treatment with EGFR-TKIs or PD-1/PD-L1 inhibitors, glutamate/glutamine, phenylalanine, n-acetyl-l-leucine, n-acetyl-d-tryptophan, D-n-valine, arachidonic acid, and linoleic acid levels were significantly increased in patients with NSCLC, whereas carnitine, stearyl carnitine, palmitoyl carnitine, linoleic carnitine, and palmitic acid levels were markedly decreased. Compared with newly diagnosed, untreated patients, there were three shared metabolic pathways (phenylalanine metabolism, glycerophospholipid metabolism, and D-glutamine and D-glutamate metabolism) in the EGFR-TKIs or PD-1/PD-L1 inhibitor-treated groups, all of which were related to lipid and amino acid metabolism. Moreover, there were significant differences in lipid metabolism (glycerophospholipid metabolism and phosphatidylinositol signaling) and amino acid metabolism (tryptophan metabolism) between the EGFR-TKI and PD-1/PD-L1 inhibitor groups. Conclusion: Our results show that EGFR-TKIs and PD-1/PD-L1 inhibitors induce changes in carnitine, amino acids, fatty acids, and lipids and alter related metabolic pathways in NSCLC patients. Endogenous metabolism changes occur due to drug action and might be indicative of antitumor therapeutic effect. These findings will provide new clues for identifying the antitumor mechanism of these two treatments from the perspective of metabolism.

Metabolomics Profiling Discriminates Prostate Cancer From Benign Prostatic Hyperplasia Within the Prostate-Specific Antigen Gray Zone.

OBJECTIVE: Prostate cancer (PCa) is the second most common male malignancy globally. Prostate-specific antigen (PSA) is an important biomarker for PCa diagnosis. However, it is not accurate in the diagnostic gray zone of 4-10 ng/ml of PSA. In the current study, the performance of serum metabolomics profiling in discriminating PCa patients from benign prostatic hyperplasia (BPH) individuals with a PSA concentration in the range of 4-10 ng/ml was explored. METHODS: A total of 220 individuals, including patients diagnosed with PCa and BPH within PSA levels in the range of 4-10 ng/ml and healthy controls, were enrolled in the study. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based non-targeted metabolomics method was utilized to characterize serum metabolic profiles of participants. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) methods were used for multivariate analysis. Receiver operating characteristic (ROC) curve analysis was performed to explore the diagnostic value of candidate metabolites in differentiating PCa from BPH. Correlation analysis was conducted to explore the relationship between serum metabolites and common clinically used fasting lipid profiles. RESULTS: Several differential metabolites were identified. The top enriched pathways in PCa subjects such as glycerophospholipid and glycerolipid metabolisms were associated with lipid metabolism. Lipids and lipid-like compounds were the predominant metabolites within the top 50 differential metabolites selected using fold-change threshold >1.5 or <2/3, variable importance in projection (VIP) > 1, and Student's t-test threshold p < 0.05. Eighteen lipid or lipid-related metabolites were selected including 4-oxoretinol, anandamide, palmitic acid, glycerol 1-hexadecanoate, dl-dihydrosphingosine, 2-methoxy-6Z-hexadecenoic acid, 3-oxo-nonadecanoic acid, 2-hydroxy-nonadecanoic acid, N-palmitoyl glycine, 2-palmitoylglycerol, hexadecenal, d-erythro-sphingosine C-15, N-methyl arachidonoyl amine, 9-octadecenal, hexadecyl acetyl glycerol, 1-(9Z-pentadecenoyl)-2-eicosanoyl-glycero-3-phosphate, 3Z,6Z,9Z-octadecatriene, and glycidyl stearate. Selected metabolites effectively discriminated PCa from BPH when PSA levels were in the range of 4-10 ng/ml (area under the curve (AUC) > 0.80). Notably, the 18 identified metabolites were negatively corrected with total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and Apo-B levels in PCa patients; and some were negatively correlated with high-density lipoprotein cholesterol (HDL-C) and Apo-A levels. However, the metabolites were not correlated with triglycerides (TG). CONCLUSION: The findings of the present study indicate that metabolic reprogramming, mainly lipid metabolism, is a key signature of PCa. The 18 lipid or lipid-associated metabolites identified in this study are potential diagnostic markers for differential diagnosis of PCa patients and BPH individuals within a PSA level in the gray zone of 4-10 ng/ml.

Vitamin D Status in Children With Short Stature: Accurate Determination of Serum Vitamin D Components Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Objective: Vitamin D is critical for calcium and bone metabolism. Vitamin D insufficiency impairs skeletal mineralization and bone growth rate during childhood, thus affecting height and health. Vitamin D status in children with short stature is sparsely reported. The purpose of the current study was to investigate various vitamin D components by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to better explore vitamin D storage of short-stature children in vivo. Methods: Serum circulating levels of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], and 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3, C3-epi] were accurately computed using the LC-MS/MS method. Total 25(OH)D [t-25(OH)D] and ratios of 25(OH)D2/25(OH)D3 and C3-epi/25(OH)D3 were then respectively calculated. Free 25(OH)D [f-25(OH)D] was also measured. Results: 25(OH)D3 and f-25(OH)D levels in short-stature subgroups 2 (school age: 7~12 years old) and 3 (adolescence: 13~18 years old) were significantly lower compared with those of healthy controls. By contrast, C3-epi levels and C3-epi/25(OH)D3 ratios in all the three short-stature subgroups were markedly higher than the corresponding healthy cases. Based on cutoff values developed by Endocrine Society Recommendation (but not suitable for methods 2 and 3), sufficient storage capacities of vitamin D in short-stature subgroups 1, 2, and 3 were 42.8%, 23.8%, and 9.0% as determined by Method 3 [25(OH)D2/3+25(OH)D3], which were lower than those of 57.1%, 28.6%, and 18.2% as determined by Method 1 [25(OH)D2+25(OH)D3+C3-epi] and 45.7%, 28.5%, and 13.6% as determined by Method 2 [25(OH)D2/3+25(OH)D3+C3-epi]. Levels of 25(OH)D2 were found to be weakly negatively correlated with those of 25(OH)D3, and higher 25(OH)D3 levels were positively correlated with higher levels of C3-epi in both short-stature and healthy control cohorts. Furthermore, f-25(OH)D levels were positively associated with 25(OH)D3 and C3-epi levels in children. Conclusions: The current LC-MS/MS technique can not only separate 25(OH)D2 from 25(OH)D3 but also distinguish C3-epi from 25(OH)D3. Measurement of t-25(OH)D [25(OH)D2+25(OH)D3] alone may overestimate vitamin D storage in children, and short-stature children had lower vitamin D levels compared with healthy subjects. Ratios of C3-epi/25(OH)D3 and 25(OH)D2/25(OH)D3 might be alternative markers for vitamin D catabolism/storage in short-stature children. Further studies are needed to explore the relationships and physiological roles of various vitamin D metabolites.

Type I IFN expression is stimulated by cytosolic MtDNA released from pneumolysin-damaged mitochondria via the STING signaling pathway in macrophages.

Pneumolysin (Ply), a major virulence factor of Streptococcus pneumoniae (S. pn), affects the immunity of host cells during infection. It has been reported that Ply is involved in S. pn standard strain D39-induced interferon-ß (IFN-ß) expression; however, other findings suggest that recombinant Ply protein is incapable of triggering IFN-ß expression. Here, we demonstrated that purified Ply was capable of initiating oxidative damage to mitochondria, resulting in the subsequent release of mitochondrial deoxyribonucleic acid (mtDNA), which mediated IFN-ß expression in macrophages. Importantly, we determined that IFN-ß expression was regulated by stimulator of interferon genes (STING) signaling in response to Ply. In conclusion, our study identified that IFN-ß production was triggered by Ply in macrophages and mtDNA released from Ply-damaged mitochondria mediated this process, through the STING pathway. This is a novel mechanism by which S. pn modulates type I IFN response in macrophages.

Protective Regulatory T Cell Immune Response Induced by Intranasal Immunization With the Live-Attenuated Pneumococcal Vaccine SPY1 via the Transforming Growth Factor-ß1-Smad2/3 Pathway.

Vaccine effectiveness is mainly determined by the mechanism mediating protection, emphasizing the importance of unraveling the protective mechanism for novel pneumococcal vaccine development. We previously demonstrated that the regulatory T cell (Treg) immune response has a protective effect against pneumococcal infection elicited by the live-attenuated pneumococcal vaccine SPY1. However, the mechanism underlying this protective effect remains unclear. In this study, a short synthetic peptide (P17) was used to downregulate Tregs during immunization and subsequent challenges in a mouse model. In immunized mice, increase in immune cytokines (IL-12p70, IL-4, IL-5, and IL-17A) induced by SPY1 were further upregulated by P17 treatment, whereas the decrease in the infection-associated inflammatory cytokine TNF-α by SPY1 was reversed. P17 also inhibited the increase in the immunosuppressive cytokine IL-10 and inflammatory mediator IL-6 in immunized mice. More severe pulmonary injuries and more dramatic inflammatory responses with worse survival in P17-treated immunized mice indicated the indispensable role of the Treg immune response in protection against pneumococcal infection by maintaining a balance among acquired immune responses stimulated by SPY1. Further studies revealed that the significant elevation of active transforming growth factor ß (TGF-ß)1 by SPY1 vaccination activated FOXP3, leading to increased frequencies of CD4+CD25+Foxp3+ T cells. Moreover, SPY1 vaccination elevated the levels of Smad2/3 and phosphor-Smad2/3 and downregulated the negative regulatory factor Smad7 in a time-dependent manner during pneumococcal infection, and these changes were reversed by P17 treatment. These results illustrate that SPY1-stimulated TGF-ß1 induced the generation of SPY1-specific Tregs via the Smad2/3 signaling pathway. In addition, SPY1-specific Tregs may participate in protection via the enhanced expression of PD-1 and CTLA-4. The data presented here extend our understanding of how the SPY1-induced acquired Treg immune response contributes to protection elicited by live-attenuated vaccines and may be helpful for the evaluation of live vaccines and other mucosal vaccine candidates.

spd1672, a novel in vivo-induced gene, affects inflammatory response in a murine model of Streptococcus pneumoniae infection.

spd1672, a novel Streptococcus pneumoniae (hereinafter S. pn) gene induced in vivo, has been identified to contribute to the virulence of S. pn; however, the role of spd1672 during host innate immune reaction against S. pn infection is unknown. In the present study, mice were infected with wild-type D39 and mutant D39Δspd1672 strains. Compared with the D39-infected mice, reduced bacterial load and attenuated inflammatory response were observed in the D39Δspd1672-treated mice. The levels of proinflammatory cytokines, including IFN-γ, TNF-α, and IL-1ß, in the blood of D39Δspd1672-infected mice were lower than that in the D39-infected group. Additionally, attenuated activation of STAT3 and AKT was observed in the D39Δspd1672-infected mice. In conclusion, our data indicated that spd1672 expression modulates the release of proinflammatory cytokines, and AKT-STAT3 signaling appears to participate in the process. In conclusion, the present study extends our understanding of the role of an in vivo-induced gene in S. pn-host interaction.

Nontypeable Haemophilus influenzae DNA stimulates type I interferon expression via STING signaling pathway.

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-ß and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-ß and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent. SUMMARY: NTHI is the leading cause of acute exacerbations of COPD (AECOPD). Since AECOPD is an immune event, it is meaningful to elucidate the mechanism under NTHI induced immune response. It has been revealed that lipooligosaccharides and protein of NTHI could induce host immune response, but the function of NTHI nuclide acid during infection is unclear. In this research, we demonstrate NTHI DNA is a trigger for I-IFN expression, and the STING/TBK1/IRF3 pathway plays an integral role in sensing NTHI DNA to induce I-IFN expression. Moreover, by long-term intrabronchial infection of LPS, we constructed a mimic COPD murine model, in which the STING expression in lung tissues were higher than healthy mice after NTHI infection, which led us to surmise that NTHI cause AECOPD by inducing I-IFN production via STING signal pathway.

The plasticizing mechanism and effect of calcium chloride on starch/poly(vinyl alcohol) films.

Starch/poly(vinyl alcohol) (PVA) films were prepared with calcium chloride (CaCl(2)) as the plasticizer. The micro morphology of pure starch/PVA film and CaCl(2) plasticized starch/PVA film was observed by scanning electron microscope. The interaction between CaCl(2) and starch/PVA molecules was investigated by Fourier transform infrared spectroscopy. The influence of CaCl(2) on the crystalline, thermal and mechanical properties of starch/PVA films was studied by X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and tensile testing, respectively. The results indicated that CaCl(2) could interact with starch and PVA molecules and then effectively destroy the crystals of starch and PVA. Starch/PVA films plasticized with CaCl(2) became soft and ductile, with lower tensile strength and higher elongation at break compared with pure starch/PVA film. The water content of starch/PVA film would increase with the addition of CaCl(2). This is an important cause of the plasticization of CaCl(2) on starch/PVA film.