Genetic variation in the tissue factor gene is associated with clinical outcome in severe sepsis patients.

INTRODUCTION: Activation of inflammation and coagulation was closely related and mutually interdependent in sepsis. Tissue factor (TF) and its endogenous inhibitor, tissue factor pathway inhibitor (TFPI) was the main regulators of the initiation of coagulation process. Altered plasma levels of TF and TFPI have been related to worse outcome in sepsis. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the TF and TFPI genes were associated with risk and outcome for patients with severe sepsis. METHODS: Seventeen SNPs in TF and TFPI were genotyped in samples of sepsis (n =577) and severe sepsis patients (n =476), and tested for association in this case-control collection. We then investigated correlation between the associated SNPs and the mRNA expression, and protein level of the corresponding gene. The mRNA levels of TF were determined using real-time quantitative reverse transcription-polymerase chain reaction and the soluble plasma levels of TF were measured using enzyme linked immunosorbent assay (ELISA) method. RESULTS: Association analysis revealed that three TF SNPs in perfect linkage disequilibrium, rs1361600, rs3917615 and rs958587, were significantly associated with outcome of severe sepsis. G allele frequency of rs1361600 in survivor patients was significantly higher than that in nonsurvivor severe sepsis patients (P =4.91 × 10(-5), odds ratio (OR) =0.48, 95% confidence interval (CI) 0.33 to 0.69). The association remained significant after adjustment for covariates in multiple logistic regression analysis and for multiple comparisons. Lipopolysaccharide-induced TF-mRNA expression levels in peripheral blood mononuclear cells from subjects carrying rs1361600 AG and GG genotypes, were significantly lower than those subjects carrying AA genotype (P =0.0012). Moreover, severe sepsis patients of GG and GA genotypes showed lower serum levels of TF than patients with AA genotype (P adj =0.02). The plasma levels of TF were also associated with outcome of severe sepsis patients (P adj =0.01). However, genotype and allele analyses did not show any significant difference between sepsis and severe sepsis patients. CONCLUSIONS: Our findings indicate that common genetic variation in TF was significantly associated with outcome of severe sepsis in Chinese Han population.

A multicenter prospective study of comprehensive metagenomic and transcriptomic signatures for predicting outcomes of patients with severe community-acquired pneumonia.

BACKGROUND: Severe community-acquired pneumonia (SCAP) results in high mortality as well as massive economic burden worldwide, yet limited knowledge of the bio-signatures related to prognosis has hindered the improvement of clinical outcomes. Pathogen, microbes and host are three vital elements in inflammations and infections. This study aims to discover the specific and sensitive biomarkers to predict outcomes of SCAP patients. METHODS: In this study, we applied a combined metagenomic and transcriptomic screening approach to clinical specimens gathered from 275 SCAP patients of a multicentre, prospective study. FINDINGS: We found that 30-day mortality might be independent of pathogen category or microbial diversity, while significant difference in host gene expression pattern presented between 30-day mortality group and the survival group. Twelve outcome-related clinical characteristics were identified in our study. The underlying host response was evaluated and enrichment of genes related to cell activation, immune modulation, inflammatory and metabolism were identified. Notably, omics data, clinical features and parameters were integrated to develop a model with six signatures for predicting 30-day mortality, showing an AUC of 0.953 (95% CI: 0.92-0.98). INTERPRETATION: In summary, our study linked clinical characteristics and underlying multi-omics bio-signatures to the differential outcomes of patients with SCAP. The establishment of a comprehensive predictive model will be helpful for future improvement of treatment strategies and prognosis with SCAP. FUNDING: National Natural Science Foundation of China (No. 82161138018), Shanghai Municipal Key Clinical Specialty (shslczdzk02202), Shanghai Top-Priority Clinical Key Disciplines Construction Project (2017ZZ02014), Shanghai Key Laboratory of Emergency Prevention, Diagnosis and Treatment of Respiratory Infectious Diseases (20dz2261100).

Role of δ2 opioid receptor in cardioprotection against hypoxia-reoxygenation injury.

Several lines of in vivo evidence demonstrated that activation of δ-opioid receptors (ORs) with agonists mimics the cardioprotective effect of ischemic preconditioning. However, the subtypes of ORs involved and the molecular and cellular mechanisms are not entirely clear. To investigate the significance of the contribution by δ ORs to cardiomyocyte survival, we used an in vitro model of hypoxia/reoxygenation (H/R) in primary cultures of neonatal rat cardiomyocytes to study the role of different δ ORs in cardiomyocyte apoptosis and the relevant downstream signaling pathway. The results showed that apoptosis in neonatal cardiomyocytes induced by H/R was reversed by δ2 OR agonist, deltorphin E but not by δ1 OR agonist DPDPE; the deltorphin E-induced cytoprotection was totally abrogated by the MEK inhibitor PD98059 and overexpression of dominant interfering form of MEK1; in contrast, overexpression of constitutive active form of MEK1 exerted a similar protective effect as deltorphin E. These results suggest that δ2 OR, but not δ1 OR, plays a key role in preventing cardiomyocytes from apoptosis during H/R injury, which is mainly mediated by the MEK/ERK1/2 pathway.

Diagnosis of sepsis with inflammatory biomarkers, cytokines, endothelial functional markers from SIRS patients.

BACKGROUND: Sepsis is a life-threatening illness with a challenging diagnosis. Rapid detection is the key to successful treatment of sepsis. To investigate diagnostic value, the plasma protein profiles of inflammatory biomarkers, cytokines, and endothelial functional markers were compared between healthy controls, SIRS, and septic patients. METHODS: The plasma protein profiles were performed by Luminex Assay in a cohort of 50 SIRS patients, 82 septic patients and 25 healthy controls. Fourteen plasma proteins were analyzed in the same cohort: IL-1ß, IL-6, IL-8, IL-10, CCL-2, VEGF, VEGF-C, VEGFR2, CD62E, CD62P, MFG-E8, ICAM-1, TFPI, Urokinase. RESULT: IL-2R, IL-6, IL-8, IL-10, CCL-2, ICAM-1, and Urokinase were significantly higher in sepsis patients than SIRS patients. VEGF, IL-1ß, CD62E, CD62P, MFG-E8, and TFPI have no statistical difference. VEGF-C, VEGFR2 were significantly different in SIRS patients than sepsis patients. Urokinase, ICAM-1, and VEGFR2 were significantly different between sepsis group and SIRS group. The AUCs of Urokinase, ICAM-1, and VEGFR2 and the combination for the diagnosis of sepsis were 0.650, 0.688, 0.643, and 0.741, respectively. CONCLUSIONS: Most patients have the higher level of several cytokines and developed endothelial cell injury in the initial phase of sepsis, Urokinase, ICAM-1, and VEGFR2 may be useful to evaluate severity and prognosis of sepsis patients.

The crystallin alpha B (HSPB5)-tripartite motif containing 33 (TRIM33) axis mediates myocardial fibrosis induced by angiotensinogen II through transforming growth factor-ß (TGF-ß1)-Smad3/4 signaling.

Myocardial fibrosis, a common pathological manifestation of cardiac remodeling (CR), often leads to heart failure (HF) and even death. The underlying molecular mechanism of the role of TRIM33 in Ang II-induced myocardial fibrosis is not fully understood. We found that TRIM33 was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. Adult mice induced by Ang II were used as in vivo models, and Ang II-induced neonatal mouse primary cardiac fibroblasts (CFs) were used as in vitro models. The level of CF fibrosis in vitro was assessed by CF proliferation, migration, activation and extracellular matrix (ECM) synthesis. In addition, Masson staining, the heart weight/body weight (HW/BW) ratio and echocardiography were used to evaluate the in vivo effect of TRIM33. TRIM33 expression was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. In in vitro experiments, we found that TRIM33 knockdown promoted Ang II-induced CF proliferation, while TRIM33 overexpression weakened Ang II-induced CF proliferation, migration, activation and collagen synthesis. Mechanistically, we showed that TRIM33, negatively regulated by HSPB5, mediated its antifibrotic effect by inhibiting the activation of TGF-ß1 and its downstream genes, Smad3 and Smad4. Finally, TRIM33 overexpression suppressed fibrosis and promoted cardiac repair and functional recovery in Ang II-induced mice. Our results clearly establish that TRIM33 limits cardiac fibrosis by hindering CF proliferation, migration, activation and collagen synthesis. Enhancing these beneficial functions of TRIM33 by a targeting vector might be a novel therapeutic strategy for CR.

Aetiology of severe community acquired pneumonia in adults identified by combined detection methods: a multi-centre prospective study in China.

Severe Community Acquired Pneumonia (SCAP) challenges public health globally. Considerable improvements in molecular pathogen testing emerged in the last few years. Our prospective study combinedly used traditional culture, antigen tests, PCR and mNGS in SCAP pathogen identification with clinical outcomes. From June 2018 to December 2019, we conducted a multi-centre prospective study in 17 hospitals of SCAP patients within 48 hours of emergency room stay or hospitalization in China. All clinical data were uploaded into an online database. Blood, urine and respiratory specimens were collected for routine culture, antigen detection, PCR and mNGS as designed appropriately. Aetiology confirmation was made by the local attending physician group and scientific committee according to microbiological results, clinical features, and response to the treatment. Two hundred seventy-five patients were included for final analysis. Combined detection methods made identification rate up to 74.2% (222/299), while 14.4% (43/299) when only using routine cultures and 40.8% (122/299) when not using mNGS. Influenza virus (23.2%, 46/198), S. pneumoniae (19.6%, 39/198), Enterobacteriaceae (14.6%, 29/198), Legionella pneumophila (12.6%, 25/198), Mycoplasma pneumoniae (11.1%, 22/198) were the top five common pathogens. The in-hospital mortality of patients with pathogen identified and unidentified was 21.7% (43/198) and 25.9% (20/77), respectively. In conclusion, early combined detection increased the pathogen identification rate and possibly benefitted survival. Influenza virus, S. pneumoniae, Enterobacteriaceae was the leading cause of SCAP in China, and there was a clear seasonal distribution pattern of influenza viruses. Physicians should be aware of the emergence of uncommon pathogens, including Chlamydia Psittaci and Leptospira.

LPS pretreatment ameliorates multiple organ injuries and improves survival in a murine model of polymicrobial sepsis.

OBJECTIVE: The endotoxin tolerance phenotype is characterized with decreased inflammation and increased phagocytosis. We hypothesized that endotoxin tolerance would provide protective effects on experimental sepsis with multiple organ injuries induced by cecal ligation and puncture (CLP). METHODS: Endotoxin tolerance was induced in male Sprague-Dawley rats with daily intraperitoneal injection of either 0.6 mg/kg of lipopolysaccharide (LPS) or vehicle for four consecutive days before subsequent CLP. Biochemical parameters, histological changes, inflammatory cytokine production, and lung tissue nuclear factor-κB (NF-κB) activation were assessed post-CLP. In a separate experiment, survival rate was monitored for 7 days after CLP. RESULTS: In vehicle-treated animals, CLP caused multiple organ injuries confirmed by the biochemical variables and histological examination. This was accompanied by an early activation of NF-κB in the lung and a substantial increase in plasma levels of tumor necrosis factor-α, interleukin-6, and interleukin-10. In contrast, pretreatment with LPS not only alleviated the development of multiple organ injuries after CLP, but also decreased sepsis-induced activation of pulmonary NF-κB and reduced plasma cytokines production. In addition, LPS pretreatment improved the survival in rats subjected to CLP. CONCLUSIONS: The beneficial effects of endotoxin tolerance indicate the potential of immunomodulatory strategies in the management of severe sepsis.

[Predictors of peri-operative mortality in patients with aortic dissection].

OBJECTIVE: To analyze the peri-operative risk factors of mortality in patients with aortic dissection (AD). METHODS: Between January 2003 and June 2008, 361 AD patients at our hospital were enrolled. Their demographics, history, clinical characteristics and laboratory examinations were retrospectively analyzed. Twenty pre-operative variables were analyzed to identify the predictors of perioperative mortality of AD patients by the analyses of univariate and multivariate logistic regression. RESULTS: The analysis of univariate logistic regression showed that history of hypertension [odds ratio (OR) 0.465, 95% confidence interval (CI) 0.229 - 0.947, P = 0.035], Stanford type A (OR 2.758, 95%CI 1.054 - 7.213 P = 0.039), acute course (OR 7.897, 95%CI 1.874 - 33.275 P = 0.005), neurological symptoms (OR 0.275, 95%CI 0.140 - 0.541, P < 0.001) and operation or not (OR 8.206, 95%CI 4.205 - 16.012, P < 0.001) had a higher mortality in AD patients. The multivariate analysis revealed that acute course (OR 8.178, 95%CI 1.796 - 37.242, P = 0.007), Stanford type A (OR 3.236, 95%CI 1.104-9.487 P = 0.032), neurological symptoms (OR 0.350, 95%CI 0.159 - 0.770, P = 0.009) and operation or not (OR 9.429, 95%CI 4.456 - 19.952, P < 0.001) were significant independent predictors of perioperative mortality in AD patients. CONCLUSION: History of hypertension, acute course, Stanford A and positive neurological symptoms are the independent predictors of perioperative mortality in AD patients. Operation or not is a determinant of patient outcome.

TMT-Based Quantitative Proteomic Analysis Identification of Integrin Alpha 3 and Integrin Alpha 5 as Novel Biomarkers in Pathogenesis of Acute Aortic Dissection.

BACKGROUND: Acute aortic dissection (AAD) is a devastating cardiovascular disease with a high rate of disability and mortality. This disease often rapidly progresses to fatal multiple organ hypoperfusion, and the incidence has been increasing in recent years. However, the molecular mechanisms have yet to be clarified. This study is aimed at identifying the differential abundance proteins (DAPs) of aortic arch tissues in patients with AAD by proteomics and select possible proteins involved in AAD pathogenesis. METHODS: The fresh aortic arch tissues obtained from 5 AAD patients and 1 healthy donor were analyzed by amine-reactive tandem mass tag (TMT) labelling and mass spectrometry; then, the pathological sections of another 10 healthy donors and 20 AAD patients were chosen to verify the proteomic results by immunohistochemistry. RESULTS: Of 809 proteins identified by proteomic analysis, 132 differential abundance proteins (DAPs) were screened, of which 100 proteins were significantly downregulated while 32 upregulated. Among 100 downregulated proteins, two proteins with known function, integrin alpha 3 (ITGA-3) and ITGA-5, were selected as target proteins involved in AAD pathogenesis. Two target DAPs were verified by immunohistochemisty, and the results showed that the integrated option density (IOD) of ITGA-3 and ITGA-5 in AAD patients was significantly lower than that in healthy donors, which were consistent with the proteomic results (P < 0.001). CONCLUSION: ITGA-3 and ITGA-5 represent novel biomarkers for the pathogenesis of AAD and might be a therapeutic target in the future.

p38 MAPK inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of PMN accumulation.

BACKGROUND: In vitro evidence suggests that the p38 mitogen-activated protein kinase (p38 MAPK) plays a crucial role in PMN activation and inflammatory cytokine production. However, the effect of p38 MAPK on myocardial reperfusion injury, a pathologic condition involving a typical inflammatory response, has not been fully examined. In the present study, we investigated the effect of SB 239063, a specific p38 MAPK inhibitor, on myocardial injury in a murine ischemia/reperfusion (I/R) model and elucidated the mechanism by which p38 MAPK inhibitor may exert its protective effect against I/R injury. METHODS AND RESULTS: I/R resulted in a significant myocardial injury (myocardial infarct 45 +/- 2.9%) and marked PMN accumulation (myeloperoxidase activity 1.03 +/- 0.16 U/100 g tissue). Administration of SB 239063 significantly inhibited the myocardial inflammatory response as evidenced by reduced PMN accumulation in I/R myocardial tissue (0.62 +/- 0.008 U/100 g tissue, P<0.01 vs. vehicle), and markedly attenuated myocardial reperfusion injury (myocardial infarct size: 28 +/- 2.4%, P<0.01 vs. vehicle). Moreover, treatment with SB 239063 significantly attenuated I/R-induced P-selectin and ICAM-1 upregulation (13.8 +/- 2.7 vs. 23.9 +/- 3.1%, and 29.4 +/- 1.6 vs. 56.3 +/- 4.8%, respectively P<0.01). In addition, pre-treatment with R15.7, a monoclonal antibody against CD 18 adhesion molecule on PMN surface that virtually abolished PMN accumulation in ischemic-reperfused myocardial tissue, significantly, but not completely, blocked the cardioprotection exerted by SB 239063. CONCLUSION: These results demonstrated for the first time that p38 MAPK activation plays a significant role in adhesion molecule upregulation on ischemia-reperfused endothelial cells and is an important signaling step in the pathogenesis of PMN-mediated tissue injury.

[Effects of glucose-insulin-potassium cocktail on cardiac myocyte death and post-ischemic recovery cardiac functional recovery: the critical role of insulin].

OBJECTIVE: To study the effect of glucose-insulin-potassium (GIK) cocktail on cardiac myocyte death (i.e., necrosis and apoptosis) and cardiac functional recovery following myocardial ischemia/reperfusion (MI/R), and to further investigate the role of insulin in the GIK-induced cardioprotective effect. METHODS: Male Sprague-Dawley rats were subjected to 30 min myocardial ischemia followed by reperfusion for 4 h (for cardiac function and cardiomyocyte apoptosis study) or 6 h (for myocardial infarction measurement). Anesthetized rats were randomly treated with continuous infusions of saline, GIK (Glucose: 200 g/L, Insulin: 60 U/L and KCl: 60 mmol/L), GK or insulin at 4 ml.kg(-1).h(-1), beginning 5 min before reperfusion and continuing through the 4-h reperfusion. Arterial blood pressure, ECG and left ventricular pressure were monitored throughout the experiment. Myocardial DNA fragmentation and myocardial infarction were determined at the end of reperfusion. RESULTS: MI/R caused significant cardiac dysfunction and myocardial death (both necrosis and strong DNA ladder formation). Compared with the vehicle treated rats, the GIK-treated rats showed protection against MI/R injury as evidenced by reduced myocardial infarction [(41.3 +/- 8.3)% vs. (54.4 +/- 10.4)% of vehicle, P < 0.05, n = 10], marked decrease of DNA fragmentation, and improved recovery of cardiac systolic/diastolic function at the end of reperfusion [left ventricular developed pressure: (94 +/- 6) mm Hg vs. (86 +/- 5) mm Hg of vehicle, P < 0.05; +LVdP/dt(max): (2 940 +/- 114) mm Hg/s vs. (2 733 +/- 132) mm Hg/s, P < 0.05; -LVdP/dt(max): (2 629 +/- 156) mm Hg/s vs. (2 463 +/- 133) mm Hg/s, P < 0.05]. Insulin exerted the similar cardioprotective effects with GIK; whereas the rats receiving GK failed to show any significant, cardioprotection against MI/R injury. CONCLUSIONS: GIK exerts cardioprotective effect against postischemic myocardial injury. Insulin, mainly through its anti-apoptotic effect, plays a critical role in the GIK-elicited myocardial protection in MI/R.

[Anti-apoptotic effect of insulin in myocardial ischemia-reperfusion and its principal signaling pathway].

OBJECTIVE: To investigate the signaling pathway involved in the insulin-elicited anti-apoptotic effect during myocardial ischemia and reperfusion (MI/R) in vivo. METHODS: Male Sprague-Dawley rats were anesthetized and subjected to 30 min of myocardial ischemia followed by 4h-reperfusion. Rats were randomly treated with intravenous infusion of saline (vehicle, 4 ml.kg(-1).h(-1)), insulin (60 U/L), or insulin + wortmannin 5 min before reperfusion and continuing throughout the 4h-reperfusion period. Cardiac myocyte apoptosis was determined both qualitatively and quantitatively by DNA laddering and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) methods. Myocardial nitric oxide (NO) was measured by using NO-specific chemiluminescence detector. Activations of Akt and p38 mitogen-activated protein kinase (MAPK) were determined by kinase activity assays using corresponding kinase activity assay kits (Cell Signaling). RESULTS: In the vehicle-treated rats, MI/R caused significant cardiac myocyte apoptotic death. Treatment with insulin produced a significant anti-apoptotic effect as evidenced by a marked reduction of apoptotic index [(8.0 +/- 2.9)% vs. (19.3 +/- 4.6)% of vehicle, P < 0.01] and decreased formation of myocardial DNA fragmentation. In addition, insulin treatment produced 2.7-fold increase (P < 0.01) of myocardial Akt activity and 28% increase of myocardial NO production (P < 0.05), while p38 MAPK activity changed insignificantly as compared with that of vehicle (P > 0.05). Both insulin-induced Akt activation and anti-apoptotic effect could be abrogated by wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor. CONCLUSION: In vivo treatment with insulin at the initial of reperfusion significantly reduced postischemic apoptotic death via the PI3-kinase-Akt signaling pathway. Akt, but not p38 MAPK, activation plays a key role in the insulin-induced anti-apoptotic effect in MI/R.

Complementary and alternative medicine is expected to make greater contribution in controlling the prevalence of influenza.

Influenza pandemics are a serious threat to public health in today's world. In the past 10 years, the outbreak of three forms of severe influenza--H5N1, H1N1, and H7N9--has caused tremendous loss of life and property. In order to better cope with pandemics, antivirals such as oseltamivir are being stockpiled in great quantities, placing a substantial burden on government budgets and potentially resulting in massive waste because of the uncertainty as to when an influenza pandemic will strike and whether emerging virus strains will be resistant to the stockpiled drugs. Complementary and alternative medicine (CAM) is generally available, affordable, and commonly used in China and many other countries and CAM has a long track record of fighting influenza. The Chinese Government appropriated funds to intensively investigate herbal medicines in accordance with the principles of evidence-based medicine in order to identify effective, inexpensive, and easily stockpiled medicines. Thus far, several drugs including Shufeng Jiedu capsules, Lianhua Qingwen capsules, Maxing Shigan decoction, Yinqiao powder, and Jinhua Qinggan granules have demonstrated effectiveness in fighting influenza. In the future, CAM is expected to make greater contribution in controlling the prevalence of influenza pandemics.