脓毒症患者血清H2S、GDF-15、PTX-3水平与凝血功能、炎症指标及病情评分的相关性分析

摘要 目的：探讨脓毒症患者血清硫化氢（H₂S）、生长分化因子-15（GDF-15）、穿透素-3（PTX-3）水平与其凝血功能、炎症指标及病情评分的相关性。方法：随机选取我院2018年2月～2020年2月收治的脓毒症患者52例作为脓毒症组,另选取我院同期收治的脓毒症休克患者46例作为休克组以及同期于我院进行体检的健康者50例作为对照组。检测三组血清H₂S、GDF-15、PTX-3水平以及凝血功能、炎症指标,其中凝血功能指标包括血小板计数（PLT）、部分凝血活酶时间（APTT）、凝血酶原时间（PT）、纤维蛋白原（FIB）。炎症指标包括降钙素原（PCT）、C反应蛋白（CRP）。采用急性生理学和慢性健康状况评分系统Ⅱ（APACHEⅡ）、快速序贯器官功能（qSOFA）评分对脓毒症组、休克组患者病情进行评分。分析血清H2S、GDF-15、PTX-3与患者凝血功能、炎症指标及病情评分的相关性。结果：脓毒症组、休克组的血清H₂S、PLT均低于对照组,且休克组低于脓毒症组（P＜0.05）。脓毒症组、休克组的血清GDF-15、PTX-3、APTT、PT、FIB、PCT、CRP均高于对照组,且休克组高于脓毒症组（P＜0.05）。脓毒症组APACHEⅡ、qSOFA评分均显著低于休克组（P＜0.05）。血清H2S与PLT呈正相关（P＜0.05）,与APTT、PT、FIB、PCT、CRP、APACHEⅡ评分、qSOFA评分呈负相关（P＜0.05）。血清GDF-15、PTX-3与PLT呈负相关（P＜0.05）,与APTT、PT、FIB、PCT、CRP、APACHEⅡ评分、qSOFA评分呈正相关（P＜0.05）。结论：脓毒症患者的血清H₂S明显下降,而血清GDF-15、PTX-3增高,三者与凝血功能、炎症以及病情评分均存在密切关联,这可能是影响脓毒症进展的重要原因之一。     

Tbc1d15–17 regulates synaptic development at the Drosophila neuromuscular junction

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15–17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15–17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15–17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15–17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15–17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15–17 and its potential substrate Rab7 in regulating synaptic development.     

Cellular mechanisms and intracellular signaling pathways for the modulation of eNOS in pulmonary arteries by 15-HETE

The 15-hydroxyeicosatetraenoic acid (15-HETE), a lipid metabolite and vasoconstrictor, plays an important role in hypoxic contraction of pulmonary arteries (PAs) through working on smooth muscle cells (SMCs). Previous studies have shown that vascular endothelium is also involved in PAs tone regulation. However, little is known as to how the pulmonary artery endothelial cells (PAECs) are related to the 15-HETE-induced vasoconstriction and that which intracellular signaling systems are critical. To test this hypothesis, we examined PAs constriction in isolated rat PAs rings, the expression and activity of endothelial nitric oxide synthase (eNOS) with western blot, and nitric oxide (NO) production using the DAF-FM DA fluorescent indicator. The results showed that the 15-HETE-induced PAs constriction was diminished in endothelium-intact rings. In the presence of the eNOS inhibitor L-NAME, vasoconstrictor responses to KCl were greater than the control. The activation of eNOS was activated by Ca²⁺ released from intracellular stores and the PI3K/Akt pathway. Phosphorylations of the eNOS at Ser-1177 and Akt at Ser-473 were necessary for their activity. A prolonged 15-HETE treatment (30?min) led to a decrease in NO production by phosphorylation of eNOS at Thr-495, leading to augmentation of PAs constriction. Therefore, 15-HETE initially inhibited the PAs constriction through the endothelial NO system, and both Ca²⁺ and the PI3K/Akt signaling systems are required for the effects of 15-HETE on PAs tone regulation.     

Macrophage inhibitory cytokine‐1 is associated with cognitive impairment and predicts cognitive decline – the Sydney Memory and Aging Study

Higher levels of macrophage inhibitory cytokine‐1, also known as growth differentiation factor 15 (MIC‐1/GDF15), are associated with adverse health outcomes and all‐cause mortality. The aim of this study was to examine the relationships between MIC‐1/GDF15 serum levels and global cognition, five cognitive domains, and mild cognitive impairment (MCI), at baseline (Wave 1) and prospectively at 2 years (Wave 2), in nondemented participants aged 70–90 years. Analyses were controlled for age, sex, education, Framingham risk score, history of cerebrovascular accident, acute myocardial infarction, angina, cancer, depression, C‐reactive protein, tumor necrosis factor‐α, interleukins 6 and 12, and apolipoprotein ε4 genotype. Higher MIC‐1/GDF15 levels were significantly associated with lower global cognition at both waves. Cross‐sectional associations were found between MIC‐1/GDF15 and all cognitive domains in Wave 1 (all P < 0.001) and between processing speed, memory, and executive function in Wave 2 (all P < 0.001). Only a trend was found for the prospective analyses, individuals with high MIC‐1/GDF15 at baseline declined in global cognition, executive function, memory, and processing speed. However, when categorizing MIC‐1/GDF15 by tertiles, prospective analyses revealed statistically significant lower memory and executive function in Wave 2 in those in the upper tertile compared with the lower tertile. Receiver operating characteristics (ROC) analysis was used to determine MIC‐1/GDF15 cutoff values associated with cognitive decline and showed that a MIC‐1/GDF15 level exceeding 2764 pg/ml was associated with a 20% chance of decline from normal to MCI or dementia. In summary, MIC‐1/GDF15 levels are associated with cognitive performance and cognitive decline. Further research is required to determine the pathophysiology of this relationship.     

Increased dosage of Ink4/Arf protects against glucose intolerance and insulin resistance associated with aging

Recent genome‐wide association studies have linked type‐2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up‐regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type‐2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age‐dependent alterations of insulin and glucose homeostasis using Super‐Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age‐matched wild‐type controls, Super‐Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super‐Ink4/Arf compared with wild‐type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)‐PI3K‐AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super‐Ink4/Arf mice showed a tendency toward increased ¹⁸F‐fluorodeoxyglucose uptake in skeletal muscle compared with wild‐type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super‐Ink4/Arf mice (P < 0.008) but not in wild‐type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age‐dependent insulin resistance and glucose intolerance.     

Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

A PPAR-γ ligand, 15-deoxy-Δ12,14-prostaglandin J2, inhibited gastric mucosal injury induced by ischemia–reperfusion in rats

Abstract

Introduction: Recent studies have demonstrated the anti-inflammatory action of 15-deoxy-Δ12,14-prostaglandin J₂ (15d-PGJ₂), a derivative of the PGD₂ metabolic pathway. Acute inflammation, including neutrophil activation, plays a critical role in the pathogenesis of ischemia–reperfusion (I/R). The aim of the present study was to determine the effect of 15d-PGJ₂ on I/R-induced gastric mucosal injury in rats.

Methods: Gastric mucosal damage was induced in male Wistar rats by clamping the celiac artery for 30 min followed by reperfusion. 15d-PGJ₂ (0.01–1.0 mg/kg) was given to the rats intraperitoneally 1 h before the vascular clamping. The area of gastric mucosal erosions (erosion index) was measured. Thiobarbituric acid reactive substances (TBARS) and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expression of tumor necrosis factor-α (TNF-α) in gastric mucosa was measured by ELISA. In addition, to elucidate whether the protective effects of 15d-PGJ₂ are related to the activation of the PPAR-γ receptor, we also investigated the effects of a PPAR-γ antagonist, GW9662.

Results: After 60 min of reperfusion, the area of gastric erosion index had significantly increased from the mean basal levels. The increase in the erosion index was significantly inhibited by pretreatment with 15d-PGJ₂ in a dose-dependent manner. On the other hand, GW9662 reversed the protective effect of 15d-PGJ₂. The concentration of TBARS and MPO activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with 15d-PGJ₂ significantly reduced these increases. The TNF-α content was significantly higher in the I/R group than in the sham-operated group. However, the increase in TNF-α was significantly inhibited by pretreatment with 15d-PGJ₂.

Conclusions: 15d-PGJ₂ significantly inhibited the severity of acute gastric mucosal injury induced by I/R in rats through PPAR-γ-dependent mechanisms. This effect may be due, in part, to a reduction in the infiltration of neutrophils into the gastric mucosa, possibly via the inhibition of inflammatory cytokine.     

Apparent superoxide dismutase-like activity of immunoglobulin

Summary

Using various superoxide generating systems and nitroblue tetrazolium or cytochrome c as superoxide detector molecules it is possible to assess the superoxide dismutase activity of proteins. Intact antibodies raised to different antigens, the Fab’ fragment of anti-TNF [M632] and well-characterized recombinant Fv fragments of the murine antibody NQ11.7.22 appear to possess superoxide dismutase (SOD)-like activity.

Kinetic characteristics of the SOD-like activity of NQ11.7.22-Fv fragments suggest an enzymatic property and these fragments behave in an analogous manner to human erythrocyte Cu-Zn SOD. Furthermore, the SOD-like activity of the NQ11.7.22-Fv fragment is affected by certain single-point mutations in the amino acid composition and has a pH optimum of 6.2–6.6 which is unlike Cu-Zn SOD (pH 7.8–8.2). A change in tyrosine at the 32 position in the heavy chain and histidine at position 27 of the light chain of the NQ11.7.22-Fv fragment results in a profound reduction in SOD-like activity. Tyrosine at the 32 position in the heavy chain is known to play a significant role in antigen binding suggesting that the SOD-like activity occurs at the antigen-binding site itself. Single-point mutations at the periphery of the antigen combining site on the NQ11.7.22-Fv fragment had little or no effect on SOD-like activity.

Further studies show that immunoglobin (lgG-1), a commercially available murine monoclonal antibody, can also enhance the generation of hydrogen peroxide, the product of superoxide dismutation, when present in superoxide producing systems. The generation of hydrogen peroxide was increased by low pH (pH 6.25) with lgG-1 but reduced with Cu-Zn SOD.     

Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

Rituximab (RTX), a chimeric IgG1 monoclonal antibody directed against the CD20 antigen, has revolutionized the treatment of B-cell malignancies. Nevertheless, the relapsed/refractory rates are still high. One strategy to increase the clinical effectiveness of RTX is based on antibody-cytokine fusion protein (immunocytokine; ICK) vectorizing together at the tumor site the antibody effector activities and the cytokine co-signal required for the generation of cytotoxic cellular immunity. Such ICKs linking various antibody formats to interleukin (IL)-2 are currently being investigated in clinical trials and have shown promising results in cancer therapies. IL-15, a structurally-related cytokine, is now considered as having a better potential than IL-2 in antitumor immunotherapeutic strategies. We have previously engineered the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. Compared with IL-15, RLI displayed better biological activities in vitro and higher antitumor effects in vivo in murine and human cancer models. In this study, we investigated the advantages of fusing RLI to RTX. Anti-CD20-RLI kept its binding capacity to CD20, CD16 and IL-15 receptor and therefore fully retained both antibody effector functions (ADCC and CDC), and the cytokine potential of RLI. In a severe combined immunodeficiency (SCID) mouse model of disseminated residual lymphoma, anti-CD20-RLI was found to induce long-term survival of 90% of mice up to at least 120 days whereas RLI and RTX, alone or in combination, just delayed the disease onset (100% of death at 28, 40 and 51 days respectively). These findings suggest that such ICK could improve the clinical efficacy of RTX, particularly in patients with refractory B-cell lymphoma.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.