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P–n junctions enhance the anti-infection ability of micro-pyramid structured surfaces. The synergetic effect of mechanical rupture and electric injury is supposed to be responsible for the potent antibacterial activity.

Neutrophils release neutrophil extracellular traps (NETs) to capture and kill pathogens, but excessive NET release can damage the surrounding tissues. Myeloperoxidase (MPO) and neutrophil elastase (NE) are thought to be important in promoting histone depolymerization and DNA breakage in the nucleus. However, the detailed path by which MPO and NE enter the nucleus is unknown. In the present study, we observed that delayed fusion of azurophilic granules with the nuclear membrane 15-20 min after extracellular degranulation in activated neutrophils. In a subsequent experiment, we further demonstrated that this fusion leads to MPO entry into the nucleus and promotes nuclear histone depolymerization and DNA breakage, a process called 'targeted nuclear degranulation'. This process can be effectively inhibited by dexamethasone and accompanied by the continuous low levels of MPO in the nucleus after PMA stimulation. Meanwhile, we found that 'targeted nuclear degranulation' is dependent on the CD44 translocation and subsequent redistribution of CD44 / ERM (Ezrin/Radixin/Moesin) / F-actin complexes, which guides the movement of azurophilic granules towards the nucleus. Application of ERM phosphorylation inhibitors and importin activity inhibitors significantly reduced the complexes formation and redistribution. Taken together, these findings indicate for the first time that delayed 'targeted nuclear degranulation' after neutrophil activation is a key mechanism of NET formation. CD44/ERM/F-actin complex mediates this process, which providing targets with promising prospects for the precise regulation of NET formation.

Background Early detection, timely diagnosis and rapid response are essential for case management and precautions of burn-associated sepsis. However, studies on indicators for early warning and intervention have rarely been conducted. This study was performed to better understand the pathophysiological changes and targets for prevention of severe burn injuries. Methods We conducted a multi-center, prospective multi-omics study, including genomics, microRNAomics, proteomics and single-cell transcriptomics, in 60 patients with severe burn injuries. A mouse model of severe burn injuries was also constructed to verify the early warning ability and therapeutic effects of potential markers. Results Through genomic analysis, we identified seven important susceptibility genes (DNAH11, LAMA2, ABCA2, ZFAND4, CEP290, MUC20 and ENTPD1) in patients with severe burn injuries complicated with sepsis. Through plasma miRNAomics studies, we identified four miRNAs (hsa-miR-16-5p, hsa-miR-185-5p, hsa-miR-451a and hsa-miR-423-5p) that may serve as early warning markers of burn-associated sepsis. A proteomic study indicated the changes in abundance of major proteins at different time points after severe burn injury and revealed the candidate early warning markers S100A8 and SERPINA10. In addition, the proteomic analysis indicated that neutrophils play an important role in the pathogenesis of severe burn injuries, as also supported by findings from single-cell transcriptome sequencing of neutrophils. Through further studies on severely burned mice, we determined that S100A8 is also a potential early therapeutic target for severe burn injuries, beyond being an early warning indicator. Conclusions Our multi-omics study identified seven susceptibility genes, four miRNAs and two proteins as early warning markers for severe burn-associated sepsis. In severe burn-associated sepsis, the protein S100A8 has both warning and therapeutic effects.

Extensive necrosis of lower extremity muscles through the clinical practice of limb salvage after a case of high level crushing injury. A case of car accident injury complicated with necrotizing fasciitis, myositis, and septic shock was admitted to our hospital. The pathogenic factors, clinical characteristics, and surgical repair of this case were analyzed. Septic shock, pulmonary infection and atelectasis, and skin and soft tissue injury of lower limbs were all effectively treated, and limbs were saved successfully. After wound healing, the patient was discharged from hospital and entered the follow-up rehabilitation treatment. Although there is no direct trauma to the distal extremity below the cross-section of both lower extremities, there are still hypoxic ischemic changes, which can easily be ignored in the early stage. If not treated in time, myofascial and osteofascial compartment syndrome, necrotizing fasciitis, myositis, and sepsis are often secondary in the later stage, which should be warned against. During surgical debridement, attention should be paid to the protection of the source artery, and debridement and surgical exploration should be carried out according to the trend of blood vessels. The interecological muscle tissue between the intersections should be kept as far as possible, and the main nerves, blood vessels, and musculocutaneous perforators should be kept to ensure the blood supply of the skin flap.

To assess the safety and efficacy of antimicrobial peptide PL-5 (Peceleganan) spray in the treatment of wound infections.Antimicrobial peptide PL-5 spray is a novel topical antimicrobial agent.We conducted a multicenter, open-label, randomized, controlled phase IIb clinical trial to evaluate the efficacy and safety of PL-5 spray, as compared with silver sulfadiazine, in patients with skin wound infections. The primary efficacy outcome was the clinical efficacy rate on the first day after ending the treatment (D8). The secondary efficacy outcome was the clinical efficacy rate on the fifth day posttreatment (D5), the bacteria clearance rate, and the overall efficacy rate at the mentioned 2 time points. The safety outcomes included adverse reactions and pharmacokinetic analysis posttreatment.A total of 220 patients from 27 hospitals in China were randomly assigned to 4 groups. On D8, the efficacy rate was 100.0%, 96.7%, 96.7% for the 1‰ PL-5, 2‰ PL-5, 4‰ PL-5 groups, respectively, as compared with 87.5% for the control group. The efficacy rate among the 4 groups was significantly different (P0.05). On D5, the efficacy rate was 100.0%, 93.4%, 98.3% for the 1‰ PL-5, 2‰ PL-5, 4‰ PL-5 groups, respectively, as compared with 82.5% for the control group. The efficacy rate among the 4 groups was significantly different (P0.05). The blood concentration of PL-5 was not detectable in pharmacokinetic analysis. No severe adverse event related to the application of PL-5 was reported.Antimicrobial peptide PL-5 spray is safe and effective for the treatment of skin wound infections.ChiCTR2000033334.

Objective This article aims to pay attention to the latest research on the expression, activation and function of hypoxia-inducible factor-2α (HIF-2α) under hypoxia and non-hypoxia conditions, and summarizes the current knowledge about the interaction between hypoxia-inducible factor-2 and angiogenesis, hoping to understand its actions in physiology and disease, with the goal of providing a new strategy for the diagnosis and treatment of wounds. Background Wound healing is a complex and continuous process, involving coagulation, inflammation, angiogenesis, new tissue formation and extracellular matrix remodeling. Of these, angiogenesis is an essential step. One of the main reasons for non-healing or delayed healing of wounds in peripheral vascular diseases and diabetes is the reduced ability to regenerate microvessels through the process of angiogenesis, which has become the focus of new methods for treating chronic wounds. HIF-2α regulates many aspects of angiogenesis, including vascular maturation, cell migration, proliferation and metastasis. Methods Throughout extensive search of PubMed, summarize the medical research on HIF-2α to 2020. Conclusions HIF-2α is necessary for normal embryonic development by stimulating the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF). It is essential for the formation of new blood vessels in physiological and pathophysiological environments. Targeting HIF-2α in wound healing has much clinical significance for tissue repair.

Background Inflammation is an important factor in pathological scarring. The role of neutrophils, one of the most important inflammatory cells, in scar hyperplasia remains unclear. The purpose of this article is to study the correlation between neutrophil extracellular traps (NETs) and scar hyperplasia and identify a new target for inhibiting scar hyperplasia. Methods Neutrophils were isolated from human peripheral blood by magnetic-bead sorting. NETs in plasma and scars were detected by enzyme-linked immunosorbent assays (ELISAs), immunofluorescence and flow cytometry. Immunohistochemistry was used to assess neutrophil (CD66B) infiltration in hypertrophic scars. To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry. Results We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs (p 0.05]. However, inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts into myofibroblasts. In vitro, NETs were found to be mediated by Toll-like receptor 9 (TLR-9) in fibroblasts and further phosphorylated nuclear factor Kappa-B (NF-κB). We found that IL-6, which is downstream of NF-κB, was increased in fibroblasts. Additionally, IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion. Conclusions Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway, thereby providing a new target for regulating hypertrophic scars.

To reveal the systematic response of neutrophils to sepsis and to study the hub lncRNAs in sepsis. Neutrophils taken from the femur and tibia of male C57 BL/6 mice were used in this study. And neutrophils were treated for 0 h, 0.5 h, 1 h, and 4 h with or without 1 µg/mL lipopolysaccharide (LPS) for further chip detection. In addition, cecal ligation and perforation were used to simulate sepsis. Here, we used different bioinformatics analyses, including differential expression analysis, weighted gene co-expression network analysis (WGCNA), and gene regulatory network analysis, to analyze the systemic response of neutrophils to sepsis. We identified nine modules and found hub lncRNAs in each module. The blue and pink modules were closely related to the inflammatory state of sepsis. Some hub lncRNAs (NONMMUT005259, KnowTID_00004196, and NR_003507) may have functions related to the inflammatory state in sepsis. Based on a new biological approach, our research results revealed the systemic-level response of neutrophils to sepsis and identified several hub lncRNAs with potential regulatory effects on this condition.

Background Low-density neutrophils (LDNs) have been described in tumors and various autoimmune diseases, where they exhibit immune dysfunction and alter disease progression. Nevertheless, LDNs have been rarely reported in sepsis. Methods We studied sepsis patients admitted to the intensive care unit. WrightGiemsa stain assay and Transmission electron microscopy were performed to detect the morphology of neutrophils. Flow cytometry was used to analyze the number and function of LDNs. Concentration of cytokines was measured using ELISA. Neutrophil chemotaxis was examined using an under-agarose chemotaxis model. Results We found that LDNs were significantly elevated in patients with sepsis. Phenotypes and morphological characteristics suggest that LDNs may be formed by mixtures of neutrophils at various maturation stages. In vitro experiments showed that LDN formation was closely associated with neutrophil degranulation. We preliminarily discussed changes in immune function in LDNs. Compared with high-density neutrophils, expression levels of CXC chemokine receptor 4 on LDN surfaces were increased, phagocytotic capacity was decreased, and life span was prolonged. The chemotactic ability of LDNs was significantly reduced, possibly related to the increased expression of P2X1. Conclusions These data suggest that LDNs are essential components of neutrophils in sepsis. To clarify the source and dysfunction mechanism of LDN in sepsis may be helpful for the diagnosis and treatment of sepsis in the future.

To investigate the effect of neutrophils on T lymphocyte function in septic mice and the role of CD80/cytotoxic T lymphocyte antigen-4 (CTLA-4) signaling pathway in this modulated effects.(1) In vivo experiment: 6-8 weeks old male C57BL/6 mice were divided into sham operation group (Sham group, n = 20), Sham+CTLA-4 antibody treatment group (Sham+aCTLA-4 group, n = 20), cecum ligation and perforation (CLP) induced sepsis model group (CLP group, n = 30) and CLP+CTLA-4 antibody treatment group (CLP+aCTLA-4 group, n = 30) according to the random number table. CLP was used to reproduce mouse sepsis model. The mice in the Sham group were treated identically but their cecums were neither punctured nor ligated. In CTLA-4 antibody treatment groups, 50 μg CTLA-4 antibody was injected intraperitoneally 6 hours and 24 hours after the operation. Forty-eight hours after operation, 6 mice in Sham group and Sham+aCTLA-4 group, 14 mice in CLP group and CLP+aCTLA-4 group were randomly selected to detect the expression of CD69 in spleen. At the same time, spleen, bone marrow and peripheral blood were collected, and the expression of CD80 on neutrophils was detected by flow cytometry. The expression of CTLA-4 on the surface of T lymphocytes in spleen was detected by immunofluorescence and flow cytometry. The remaining mice in each group were used to observe the 96-hour survival after operation. (2) In vitro experiment 1: neutrophils were extracted from bone marrow of healthy mice and stimulated with LPS (1 mg/L) for 4, 8 and 12 hours respectively. The control group was added with the same amount of phosphate buffer saline (PBS) at each time point, and the expression of CD80 was detected at each time point. (3) In vitro experiment 2: splenic T lymphocytes of healthy mice were extracted and divided into PBS control group, LPS group (final concentration of LPS 1 mg/L), neutrophil group and neutrophil+LPS group. In the latter two groups, the co-culture model of neutrophils and T lymphocytes was established, and then the corresponding treatment was given to detect the expression of CTLA-4 on the surface of T lymphocytes. With the above four groups as controls, CTLA-4 antibody treatment groups (final concentration of CTLA-4 antibody 50 mg/L) were set up respectively. After 48 hours, the level of interleukin-2 (IL-2) in the cell supernatant was detected by enzyme linked immunosorbent assay (ELISA).(1) Results of in vivo experiment: compared with Sham group, the expression of CD80 on neutrophils in spleen, bone marrow and peripheral blood was significantly up-regulated, while the expression of CTLA-4 on the surface of T lymphocytes was significantly increased [(9.98±0.84)% vs. (3.48±0.64)%, P0.05]. It suggested that neutrophils may affect T lymphocytes function through CD80/CTLA-4 pathway in sepsis. Compared with CLP group, CTLA-4 antibody could significantly improve the 96-hour cumulative survival rate of CLP mice (56.25% vs. 18.75%, P0.05), and increase the expression of CD69 on the surface of T lymphocytes. It suggested that CTLA-4 antibodies might increase T lymphocytes activation in sepsis and improve survival. (2) Results of in vitro experiment: with the prolongation of LPS stimulation, the expression of CD80 on neutrophils gradually increased in time-dependent manner as compared with PBS control group [4 hours: (6.35±0.40)% vs. (3.41±0.40)%, 8 hours: (8.57±0.64)% vs. (3.09±0.27)%, 12 hours: (19.83±1.06)% vs. (5.16±0.36)%, all P0.05]. Compared with PBS control group, the expression of CTLA-4 on CD4Neutrophils mediate T lymphocytes dysfunction in sepsis, and the CD80/CTLA-4 pathway plays an important role. The CTLA-4 antibody improves survival and T lymphocytes function in sepsis mice, which may be a new method of immunotherapy for sepsis.

Background Burn shock caused by vascular leakage is one of the main causes of high mortality in severe burn injury. However, the pathophysiological mechanism of vascular leakage is still unclear. The purpose of this study was to explore the molecular mechanism of vascular leakage in the early stage of severe burn and provide a new target for the treatment of severe burns. Methods Neutrophils were isolated from human peripheral blood by magnetic beads sorting. ELISA was used to detect neutrophil-derived granule proteins and glycocalyx injury products in plasma. The vascular leakage and neutrophil movement were assessed by in vivo laser confocal imaging in mice, and high-quality video were provided. Adhesion-related molecules were investigated by qRT-PCR. The damage to glycocalyx of mice vascular endothelial cells was observed by transmission electron microscope and scanning electron microscope. Proteomic analysis, flow cytometry and immunofluorescence were used to further study the relationship between human peripheral blood neutrophil-derived hypochlorite (HOCl) and CD44 of human vascular endothelial cells. Results In this study, we found that rapidly increasing activated neutrophils secrete heparin binding protein (HBP) and myeloperoxidase (MPO) after severe burn injury. Increased HBP triggers vascular leakage with synergy of MPO, results in systemic edema and burn shock. Furthermore, we found that the MPO catalytic product HOCl but not MPO triggers CD44 extracellular domain shedding from vascular endothelial cells to damage the glycocalyx. Damage to the glycocalyx results in firm adhesion of neutrophils and increases vascular leakage. However, MPO inhibitors partially protect the glycocalyx of vascular endothelial cells. The combination of HBP and MPO inhibitors markedly reduces vascular leakage and systemic edema in the early stage of severe burns. Conclusions Taken together, these data reveal that neutrophil-derived HBP and MPO play an important synergies role in triggering vascular leakage at the early stage of severe burns. Targeted intervention in these two biomolecules may introduce new strategies for helping to reduce large amount of fluid loss and subsequent burn shock.

Objectives To evaluate the value of chemotactic function of neutrophils in patients with severe infections. Methods A computer vision‐based cellular chemotaxis analysis platform was established for the dynamic assessment of neutrophil chemotaxis. Fifty‐three patients in the intensive care unit were eligible for the study. In parallel, 142 healthy volunteers were recruited to detect and establish the normal values for chemotactic function. Four chemotactic function indicators were determined–chemotaxis distance (CD), chemotaxis cell ratio (CCR), chemotaxis index (CI) and maximum speed of chemotaxis (Vmax). The chemotaxis function scores (CFS) were calculated for further correlation analysis with clinical data. Results The normal ranges of indicators were established as CD ≥ 1755.85 µm, CCR ≥ 3.34%, CI ≥ 39.63, Vmax ≥ 14.63 µm min−1 and CFS ≥ 15. We found that the chemotactic function of neutrophils in patients suffering from infections was significantly impaired. The mean values of CD, CCR, CI, Vmax and CFS were 1452.8 µm (P, In this study, we focused on patients with severe infection in the intensive care unit (ICU) and accurately evaluated their neutrophil chemotactic function. We developed a platform, the ‘Cell Chemotaxis Analysis Platform based on Computer Vision’ (CCAP), and critically assessed the chemotactic function of neutrophils in 53 ICU patients with severe infection by the improved agarose gel chemotactic model and detected the expression of P2RX1 on the surface of PMN synchronously. For the first time, we accurately revealed the dynamic changes in PMN chemotactic function in patients with severe infection and provided novel evaluation indexes and a potential biomarker for clinical treatment.

Background: Antimicrobial Peptide PL-5 (Peceleganan) Spray is a novel topical antimicrobial agent targeted on the treatment of skin wound infections. Methods: We conducted a multicenter, open-label, randomized, controlled phase IIb clinical trial to evaluate the efficacy and safety of PL-5 Spray, as compared with silver sulfadiazine, in patients with skin wound infections. The primary efficacy outcome was the clinical efficacy rate on the first day after ending the treatment (D8). The secondary efficacy outcome was the clinical efficacy rate on the fifth day post-treatment (D5), the bacteria clearance rate and the overall efficacy rate at the mentioned two timepoints. The safety outcomes included adverse reactions and pharmacokinetic analysis post-treatment. Findings: Between Jul 29, 2020, and May 10, 2021, a total of 220 patients from 27 hospitals in China were randomly assigned to 4 groups. On D8, the efficacy rate was 91·6%, 83·6%, 85·0% for the 1 ‰ PL-5, 2 ‰ PL-5, 4 ‰ PL-5 groups respectively, as compared with 60% for the control group. The efficacy rate between each PL-5 group and the control group was significantly different (P

Additional file 1. Supplemental data (Fig. S1-S6).

Background Extensive burn injury results in a complex immune response that is associated with mortality and prognosis. Studies on acquired immune and the development of sepsis in burn patients have been reported. However, one of the main cells in innate immune, neutrophil dysfunction in the burn shock stage has not been thoroughly characterized. Methods Neutrophil chemotaxis, expression of neutrophil surface markers (P2 × 1 receptor, [P2RX1]), degranulation (myeloperoxidase [MPO], heparin-binding protein [HBP], matrix metalloproteinase-9 [MMP-9] and neutrophil elastase [NE]), oxidative burst capacity, neutrophil extracellular trap (NET) generation, phagocytosis and apoptosis were measured in 18 patients with major burns (≥30% total body surface area [TBSA]) within 48 hours after burn injury. In addition, circulating neutrophils and vascular permeability in mice model with 30% TBSA third-degree burns were also observed and investigated. Results Neutrophil functions were reduced considerably in burn shock stage, which was characterized by decreased chemotaxis, phagocytosis and abnormal bactericidal function. Increased release of heparin-binding protein (HBP) and the expression of P2RX1 on the neutrophil surface are related to fluid leakage and decreased chemotaxis during burn shock stage, respectively. The combination of HBP concentration in plasma and P2RX1 expression on neutrophils gives a better prediction of neutrophil dysfunction in burn-injured patients. Conclusion Neutrophil dysfunction plays a key role in the development of burn injury. Targeting the restoration of neutrophil function may be a feasible therapeutic intervention to help reduce fluid loss during shock and the severity of subsequent infection.

To investigate the effect of heparin-binding protein (HBP) on the damage of vascular permeability in early burn.(1) Clinical research: 12 patients with severe burns admitted to Suzhou Hospital of Nanjing Medical University from January 1st to August 30th in 2019 were enrolled. Eight patients with severe trauma admitted to the same hospital during the same period were also enrolled as controls to explain the specificity of burn injury. Whole blood samples were obtained within 0.5 hour after admission. The white blood cell count (WBC), absolute value and ratio of neutrophils, and serum HBP levels were measured. Serum samples of 12 patients with severe burn were collected within 9 days after admission, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of metabolism products of glycocalyx including syndecan-1 and hyaluronic acid (HA). The correlation between HBP and neutrophils ratio, syndecan-1 and HA were analyzed by linear correlation. (2) Basic research: a 30% total body surface area (TBSA) III degree angle burn model of Sprague-Dawley (SD) rat aged 6-8 weeks was prepared. In low molecular weight heparin (LMWH) intervention group (n = 5), 200 U/kg LMWH was injected subcutaneously immediately and every 2 hours after injury for 4 times in total; the burn group (n = 5) was given the same amount of normal saline. No intervention was given to the normal control group (n = 5). The peripheral venous blood was collected at 0, 2, 4, and 8 hours after injury, and the serum levels of HBP, syndecan-1 and HA were measured; the injury of glycocalyx on pulmonary vascular endothelial cells was observed under transmission electron microscope.(1) Clinical research results: the WBC, neutrophils absolute value and ratio, and HBP levels were increased in 12 patients with severe burn and 8 patients with severe trauma. There was no significant difference in the WBC, absolute value and ratio of neutrophils between severe burn and severe trauma patients [WBC (×10The massive exudation of body fluids and the significant increase of vascular permeability in patients in early burns may be related to the destruction of the glycocalyx on endothelial cells by HBP released from increased neutrophils.

Purinergic signaling is that nucleotides (especially ATP) and adenosine are utilized as transmitter molecules, which play an important role in the immune system. In the extracellular ventricle, ATP plays a significant role of pro-inflammatory molecules mainly through activating P2 receptors, while adenosine plays the role of anti-inflammatory molecules mainly through activating P1 receptors. As we know，neutrophils are the most abundant immune cells in our circulation and have become an essential part of coordinating a series of complex events during inflammatory diseases. However, due to the destruction of inflammatory substances from neutrophils, the activation of neutrophils is fine-tuned, and purinergic signaling is associated with this process. As a matter of fact, altering the balance between P2 and P1 signals is of great importance for neutrophils to exert immune activities properly. Here, we review the role of purinergic signaling in regulatory function of neutrophils during inflammatory disease, and then discuss the potential contribution of targeted purinergic signals in the treatment of the neutrophil during inflammatory diseases.

Supplemental material, sj-pdf-1-ini-10.1177_1753425920951985 for Metabolomic analysis of spontaneous neutrophil apoptosis reveals the potential involvement of glutathione depletion by Xiong Yuyun, Yu Fan, Wei Weiping, Yin Qing and Sun Bingwei in Innate Immunity

While neutrophils have dutifully performed their function in injury and infection, the recent works have found that cytotoxicity and/or cytostatic of neutrophils has also been observed in tumor. Till now the molecular players that participate in this neutrophils antitumoral effect remain unclear. In the current study, we find that neutrophils from healthy donors have potent suppression to tumor cell lines by physical contact. Importantly, these suppression activities seem to be cancer cell-specific which is not observed in the normal cells. Further observations show that neutrophils mediated tumor cell lines growth inhibitory effect through early cell cycle arrested. Treatment with an antagonist Fas receptor in A549 cell line or knocking out of the Fas gene in A549 cell line recovers tumor cells cycle and lessen neutrophils anti-tumor effect. The interaction between neutrophils and A549 cell line through Fas ligand /Fas regulates the expression of cell cycle checkpoint proteins, leading to early cell cycle arrest. This phenomenon is also seen in other 3 tumor cell lines. Taken together, our results identified a new role of Fas ligand /Fas interaction in neutrophils antitumoral effect in tumors via arresting cell cycle.

Although the neutrophil recruitment cascade during inflammation has been well described, the molecular players that halt neutrophil chemotaxis remain unclear. In this study, we found that lipopolysaccharide (LPS) was a potent stop signal for chemotactic neutrophil migration. Treatment with an antagonist of the ATP receptor (P2X1) in primary human neutrophils or knockout of the P2X1 receptor in neutrophil-like differentiated HL-60 (dHL-60) cells recovered neutrophil chemotaxis. Further observations showed that LPS-induced ATP release through connexin 43 (Cx43) hemichannels was responsible for the activation of the P2X1 receptor and the subsequent calcium influx. Increased intracellular calcium stopped neutrophil chemotaxis by activating myosin light chain (MLC) through the myosin light chain kinase (MLCK)-dependent pathway. Taken together, these data identify a previously unknown function of LPS-induced autocrine ATP signaling in inhibiting neutrophil chemotaxis by enhancing MLC phosphorylation, which provides important evidence that stoppage of neutrophil chemotaxis at infectious foci plays a key role in the defense against invading pathogens.

To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients.Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep II to III degrees, burn index ≥ 20%, age20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot.Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P0.05).Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis.(1) In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg×kg(1) In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice. (2) In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.

Sepsis, an intractable clinical syndrome, is often accompanied by severe vascular endothelial injury and barrier dysfunction. Previous evidence has shown that the endogenous repair mechanism of damaged vascular endothelium requires the proliferation of local endothelial cells (ECs), but processes of re-endothelialization and angiogenesis after endothelial injury are also affected by bone marrow-derived endothelial progenitor cells (EPCs). EPC mobilization has been linked to the mechanism of vascular endothelial repair in various chronic diseases. However, the potential value of EPC mobilization in the treatment of sepsis has not been explored. Literature review was done to summarize the mobilization mechanism of EPC and to describe the cytokines and treatments related to EPC mobilization. Additionally, we summarize what is known about the mechanisms of endothelial damage and repair in sepsis. During sepsis, many endotoxins and inflammatory factors can damage ECs, resulting in increased vascular permeability and microcirculatory disorders. EPCs can serve as a source of ECs. Increasing evidence suggests that various cytokines and medicines can induce EPC mobilization. EPC mobilization plays an important role in endothelial repair; this may guide the discovery of novel methods to treat sepsis.

The purpose of the present study was to investigate the effect of carbon monoxide (CO) released from CO-releasing molecule 2 (CORM-2) on mice with acute pancreatitis (AP). To perform the investigation, a mouse AP model was established using caerulein. The mice were treated with or without CORM-2. The survival rate of the mice in the different groups was analyzed, and serum amylase and lipase levels were measured to assess the degree of pancreatic injury. The severity of AP was also evaluated by histological examination, and histopathological scoring of the pancreatic damage was performed. Pancreatic cell apoptosis was analyzed using a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay. The function of the lung and liver was also assessed in the present study. Furthermore, the role of CORM-2 on oxidative stress, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression, pro-inflammatory cytokine production, and nuclear factor (NF)-κB activation in the pancreas of AP mice was determined. The results demonstrated that CORM-2 reduced the mortality, pancreatic damage, and lung and liver injury of AP mice. CORM-2 administration also reduced systemic and localized inflammatory cell factors. Furthermore, treatment with CORM-2 inhibited the expression of ICAM-1 and VCAM-1, and the activation of NF-κB and phosphorylated inhibitor of NF-κB subunit α, in the pancreas of AP mice. These results indicated that CO released from CORM-2 exerted protective effects on AP mice, and the beneficial effects were likely due to inhibition of NF-κB pathway activation.

Machining accuracy is a very important index to measure the performance of NC machine tools, and how to improve the machining accuracy grabs the attention of both manufacturers and users. Since the geometric errors are intercoupling and are one of the main factors affecting machining accuracy of machine tools, in this paper, a new method for extracting key geometric errors based on the screw theory and extended Fourier amplitude sensitivity test (EFAST) method was proposed. First, screw theory was introduced, and the machining accuracy modeling of a three-axis vertical machining center (VMC) was established. Then, in order to analyze the coupling effects between geometric errors by high-dimensional nonlinear model, EFAST global sensitivity analysis method was adopted to help in extracting the main geometric errors. An illustration example on a three-axis VMC shows that the proposed method is effective and can be helpful to improve the geometric accuracy of machine tool.

Error compensation technique is a recognized and cost-effective method to improve machining accuracy of machine tools. In this article, a new compensation method for geometric error is proposed based on Floyd algorithm and product of exponential screw theory. Based on topological structure and measured data, volumetric geometric error modeling is established by product of exponential screw theory. Then, the improved Floyd minimum-distance method was used to establish an error compensation model by adjusting weight unceasingly. In order to verify the effectiveness and generality of the method proposed in this article, two experiments were designed. A total of 5 five-axis machining centers of the same type with different use time were selected to carry out the simulation experiments. Results show that the Floyd method can provide higher compensation precision, that is, Floyd algorithm compensation method can keep positioning errors within the range [−8 µm, 9 µm]. In addition, roundness error, coaxial error, and surface roughness were reduced in the actual machining experiments of two machined conical tables. Therefore, it can be seen that the proposed compensation method is effective to improve machining accuracy of machine tools.

// Xu Wang 1 , Weiting Qin 1 , Yisen Zhang 1 , Huafeng Zhang 1 , Bingwei Sun 1 1 Department of Burn and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang, Jiangsu Province, China Correspondence to: Bingwei Sun, email: sunbinwe@hotmail.com Keywords: neutrophil, endotoxin, chemotaxis, membrane receptor, p38 Received: August 02, 2016 Accepted: September 10, 2016 Published: September 17, 2016 ABSTRACT Neutrophils are the most abundant leukocytes in peripheral blood and play critical a role in bacterial infection, tumor immunity and wound repair. Clarifying the process of neutrophil chemotaxis to target sites of immune activity has been a focus of increased interest within the past decade. In bacterial infectious foci, neutrophils migrate toward the bacterial-derived chemoattractant N-formyl-Met-Leu-Phe (fMLP) and ignore other intermediary chemoattractants to arrive at the area of infection. Using an under agarose chemotaxis assay, we observed that the bacterial fMLP-induced neutrophil chemotaxis signal overrode interleukin 8 (IL-8)- and leukotriene B4 (LTB4)-induced chemotaxis signals. Moreover, in the presence of bacterial lipopolysaccharide (LPS), the fMLP-induced hierarchical chemotaxis signal was enhanced. Further studies revealed that LPS increased the membrane expression of the fMLP receptor, formyl peptide receptor 1 (FPR1). However, expression levels of the membrane receptors for IL-8 and LTB4 were decreased by LPS administration. A human Phospho-mitogen-activated protein kinase (MAPK) proteome array showed that the p38 pathway was significantly activated by LPS stimulation. Moreover, p38 was responsible for the altered expression of neutrophil membrane chemoattractant receptors. Inhibition of neutrophil p38 restored LPS-improved hierarchical chemotaxis. Taken together, these data indicate that endotoxin promotes neutrophil hierarchical chemotaxis via the p38-membrane receptor pathway.

Activation of coagulation occurs in sepsis and contributes to the development of thrombosis. Platelet α-granule exocytosis plays an important role in septic coagulation abnormalities. The present study aimed to investigate the effects and the underlying mechanisms of exogenous carbon monoxide, carbon monoxide-releasing molecules II (CORM-2)-liberated CO, on suppressing platelet α-granule exocytosis in sepsis. It was shown that CORM-2 weakened α-granule membrane fusion with platelet plasma membrane and attenuated α-granule contents exocytosis in LPS-Induced platelet. Further studies revealed that CORM-2 suppressed the expression of integrin αIIbβ3 in platelets stimulated by LPS. This was accompanied by a decrease in production and phosphorylation of PKCθ and Munc18a, SNARE complex assembly and subsequently platelet α-granule exocytosis. Taken together, we suggested that the potential mechanism of suppressive effect of CORM-2 on LPS-induced platelet SNAREs complex assembly and α-Granule Exocytosis might involve integrin αIIbβ3-mediated PKCθ/Munc18a pathway activation.

Pyroptosis is a caspase-1 or caspase-4/5/11-dependent programmed cell death associated with inflammation, which is initiated by inflammasomes or cytosolic LPS in innate immunity. Sepsis is a life-threatening organ dysfunction caused by an imbalance in the body's response to infection. It is a complex interaction between the pathogen and the host's immune system. Neutrophils play the role of a double-edged sword in sepsis, and a number of studies have previously shown that regulation of neutrophils is the most crucial part of sepsis treatment. Pyroptosis is one of the important forms for neutrophils to function, which is increasingly understood as a host active immune response. There is ample evidence that neutrophil pyroptosis may play an important role in sepsis. In recent years, a breakthrough in pyroptosis research has revealed the main mechanism of pyroptosis. However, the potential value of neutrophil pyroptosis in the treatment of sepsis did not draw enough attention. A literature review was performed on the main mechanism of pyroptosis in sepsis and the potential value of neutrophils pyroptosis in sepsis, which may be suitable targets for sepsis treatment in future.

The present study aimed to investigate the effect of carbon monoxide (CO)-releasing molecule-2 (CORM-2) on pancreatic function in sepsis-model mice. To perform the present investigation, mice were rendered septic by cecal ligation and puncture (CLP). Then, mice were either treated with or without CORM-2 (8 mg/kg, intravenous) for different durations (6, 12 and 24 h) immediately following CLP. The levels of serum amylase and lipase, tumor necrosis factor α, interleukin-1β and interleukin-6 in addition to myeloperoxidase (MPO) activity in pancreatic tissues were determined at 6, 12 and 24 h post-CLP. Histological scores and the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), nuclear factor-κB (NF-κB) and phosphorylated inhibitor of κB (p-IκB-α) in the pancreas were also evaluated at 24 h post-CLP. The results of the present study revealed that compared with CLP-alone group, CORM-2 treatment significantly (P