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Neutrophils accumulate in the inflammatory mucosa of patients with inflammatory bowel disease (IBD), and excessive release of NETs (neutrophil extracellular traps may be one of the important factors that cause IBD progression. However, the specific mechanism underlying vascular injury caused by NETs remains unclear. Immunofluorescence, ELISA, and flow cytometry were used in this study to detect the expression of NETs and DNase in the tissue and peripheral blood samples of patients with IBD. DSS mouse model was used to detect colon injury and vascular permeability. We found that NETs and DNase levels increased in the colon of patients with IBD. We found an increase in the activity of NET-related MPO released by DNase. DNase released NET-related proteins and damaged vascular endothelial cells in vitro. In DSS mouse model, the synchronous increase of DNase and NETs in the colon leads to an increase in vascular injury markers (CD44, sTM). DNase aggravated colon injury and increased vascular permeability in vivo, which was inhibited by gentamicin sulfate (GS). GS does not reduce the expression of DNase, but rather reduces the release of NET-related proteins to protect vascular endothelium by inhibiting DNase activity. MPO and histones synergistically damaged the vascular endothelium, and vascular injury can be improved by their active inhibitors. We further found that H2 O2 is an important substrate for MPO induced vascular damage. In conclusion, in IBD, DNase, and NET levels increased synchronously in the lesion area and released NET-related proteins to damage the vascular endothelium. Therefore, targeting DNase may be beneficial for the treatment of IBD.
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Traumatismos Abdominales , Trampas Extracelulares , Enfermedades Inflamatorias del Intestino , Lesiones del Sistema Vascular , Animales , Ratones , Humanos , Desoxirribonucleasas , Células Endoteliales , Modelos Animales de EnfermedadRESUMEN
Although trace metals in strawberry production system have attracted growing attention, little is known about metal fractionation in soil for strawberry cultivation. We hypothesized that the metal fractions in soil influenced by strawberry production had significant effect on food chain transport of metals and their risk in soil. Here, samples of strawberries and soil were gathered in the Yangtze River Delta, China to verify the hypothesis. Results showed that the acid-soluble Cr, Cd, and Ni in soil for strawberry cultivation were 21.5%-88.3% higher than those in open field soil, which enhanced uptake and bioaccessible levels of these metals in strawberries. Overall, the ecological, mobility, and health risks of Pb, Zn, Ni, and Cu in soil were at a low level. However, the ecological risk of bioavailable Cd, mobility risk of Cd, and cancer risk of bioavailable Cr in over 70% of the soil samples were at moderate, high, and acceptable levels, respectively. Since the increased acid-soluble Cr and Ni in soil were related to soil acidification induced by strawberry production, nitrogen fertilizer application should be optimized to prevent soil acidification and reduce transfer of Cr and Ni. Additionally, as Cd and organic matter accumulated in soil, the acid-soluble Cd and the ecological and mobility risks of Cd in soil were enhanced. To decrease transfer and risk of Cd in soil, organic fertilizer application should be optimized to mitigate Cd accumulation, alter organic matter composition, and subsequently promote the transformation of bioavailable Cd into residual Cd in soil.
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Fragaria , Contaminantes del Suelo , Suelo , Fragaria/química , Fragaria/crecimiento & desarrollo , Contaminantes del Suelo/análisis , Medición de Riesgo , China , Suelo/química , Cadena Alimentaria , Monitoreo del Ambiente/métodos , Agricultura/métodos , Metales/análisis , Metales Pesados/análisisRESUMEN
A collaborative assessment approach, including impact index of comprehensive quality (IICQ), food pollution index (FPI), and single factor pollution index (PI), was used to simultaneously select priority metal pollutants and assess metal contamination status in the plastic-shed soil (PSS)-vegetable system of the industrial towns situated in the Yangtze River Delta, China. Overall, significant Cr increment as well as Cd and Cu pollution in PSS existed, which was related to anthropogenic activities, especially industrial wastewater irrigation. The evaluation using PI and FPI demonstrated that priority metal pollutants were Cu and Cd in PSS while Cr and Cd in vegetables. Additionally, the estimation using IICQ method revealed that 23.3% and 13.3% of the sampling sites were sub-moderately and heavily contaminated by metals, respectively. These sites especially with heavy pollution need priority pollution management. These data will be beneficial to metal pollution control in PSS-vegetable system around industrial areas.
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Monitoreo del Ambiente , Contaminación de Alimentos , Metales Pesados , Plásticos , Contaminantes del Suelo , Verduras , Verduras/química , Monitoreo del Ambiente/métodos , China , Contaminantes del Suelo/análisis , Plásticos/análisis , Metales Pesados/análisis , Contaminación de Alimentos/análisisRESUMEN
Growing concern has been paid to metals in soil-strawberry system. In contrast, few attempts have been made to investigate bioaccessible metals in strawberries and further assess health risk based on bioaccessible metals. Moreover, the connections between soil parameters (e.g. soil pH, organic matter (OM), total and bioavailable metals) and metal transfer in soil-strawberry-human system still need to be systematically investigated as well. Considering that strawberries are extensively grown under plastic-shed conditions in China, a total of 18 paired plastic-shed soil (PSS) and strawberry samples were taken from the strawberry bases located in the Yangtze River Delta of China as a case study to assess accumulation status, migration and health risk of Cd, Cr, Cu, Ni, Pb, and Zn in the PSS-strawberry-human system. Overall, heavy application of organic fertilizers induced accumulation and contamination of Cd and Zn in PSS. In particular, 55.6% and 44.4% of PSS samples had considerable and moderate ecological risk caused by Cd, respectively. Despite no metal pollution in strawberry, PSS acidification mainly caused by high nitrogen input promoted Cd and Zn uptake by strawberry and enhanced bioaccessible concentrations of Cd, Cu, and Ni. In contrast, the increased soil OM caused by organic fertilizer application decreased Zn migration in PSS-strawberry-human system. Additionally, bioaccessible metals in strawberries induced limited non-cancer and cancer risk. To mitigate accumulation of Cd and Zn in PSS and metal transfer in the food chain, feasible fertilization strategies should be developed and carried out.
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Fragaria , Metales Pesados , Contaminantes del Suelo , Oligoelementos , Humanos , Suelo , Metales Pesados/análisis , Cadmio , Contaminantes del Suelo/análisis , Oligoelementos/análisis , China , Monitoreo del Ambiente , Medición de RiesgoRESUMEN
Chronic diabetic wounds are primarily caused by infection, inflammation, and angiogenesis-related disorders. An ideal approach for treating chronic diabetic wounds is by combining anti-infection strategies, immune microenvironment regulation, and angiogenesis promotion. Vascular endothelial growth factor (VEGF) can promote the proliferation and migration of vascular endothelial cells, thereby promoting angiogenesis. However, the low stability and inability to target lesions limit its application. Polymorphonuclear neutrophil-derived exosomes (PMNExo) exhibit good delivery properties and can be used for the therapeutic delivery of VEGF. Furthermore, they retain the antibacterial ability of polymorphonuclear neutrophils (PMNs). Nonetheless, low PMNExo generation impedes its therapeutic applications. In this study, we prepared exosome mimetics (EM) from PMNs using the extrusion process; as a result, exosome yield significantly improved. To increase the residence of exosomes, an extracellular matrix (ECM) hydrogel, a thermosensitive material that can function as an in situ gel in vivo, was used as an exosome carrier. The active peptides in the ECM regulated the immune microenvironment of the wound. In summary, we loaded ECM with VEGF-encapsulated activated neutrophil exosome mimetics (aPMNEM) to develop VEGF-aPMNEM-ECM hybrid hydrogel for treating chronic wounds. The hydrogel accelerates the regeneration of chronic diabetic wounds. Our study provides a prospective therapy platform involving cytokines for treating different diseases.
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Diabetes Mellitus , Exosomas , Neutrófilos , Factor A de Crecimiento Endotelial Vascular , Hidrogeles/farmacología , Células Endoteliales , Cicatrización de Heridas , Antibacterianos/farmacología , Matriz ExtracelularRESUMEN
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.
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Trampas Extracelulares , Sepsis , Actinas , Animales , Humanos , Receptores de Hialuranos , Ratones , Activación Neutrófila , Neutrófilos , PeroxidasaRESUMEN
BACKGROUND: Despite many advances in treatment, the prognosis of patients with sepsis still remains poor. Polymorphonuclear leukocytes (PMNs) are the first line of defense against infection. This study aimed to reveal the reason and mechanism of the production of PD-L1+ PMNs in sepsis. METHODS: Cecal ligation and perforation mouse model was established to simulate sepsis. And PMNs were treated for 4 h, 12 h with or without 100 ng/mL (IFN-γ) for further gene sequencing. PD-L1, PD-1, Ly6G, and CD3 were detected by multiplexed immunofluorescence. In addition, expression of PD-L1 and function of PMNs were assessed by flow cytometry. Serum and cell culture supernatant were measured with ELISA assays. Western blot was used to verify the JAK2/STAT1 pathway. RESULTS: Our study demonstrates that PMNs are the main immune cells with high expression of PD-L1 during sepsis, and these cells, therefore, play a critical role in immunosuppression. In vivo studies demonstrated a specific interaction between PD-L1+ PMNs and PD-1+ T cells. In vitro studies further demonstrated that IFN-γ induced the production of PD-L1+ PMNs through the JAK2/STAT1 pathway. In addition, Fedratinib, an inhibitor of Jak2, was shown to significantly reduce the expression of PD-L1 in neutrophils. CONCLUSIONS: These data demonstrate that secretion of IFN-γ by splenic T lymphocytes induces the production of PD-L1 + PMNs through the JAK2/STAT1 pathway in sepsis.
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Neutrófilos , Sepsis , Animales , Humanos , Interferón gamma/metabolismo , Ratones , Bazo/metabolismo , Linfocitos TRESUMEN
BACKGROUND: Although the immune function of neutrophils in sepsis has been well described, the heterogeneity of neutrophils remains unclear during the process of sepsis. METHODS: In this study, we used a mouse CLP model to simulate the clinical scenario of patients with sepsis, neutrophil infiltration, abnormal distribution and dysfunction was analyzed. LPS was used to stimulate neutrophils in vitro to simulate sepsis; single-cell gene sequencing technology was used to explore the immunological typing. To explore the immunological function of immunosuppressive neutrophils, PD-L1 knockout neutrophils were cocultured with lymphocytes from wild-type mice. RESULTS: We found that neutrophils presented variant dysfunction at the late stage of sepsis, including inhibition of apoptosis, seriously damaged chemotaxis and extensive infiltration into the tissues. Single-cell RNA sequencing revealed that multiple subclusters of neutrophils were differentiated after LPS stimulation. The two-dimensional spatial distribution analysis showed that Foxp3+ T cells were much closer to Ly-6G than the CD4+ and CD8+ cells, indicating that infiltrated neutrophils may play immunomodulatory effect on surrounding T-regs. Further observations showed that LPS mediates PD-L1 over expression through p38α-MSK1/-MK2 pathway in neutrophils. The subsets of highly expressed PD-L1 exert immunosuppressive effect under direct contact mode, including inhibition of T cell activation and induction of T cell apoptosis and trans-differentiation. CONCLUSIONS: Taken together, our data identify a previously unknown immunosuppressive subset of neutrophils as inhibitory neutrophil in order to more accurately describe the phenotype and characteristics of these cells in sepsis.
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Heterogeneidad Genética , Neutrófilos/clasificación , Sepsis/sangre , Animales , Modelos Animales de Enfermedad , Recuento de Leucocitos/métodos , Recuento de Leucocitos/estadística & datos numéricos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa/métodos , Sepsis/genéticaRESUMEN
The management of granulocytopenia-associated infections is challenging, and a high mortality rate is associated with traditional supportive therapies. Neutrophils-the primary defenders of the human immune system-have potent bactericidal capabilities. Here, we investigated the dynamic in vivo distribution of neutrophil transfusion and their impact on the treatment outcome of severe granulocytopenic infections. We transfused 89Zr-labeled neutrophils in the C57BL/6 mice and observed the dynamic neutrophil distribution in mice for 24 h using the micro-positron emission tomography (Micro-PET) technique. The labeled neutrophils were predominantly retained in the lungs and spleen up to 4 h after injection and then redistributed to other organs, such as the spleen, liver, and bone marrow. Neutrophil transfusion did not elicit marked inflammatory responses or organ damage in healthy host mice. Notably, allogeneic neutrophils showed rapid chemotaxis to the infected area of the host within 1 h. Tail vein infusion of approximately 107 neutrophils substantially bolstered host immunity, ameliorated the inflammatory state, and increased survival rates in neutrophil-depleted and infected mice. Overall, massive allogeneic neutrophil transfusion had a therapeutic effect in severe infections and can have extensive applications in the future.
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Trasplante de Células Madre Hematopoyéticas , Neutrófilos , Ratones , Humanos , Animales , Neutrófilos/fisiología , Tasa de Supervivencia , Ratones Endogámicos C57BL , Médula ÓseaRESUMEN
The early diagnosis of neonatal sepsis is crucial as it remains a prevalent cause of neonatal mortality. In this study, we conducted an analysis on the clinical data and detection indicators of 22 cases with sepsis and 62 cases without sepsis among neonates. Our findings indicate that the clinical signs observed in neonates with sepsis lack specificity. In addition, the commonly used clinical inflammatory indicators (such as leukocyte count, neutrophil-to-lymphocyte ratio [NLR], C-reactive protein [CRP], procalcitonin) exhibit limited sensitivity and specificity. Furthermore, the current clinical measures lack the assessment of inflammatory factors. Therefore, in order to enhance the accuracy of early sepsis diagnosis in neonates, we have employed a novel microfluidic-based single-cell technology platform for the analysis of 32 cytokines secreted by neutrophils at the individual cell level under various toxin stimulation conditions. We have further investigated and compared the disparities in single-cell protein secretomics between umbilical cord blood neutrophils and healthy adult peripheral neutrophils within an in vitro sepsis model. Our findings indicate that in a resting state UCB neutrophils exhibited lower polyfunctionality compared with healthy adult blood neutrophils, and notable variations in cytokine secretion profiles were detected between the two groups. However, the polyfunctionality of UCB neutrophils significantly increased and surpassed that of healthy adult neutrophils when exposed to alpha-hemolysin or lipopolysaccharide. UCB neutrophils secreted a wide range of chemokines and inflammatory factors, among which GM-CSF and IL-18 were the most significant. Furthermore, we initially categorized the functional subgroups of neutrophils by considering the secretion of five primary cytokines by neutrophils (GM-CSF, IL-18, IL-8, MIP-1ß, and MIF). The current study, for the first time, examined in detail the heterogeneity of protein secretion and the functional diversity of UCB neutrophils stimulated by different antigens. Moreover, new insight into neonatal sepsis, early diagnosis, and wider clinical applications of UCB neutrophils are provided by these data.
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Objective: To investigate the role of neutrophils in colon cancer progression. Methods: Genetic data from 1,273 patients with colon cancer were procured from public databases and categorized based on genes linked to neutrophils through an unsupervised clustering approach. Through univariate Cox regression analysis, differentially expressed genes (DEGs) influencing overall survival (OS) were identified, forming the basis for establishing a prognostic risk score (PRS) system specific to colon cancer. Additionally, the correlation between PRS and patient prognosis, immune cell infiltration, and intratumoral gene mutations were analyzed. Validation of PRS as an indicator for "pan-tumor" immunotherapy was conducted using four distinct immunotherapy cohorts. Results: The research identified two distinct subtypes of colon cancer, namely Cluster A and B, with patients in Cluster B demonstrating remarkably superior prognoses over those in Cluster A. A total of 17 genes affecting OS were screened based on 109 DEGs between the two cluster for constructing the PRS system. Notably, individuals classified under the high-PRS group (PRShigh) exhibited poorer prognoses, significantly linked with immune cell infiltration, an immunosuppressive tumor microenvironment, and increased genomic mutations. Remarkably, analysis of immunotherapy cohorts indicated that patients with PRShigh exhibited enhanced clinical responses, a higher rate of progression-free events, and improved overall survival post-immunotherapy. The PRS system, developed based on tumor typing utilizing neutrophil-associated genes, exhibited a strong correlation with prognostic elements in colon cancer and emerged as a vital predictor of "pan-tumor" immunotherapy efficacy. Conclusions: PRS serves as a prognostic model for patients with colon cancer and holds the potential to act as a "pan-tumor" universal marker for assessing immunotherapy efficacy across different tumor types. The study findings lay a foundation for novel antitumor strategies centered on neutrophil-focused approaches.
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Background: Bacterial infections pose a considerable threat to skin wounds, particularly in the case of challenging-to-treat diabetic wounds. Systemic antibiotics often struggle to penetrate deep wound tissues and topically applied antibiotics may lead to sensitization, necessitating the development of novel approaches for effectively treating germs in deep wound tissues. Neutrophils, the predominant immune cells in the bloodstream, rapidly release an abundance of molecules via degranulation upon activation, which possess the ability to directly eliminate pathogens. This study was designed to develop novel neutrophil cell engineered nanovesicles (NVs) with high production and explore their bactericidal properties and application in promoting infectious wound healing. Methods: Neutrophils were isolated from peripheral blood and activated in vitro via phorbol myristate acetate (PMA) stimulation. Engineered NVs were prepared by sequentially extruding activated neutrophils followed by ultracentrifugation and were compared with neutrophil-derived exosomes in terms of morphology, size distribution and protein contents. The bactericidal effect of NVs in vitro was evaluated using the spread plate technique, LIVE/DEAD backlight bacteria assay and observation of bacterial morphology. The therapeutic effects of NVs in vivo were evaluated using wound contraction area measurements, histopathological examinations, assessments of inflammatory factors and immunochemical staining. Results: Activated neutrophils stimulated with PMA in vitro promptly release a substantial amount of bactericidal proteins. NVs are similar to exosomes in terms of morphology and particle size, but they exhibit a significantly higher enrichment of bactericidal proteins. In vitro, NVs demonstrated a significant bactericidal effect, presumably mediated by the enrichment of bactericidal proteins such as lysozyme. These NVs significantly accelerated wound healing, leading to a marked reduction in bacterial load, downregulation of inflammatory factors and enhanced collagen deposition in a full-thickness infectious skin defect model. Conclusions: We developed engineered NVs derived from activated neutrophils to serve as a novel debridement method targeting bacteria in deep tissues, ultimately promoting infectious wound healing.
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Although hydroxyapatite (HAP) can prominently lower Cd uptake by celery from Cd-polluted soil, its high application rates in reality may lead to high cost and potential environmental risk. Therefore, we aimed to clarify whether combined amendments of HAP and another low-cost material (hydrated lime, corn straw-derived biochar, or zeolite) with reduced application rate of each single amendment could significantly decrease Cd transfer in soil-celery-human system without side effect on celery growth through a pot experiment. Results revealed that adding biochar, HAP, zeolite, or combined amendments had no obvious side effect on celery growth, while adding 0.3% hydrated lime significantly decreased fresh edible celery yield by 69.0%. Conversely, adding 0.5% HAP + 0.05% hydrated lime increased fresh edible celery yield by 39.8%. Additionally, adding HAP, zeolite, or hydrated lime rather than adding biochar effectively decreased total and bioaccessible Cd in edible celery. Similarly, HAP + hydrated lime and HAP + zeolite were much more efficient than HAP + biochar in lowering Cd transfer in soil-celery-human system. The total and bioaccessible Cd in edible celery were even reduced by over 50.0% after adding HAP + hydrated lime or HAP + zeolite at low rates. Considering the effects on celery growth and Cd transfer, HAP + hydrated lime and HAP + zeolite have the potential in remediating soil Cd contamination.
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Apium , Oryza , Contaminantes del Suelo , Zeolitas , Humanos , Cadmio/análisis , Suelo , Durapatita , Carbón Orgánico , Contaminantes del Suelo/análisisRESUMEN
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. We studied sepsis patients admitted to the intensive care unit. Wright-Giemsa 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. 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. 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.
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Neutrófilos/patología , Neutrófilos/fisiología , Sepsis/sangre , Adulto , Anciano , Degranulación de la Célula , Quimiotaxis de Leucocito , Citocinas/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Fagocitosis , Receptores Purinérgicos P2X1/metabolismo , Sepsis/diagnósticoRESUMEN
Accumulation and concomitant risk of metals in plastic-shed soil (PSS)-vegetable system around industrial areas have attracted growing public concern recently, while limited studies have focused on human bioaccessible metals in various plastic-shed vegetables and health risk calculated using bioaccessible metals. Previous studies showed that intensive farming and industrial activities could prominently affect metal migration from PSS to vegetables via altering PSS pH, total and bioavailable metal contents. In contrast, whether changes in PSS pH and metal contents control bioaccessible metals in vegetables and health risk is still unknown. For PSS management and sustainable plastic-shed vegetable production in the areas with rapid industrialization, 41 PSS and 32 plastic-shed vegetable samples were sampled from the industrial areas of Yangtze River Delta, China to systematically clarify the specific connections among anthropogenic activities, soil pH and metal contents, and metal transfer and health risk in PSS-vegetable-human system. The results indicated that Cr and Cd contents in 15.6% and 9.38% of vegetable samples exceeded the allowable limits in China. Tolerable cancer risk existed and was mainly induced by bioaccessible Cr in vegetables. Decreased PSS pH mainly caused by heavy use of nitrogen fertilizers increased bioavailable Ni, Cd, Zn, Pb, and Cu in PSS and subsequently enhanced their total and bioaccessible contents in vegetables. Prominent Cr accumulation in PSS induced by industrial wastewater irrigation exacerbated Cr uptake by vegetables, which increased bioaccessible Cr in vegetables and contributed greatly to cancer risk. To reduce transfer and health risk especially of Cd and Cr in the food chain, some appropriate measures related to source control and remediation should be proposed for preventing and mitigating PSS acidification and Cr accumulation.
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Metales Pesados , Neoplasias , Contaminantes del Suelo , Efectos Antropogénicos , Cadmio , China , Monitoreo del Ambiente , Fertilizantes/análisis , Humanos , Concentración de Iones de Hidrógeno , Plomo , Metales Pesados/análisis , Nitrógeno , Plásticos , Medición de Riesgo , Suelo/química , Contaminantes del Suelo/análisis , Verduras/química , Aguas ResidualesRESUMEN
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). Hypertrophic scars showed greater infiltration with neutrophils and NETs (p < 0.05). NETs activate fibroblasts in vitro to promote their differentiation and migration. Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice. We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs. We did not find an increase in the expression of common scarring factors [interleukin (IL)-17 and transforming growth factor-ß (TGF-ß), 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.
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The function and heterogeneity of neutrophils in neonatal umbilical cord blood (UCB) have not been characterized. In this study, we analyzed the neutrophils in UCB and healthy adults using single-cell RNA sequencing analysis for the first time. We found that neutrophils divided into six subpopulations (G2, G3, G4, G5a, G5b, and G5c) with different marker genes and different functions under homeostasis. Compared with healthy adults, neutrophils of UCB were more naïve and have more obvious degranulation and activation functions. Moreover, we found significant differences in the amount and function of G5b cells between healthy adults and UCB. The amount of G5b group in UCB was lower, but it has more degranulation, secretion and activation functions. In addition, we noted a new subset of G5c labeled by CD52, which almost did not exist in UCB. Besides, its differential genes were enriched in terms such as protein synthesis and mRNA transcription. Furthermore, uncharacteristic transcription factors ZNF-276, ZNF-319 and ZNF-354A were identified in our study. In summary, we first examined the heterogeneity and functional diversity of neutrophils in UCB, and these data provided new insights into the mechanism of neutrophil-mediated diseases of neonates and the wider use of neutrophils in UCB.
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Perfilación de la Expresión Génica , Neutrófilos , Adulto , Sangre Fetal , Humanos , Recién Nacido , Cordón UmbilicalRESUMEN
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 (P2X1 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 h 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.
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Quemaduras , Sepsis , Animales , Quemaduras/metabolismo , Permeabilidad Capilar , Humanos , Ratones , Neutrófilos , Fagocitosis , Sepsis/complicacionesRESUMEN
The pathophysiological mechanisms, especially the roles of immune cells, underlying early stages of severe burn injury have not yet been fully clarified. Here, we analyzed circulating neutrophils (PMNs) in healthy donors and early burned patients by single-cell RNA sequencing to provide a comprehensive transcriptional landscape of PMNs in heterogeneity and functional multiplicity. Circulating PMNs in the healthy donors and burned groups were divided into five subgroups (G3, G4, G5a, G5b, G5c) with different functions. The dominant subsets of PMNs in homeostasis and burn injury significantly differed between groups. In addition, cells in the same subpopulation had the same core identity markers but performed different functions in healthy and burned states. Under burned conditions, PMN activation was very evident and accompanied by clear degranulation and metabolic abnormalities. Interestingly, was found that PMN activation, degranulation, chemotaxis, phagocytosis and reactive oxygen species (ROS) production in burned patients significantly differed between day 1 and days 2 or 3, thus providing a theoretical basis for PMN interventions in early burn stages. Significantly, previously undescribed transcription factors were also identified, including ZNF-787, ZNF-467, ZNF-189, ZNF-770, ZNF-262. In conclusion, this study conducted for the first time a detailed analysis of the heterogeneity and functional multiplicity of PMNs in early stages of severe burn injuries. Our findings attempted to clarify the influence of PMN heterogeneity on the pathophysiology and related mechanisms of burn injuries, which can provide new ideas for further research in burn intervention.
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Quemaduras/inmunología , Neutrófilos/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Análisis de la Célula Individual/métodos , Adulto JovenRESUMEN
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 < 0.0001), 3.1% (P < 0.0001), 34.5 (P < 0.0001), 12.2 µm min-1 (P < 0.0001) and 9 (P < 0.0001), respectively. CD and CFS were significantly negatively correlated with the APACHE II score (rCD = -0.55, rCFS = -0.39), SOFA score (rCD = -0.68, rCFS = -0.56), procalcitonin concentration (rCD = -0.60, rCFS = -0.5) and the expression of P2RX1 (rCD = -0.76, rCFS = -0.56), respectively. CONCLUSIONS: CD, CCR, CI and Vmax can well reflect the neutrophil chemotactic function in patients with severe infections. CFS systematically indicated neutrophil function and has promising clinical application prospects.