RESUMO
Vascular occlusions in patients with coronavirus diseases 2019 (COVID-19) have been frequently reported in severe outcomes mainly due to a dysregulation of neutrophils mediating neutrophil extracellular trap (NET) formation. Lung specimens from patients with COVID-19 have previously shown a dynamic morphology, categorized into three types of pleomorphic occurrence based on histological findings in this study. These vascular occlusions in lung specimens were also detected using native endogenous fluorescence or NEF in a label-free method. The three types of vascular occlusions exhibit morphology of DNA rich neutrophil elastase (NE) poor (type I), NE rich DNA poor (type II), and DNA and NE rich (type III) cohort of eleven patients with six males and five females. Age and gender have been presented in this study as influencing variables linking the occurrence of several occlusions with pleomorphic contents within a patient specimen and amongst them. This study reports the categorization of pleomorphic occlusions in patients with COVID-19 and the detection of these occlusions in a label-free method utilizing NEF.
Assuntos
COVID-19 , Armadilhas Extracelulares , Doenças Vasculares , Masculino , Feminino , Humanos , COVID-19/complicações , COVID-19/patologia , SARS-CoV-2 , Pulmão/patologia , Neutrófilos/patologia , Doenças Vasculares/patologiaRESUMO
Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.
Assuntos
Síndromes do Olho Seco , Disfunção da Glândula Tarsal , Animais , Túnica Conjuntiva/patologia , Síndromes do Olho Seco/patologia , Inflamação/patologia , Glândulas Tarsais/patologia , Camundongos , Lágrimas/químicaRESUMO
Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization in vitro. We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI.
Assuntos
Armadilhas Extracelulares , Infarto do Miocárdio , Armadilhas Extracelulares/fisiologia , Humanos , Hipóxia/complicações , Infarto do Miocárdio/complicações , Neutrófilos/fisiologia , OxigênioRESUMO
Enniatins (ENNs) and beauvericin (BEA), known as emerging mycotoxins, are the toxic secondary metabolites produced by various Fusarium species. Most grain and grain-based products are contaminated with ENNs and BEA. Animals have been exposed to ENNs and BEA primarily due to consumption of cereal grains and cereal by-products. ENNs and BEA have been detected in animal-derived food and human breast milk, and they pose significant threats to public health. Therefore, more contamination data are urgently needed for the risk assessment of ENNs and BEA present in animal-derived food. To ensure the quality of animal-derived food, a method has been developed for the simultaneous detection of five emerging mycotoxins (viz. enniatin B, enniatin B1, enniatin A, enniatin A1, and beauvericin) in eggs by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with cold-induced liquid-liquid extraction (CI-LLE) and dispersive solid phase extraction (DSPE). The main factors governing the response, recovery, and sensitivity of the method, such as the type of extraction solvent, the temperature and duration of cold treatment in CI-LLE, the type and dosages of adsorbents, and apparatus conditions and the type of mobile phase used, were optimized during sample pretreatment and instrument analysis. The mycotoxin residues in eggs were extracted using 20 mL acetonitrile-water-acetic acid (79â¶20â¶1, v/v/v) mixture for 20 min by the vortex shock method. After mixing, the mixture was frozen for 30 min in a freezer at -40 â and centrifuged for 10 min at 10000 r/min. A 2 mL aliquot of the upper acetonitrile layer was purified by using 70 mg of C18 adsorbents. After whirling, the mixtures were centrifuged at 10000 r/min for 5 min. The purified solution was then concentrated to nearly dry in nitrogen atmosphere at 40 â. The residues were dissolved in 1.0 mL 80%(v/v) acetonitrile aqueous solution. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 µm) at a column temperature of 40 â, with a flow rate of 0.3 mL/min. The injection volume was 5 µL, and gradient elution was conducted using acetonitrile and 5 mmol/L ammonium formate solution as the mobile phases. Multiple reactions monitoring (MRM) was conducted in the positive electrospray ionization (ESI +) mode. The isotope internal standard method was used for quantification of BEA, and the matrix-matched external standard method was used for quantification of four ENNs. The results of the optimized method showed that the five analytes were completely separated by using the above-mentioned chromatographic column. Good linear relationships were obtained for the five mycotoxins in the concentration range of 0.1-50.0 µg/L; the correlation coefficient (r2) ranged from 0.9983 to 0.9997. The limits of detection (LODs) ranged from 0.05 to 0.15 µg/kg, while the limits of quantification (LOQs) ranged from 0.20 to 0.50 µg/kg. Accuracy and precision experiments were conducted by spiking egg samples with known amounts of analytes at three concentration levels (0.5, 5.0, and 25.0 µg/kg, in compliance with the current legislation) with six replicates. The average recoveries of the five analytes ranged from 81.1% to 106%, and the relative standard deviations (RSDs) were between 0.27% and 9.79%. The matrix effects of the analytes were between 2.70% and 45.1% in egg samples after pretreatment by CI-LLE coupled with DSPE. The developed method was applied to the determination of five mycotoxins in rural eggs and commercial eggs. BEA was detected in most rural egg samples, with detection rates of 30.4%. None of the four ENN residues were detected. Therefore, we can conclude that the method described herein has the advantages of sensitivity, stabilization, accuracy, good recovery, and easy operation, and is suitable for the simultaneous and rapid determination of BEA and ENN residues in eggs.
Assuntos
Depsipeptídeos/análise , Ovos/análise , Contaminação de Alimentos , Animais , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos/análise , Extração Líquido-Líquido , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
Radiotherapy and chemotherapy are the standard interventions for cancer patients, although cancer cells often develop radio- and/or chemoresistance. Hyperthermia reduces tumor resistance and induces immune responses resulting in a better prognosis. We have previously described a method to induce tumor cell death by local hyperthermia employing pegylated reduced graphene oxide nanosheets and near infrared light (graphene-induced hyperthermia, GIHT). The spatiotemporal exposure/release of heat shock proteins (HSP), high group mobility box 1 protein (HMGB1), and adenosine triphosphate (ATP) are reported key inducers of immunogenic cell death (ICD). We hypothesize that GIHT decisively contributes to induce ICD in irradiated melanoma B16F10 cells, especially in combination with radiotherapy. Therefore, we investigated the immunogenicity of GIHT alone or in combination with radiotherapy in melanoma B16F10 cells. Tumor cell death in vitro revealed features of apoptosis that is progressing fast into secondary necrosis. Both HSP70 and HMGB1/DNA complexes were detected 18 hours post GIHT treatment, whereas the simultaneous release of ATP and HMGB1/DNA was observed only 24 hours post combined treatment. We further confirmed the adjuvant potential of these released DAMPs by immunization/challenge experiments. The inoculation of supernatants of cells exposed to sole GIHT resulted in tumor growth at the site of inoculation. The immunization with cells exposed to sole radiotherapy rather fostered the growth of secondary tumors in vivo. Contrarily, a discreet reduction of secondary tumor volumes was observed in mice immunized with a single dose of cells and supernatants treated with the combination of GIHT and irradiation. We propose the simultaneous release of several DAMPs as a potential mechanism fostering anti-tumor immunity against previously irradiated cancer cells.
RESUMO
Chloramphenicols, nitroimidazoles, lincosamides, and macrolides are common antibiotics used in veterinary medicine. Overdoses of these drugs will lead to residual substances in animal-derived foods and accumulate in the body through the food chain, thereby exerting adverse effects on human health. Therefore, regulation of veterinary drug levels is imperative to ensure the quality of animal-derived foods and safeguard the health of consumers. In this study, a method based on ultra performance liquid chromatography-tandem mass spectrometry coupled with solid phase extraction (SPE-UPLC-MS/MS) was developed for the simultaneous determination of eight prohibited and restricted veterinary drugs and three metabolite residues across four categories (chloramphenicols, nitroimidazoles, lincosamides, and macrolides) in eggs, liquid milk, chicken, and freshwater fish. The main factors affecting the response, recovery, and sensitivity of the method, such as the type and pH values of the extraction solvent, dilution solution for the analytes, type of chromatographic column, and type and proportion of the mobile phase, were optimized during sample pretreatment and instrument analysis. The samples were hydrolyzed and dispersed in 0.1 mol/L phosphate buffer solutions (pH 9.0) and extracted with acetonitrile. The extract was further extracted using ethyl acetate. After centrifugation, the supernatant ethyl acetate was concentrated to near dryness in nitrogen below 40 â. The residue was dissolved in 0.3 mL methanol, followed by the addition of 5.7 mL phosphate buffer solution. After shaking, the solutions were purified and enriched on an Oasis HLB SPE column. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 µm) at a column temperature of 40 â with a flow rate of 0.4 mL/min. The injection volume was 10 µL. Gradient elution was carried out with methanol and 0.1% formic acid aqueous solution as the mobile phases. Multiple reaction monitoring (MRM) was conducted in the positive and negative electrospray ionization modes. The isotope internal standard method was used for quantitative analysis. Under optimal conditions, each analyte showed a good linear relationship in each range, and the correlation coefficient (R2) was greater than 0.99. The limits of detection (LODs) ranged from 0.050 to 0.50 µg/kg, and the limits of quantification (LOQs) ranged from 0.20 to 1.5 µg/kg. With eggs, freshwater fish, chicken, and liquid milk as the matrix samples, the recoveries in spiked blank samples were determined at different addition levels in compliance with the current legislation. The average recoveries of the 11 analytes were 65.3% to 108%. The relative standard deviations (RSDs) were between 0.40% and 21%. The matrix effects of the analytes were between 0.0124% and 46.80% in four different samples after purification on the Oasis HLB column. The practicality of the proposed approach for routine analyses of the eight prohibited and restricted veterinary drugs, and three metabolite residuals was evaluated by applying it to the determination of these compounds in animal-derived food samples. The samples, including 80 eggs, 80 chicken, 40 liquid milk, and 32 freshwater fish, were procured from a supermarket and a farm product market. The results of the positive samples were consistent with those observed with the standard methods. The method described herein is easy to operate, sensitive, and accurate. It is suitable for the simultaneous and rapid determination of various prohibited and restricted veterinary drug residues and metabolites in animal-derived foods.
Assuntos
Resíduos de Drogas/análise , Contaminação de Alimentos , Drogas Veterinárias , Animais , Cromatografia Líquida de Alta Pressão , Ovos , Peixes , Contaminação de Alimentos/análise , Leite , Produtos Avícolas , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Drogas Veterinárias/análiseRESUMO
PURPOSE: Obstructive Meibomian gland dysfunction (MGD) is one of the leading causes of evaporative dry eye disease. Meibomian glands at the eyelid secrete lipids that prevent evaporation of the aqueous tear film. The pathogenesis of obstructive MGD is incompletely understood to date. Herein, we aim to investigate the pathogenesis of obstructive MGD using murine and human samples with various forms of ocular surface inflammation. METHOD: The presence of Neutrophil extracellular Traps (NETs) was detected with immunofluorescence analysis of ocular surface discharge and biopsy samples from patients with blepharitis. Tear fluid from patients with MGD and blepharitis were evaluated for the presence of inflammatory mediators using bead based immunoassay. Murine model of allergic eye disease (AED) was performed to investigate the role of NETs in MG occlusion. RESULTS: we show that the ocular discharge from patients with blepharitis contains aggregated neutrophil extracellular traps (aggNETs). Furthermore, the ducts of human Meibomian glands affected by blepharitis were largely congested by aggNETs. Tear fluid from patients with MGD showed elevated neutrophil chemoattractants (C5a, IL6, IL8 and IL18). C5a and IL8 correlated with the degree of deficiency of tear fluid. In the murine model of allergic eye disease (AED), aggNETs accumulated in the MG leading to occlusion of their ducts and the retrograde pent-up of the fluid followed by acinar atrophy. Constraining aggNET formation by genetic or pharmacological inhibition of peptidyl arginine deiminase type 4 (PADI4) effectively reduced MG damage. CONCLUSION: We conclude that aggNETs occlude MG causing MGD after ocular surface inflammation.
Assuntos
Síndromes do Olho Seco , Armadilhas Extracelulares , Doenças Palpebrais , Animais , Humanos , Inflamação , Glândulas Tarsais , Camundongos , LágrimasRESUMO
OBJECTIVE: A investigate on the nutritional quality of the prefabricated beef steak in the market was conducted, aiming to provide scientific basis for consumers in the consumption of prefabricated beef steaks. METHODS: The basic nutriments, amino acids, fatty acids, vitamins and mineral elements of 8 kinds of prefabricated beef steaks were analyzed and evaluated. RESULTS: It was showed that the contents of crude ash(1. 3-2. 4 g/100 g edible) and crude fat(1. 3-10. 0 g/100 g edible) in the 8 kinds of prefabricated beef steaks were higher than those of raw beef, while the contents of protein(11. 4-17. 2 g/100 g edible) was lower than those of raw beef. All the 8 kinds of prefabricated beef steaks had 18 kinds of amino acids and the content of histidine in children's steak(0. 57 g/100 g edible) was the highest. With the exception of snowflake steak and bulk steak, the first limiting amino acid of other 6 kinds of prefabricated beef steaks was valine. The essential amino acid composition of the 8 kinds of prefabricated steaks was close to that of FAO/WHO standard protein. This meant they were all high quality protein sources. The proportion of saturated fatty acids(48. 0%-62. 0%) was the highest, followed by monounsaturated fatty acids(32. 2%-46. 6%) and polyunsaturated fatty acids(0. 7%-7. 6%). All the 8 kinds of prepared beef steak didn't meet the Sâ¶Mâ¶P standard recommended by American Heart Association. There were differences in the content of vitamins among 8 kinds of prefabricated beef steaks. In terms of mineral elements, the content of Na(344-689 mg/100 g edible), Ca(6-15 mg/100 g edible), Fe(1. 0-1. 9 mg/100 g edible), Cu(0. 029-0. 050 mg/100 g edible), Mn(0. 056-0. 183 mg/100 g edible) in prefabricated steak was higher than that in raw beef. CONCLUSION: There were interspecific differences in the nutritional element content and quality among different kinds of prefabricated steaks. Compared with the raw beef, the fat content was higher and protein content was lower, the composition of fatty acids was less reasonable than fish and pork.
Assuntos
Ácidos Graxos , Carne , Animais , Bovinos , Criança , Ácidos Graxos/análise , Ácidos Graxos Monoinsaturados , Humanos , Carne/análise , Valor Nutritivo , Estados UnidosRESUMO
Pentachlorophenol (PCP) was used in large quantities, and mainly for killing the intermediate host snails of schistosome in China, thereby resulting in ubiquitous PCP residue in the environment. However, studies considering the carcinogenicity of PCP for humans mainly focused on occupational workers, and the actual carcinogenicity of PCP for general population is uncertain. To investigate the association between cancer risks and PCP exposure in a community population, an ecological study was conducted in three contaminated areas along the Yangtze River. Standardized rate ratio (SRR) was calculated to represent the risk of cancer incidence, by using incidence in the low PCP exposure category as the reference group. A total of 15,962 cancer records were collected, and 76 water samples and 213 urine samples in three areas were examined. Our findings suggested that compared with the low PCP group, the high PCP group had significantly excessive incidences of various cancers related to different organs including lymph (SRR = 19.44, 95% CI = 15.00-25.19), blood (SRR = 17.24, 95% CI = 12.92-23.01), nasopharynx (SRR = 3.97, 95% CI = 3.75-4.21), gallbladder (SRR = 3.46, 95% CI = 3.09-3.87), pancreas (SRR = 3.41, 95% CI = 3.07-3.79), respiratory system (SRR = 3.41, 95% CI = 3.27-3.57) and liver (SRR = 3.31, 95% CI = 3.09-3.56). Taken together, our present study provides evidence that general community population exposed to high level of PCP exhibits a broader spectrum of increased cancer risks as compared to occupational groups.
Assuntos
Neoplasias/induzido quimicamente , Pentaclorofenol/toxicidade , Adulto , China/epidemiologia , Água Potável/química , Feminino , Humanos , Incidência , Masculino , Neoplasias/epidemiologia , Pentaclorofenol/análise , Medição de Risco , RiosRESUMO
OBJECTIVE: To study the change of airway mucus secretion under a high temperature and humidity environment, and explore the effects of hot-humid stress and acclimation on the morbidity and mortality of respiratory disease. METHODS: Forty-five BABL/c mice were randomly divided into five groups:normal group, hot-humid group I, hot-humid group â ¡, hot-humid group â ¢, hot-humid group IV, with 9 mice in each group. Mice in normal group were continuously placed in the common environment and sacrificed after 7 days. Mice in other groups were housed in a temperature-and-humidity-controlled environment (33â±0.5â, 95%±5%). Mice in hot-humid group I, hot-humid group â ¡, hot-humid group â ¢ and hot-humid group IV were sacrificed after 12 hours, 24 hours, 4 days, 7 days respectively. The protein expression of mucin 5AC(MUC5AC)ãepidermal growth factor receptor (EGFR)ãaquaporin 1(AQP1) and aquaporin 5(AQP5) in lung were tested by immunohistochemisty. RESULTS: After housed in a high temperature and humidity environment, immunohistochemisty revealed a significant increase of AQP5 12 h later, MUC5AC and EGFR 24 h later, compared with normal group(P<0.05). There was a significant decrease of MUC5AC 7 d later, compared with normal group(P<0.05). There was no significant difference in MUC5AC, EGFR and AQP5 expression among all groups at other time points. There was no difference of AQP1 in humid heat groups, compared with normal group, but a significant decrease in humid heat â ¢ and IV groups, compared with humid heat I and â ¡ groups. CONCLUSIONS: These findings indicate that hot-humid stress induces mucus hypersecretion in airways, which may be related to the up-regulation of EGFR and down-regulation of AQP5 in MUC5AC. Although acclimation mitigates above-mentioned response, a series of more complex responses may be induced if still in the hot-humid environment.
Assuntos
Aclimatação , Temperatura Alta , Umidade , Pulmão/fisiologia , Mucina-5AC/fisiologia , Muco/fisiologia , Animais , Aquaporina 1/fisiologia , Aquaporina 5/fisiologia , Receptores ErbB/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Estresse FisiológicoRESUMO
Widespread use of pentachlorophenol (PCP) in schistosomiasis endemic areas had led to ubiquitous exposure to PCP and its residues. Numerous studies had revealed that occupational PCP exposure probably increased risk of cancers, but whether long-term community-level exposure to PCP generates the similarly carcinogenic effect, seldom studies focused on it. This study was to explore the cancer risks of long-term community-level PCP exposure from drinking water in a Chinese general population. Incident (2009-2012) cancer records were identified by local government national registry. And PCP concentration of raw drinking water samples in each district was measured by GC-MS/MS analysis for further division of three PCP exposure categories by interquartile range (high vs. medium vs. low). Internal comparisons were performed, and standard rate ratio was calculated to describe the relationship between PCP exposure and cancer risks by using low-exposure group as the reference group. PCP was detected in all 27 raw drinking water samples ranging from 11.21 to 684.00 ng/L. A total of 6,750 cases (4,409 male and 2,341 female cases) were identified, and age-standardized rate (world) was 154.95 per 100,000 person-years. The cancer incidence for the high-exposure group was remarkably high. Internal comparisons indicated that high PCP exposure might be positively associated with high cancer risks in the community population, particularly for leukemia (SRR = 5.93, 95 % CI = 5.24-6.71), maligant lymphoma (SRR = 2.27, 95 % CI = 2.10-2.54), and esophageal cancer (SRR = 2.42, 95 % CI = 2.35-2.50). Long-term community-level exposure to PCP was probably associated with hemolymph neoplasm, neurologic tumors, and digestive system neoplasm.