RESUMEN
To report two novel TTN variants associated with fetal recessive titinopathy, thereby broadening the range of TTN variants that can lead to titinopathy. Clinical information on the fetus and parents was gathered, and genomic DNAs were extracted from the fetal tissue and family members' peripheral blood samples. Exome sequencing on fetal DNA was performed and following bioinformatics analysis, the suspected pathogenic variants were confirmed through Sanger sequencing. Prenatal ultrasound performed at 29 weeks of gestation revealed hydrops fetalis, decreased fetal movements, multiple joint contractures and polyhydramnios. Intrauterine fetal death was noted in the third trimester. Exome sequencing revealed compound heterozygous variants in the TTN gene: a paternally inherited allele c.101227C>T (p.Arg33743Ter) and a maternally inherited c.104254C>T (p.Gln34752Ter) allele. These variants have not been previously reported and are evaluated to be likely pathogenic according to the American College of Medical Genetics and Genomics guidelines. We report a fetus with hydrops fetalis and arthrogryposis multiplex congenita associated with a compound heterozygote in the TTN gene. Our report broadens the clinical and genetic spectrum associated with the TTN-related conditions.
Asunto(s)
Artrogriposis , Hidropesía Fetal , Embarazo , Femenino , Humanos , Hidropesía Fetal/diagnóstico por imagen , Hidropesía Fetal/genética , Exones , Artrogriposis/diagnóstico por imagen , Artrogriposis/genética , Tercer Trimestre del Embarazo , Feto/diagnóstico por imagen , Conectina/genéticaRESUMEN
Thrombin generation is pivotal to both physiological blood clot formation and pathological development of disseminated intravascular coagulation (DIC). In critical illness, extensive cell damage can release histones into the circulation, which can increase thrombin generation and cause DIC, but the molecular mechanism is not clear. Typically, thrombin is generated by the prothrombinase complex, comprising activated factor X (FXa), activated cofactor V (FVa), and phospholipids to cleave prothrombin in the presence of calcium. In this study, we found that in the presence of extracellular histones, an alternative prothrombinase could form without FVa and phospholipids. Histones directly bind to prothrombin fragment 1 (F1) and fragment 2 (F2) specifically to facilitate FXa cleavage of prothrombin to release active thrombin, unlike FVa, which requires phospholipid surfaces to anchor the classical prothrombinase complex. In vivo, histone infusion into mice induced DIC, which was significantly abrogated when prothrombin F1 + F2 were infused prior to histones, to act as decoy. In a cohort of intensive care unit patients with sepsis (n = 144), circulating histone levels were significantly elevated in patients with DIC. These data suggest that histone-induced alternative prothrombinase without phospholipid anchorage may disseminate intravascular coagulation and reveal a new molecular mechanism of thrombin generation and DIC development. In addition, histones significantly reduced the requirement for FXa in the coagulation cascade to enable clot formation in factor VIII (FVIII)- and FIX-deficient plasma, as well as in FVIII-deficient mice. In summary, this study highlights a novel mechanism in coagulation with therapeutic potential in both targeting systemic coagulation activation and correcting coagulation factor deficiency.
Asunto(s)
Coagulación Intravascular Diseminada/metabolismo , Factor V/metabolismo , Factor X/metabolismo , Factor Xa/metabolismo , Histonas/metabolismo , Animales , Coagulación Sanguínea , Humanos , Ratones , Ratones Endogámicos C57BL , Tromboplastina/metabolismoRESUMEN
The analysis of nerve agents is the focus of chemical warfare agent determination because of their extreme toxicity. A classical chemical colorimetric method, namely, the Schoenemann reaction, has been developed to detect G agents; however, it has not been utilized for VX analysis mainly because of its low peroxyhydrolysis rate. In this study, based on the mechanism of the Schoenemann reaction, a novel rapid quantitative determination method for VX was developed by optimizing the reaction conditions, such as concentrations of peroxide and the indicator, temperature, and reaction time. Using 2 ml 0.5 wt% sodium perborate as the peroxide source, 1 ml 0.1 wt% benzidine hydrochloride as the indicator, and 1 ml acetone as the co-solvent, VX and GD in ethanol or water solutions could be quantitatively analyzed within 15 min at 60°C. Further experiments based on 31P NMR spectroscopy confirmed the existence of a peroxyphosphate intermediate during the GD assay. This quantitative colorimetry system for VX and GD analysis can be developed as a portable device for the water samples in fieldwork applications.
Asunto(s)
Sustancias para la Guerra Química , Compuestos Organotiofosforados , Colorimetría , Sustancias para la Guerra Química/análisis , Compuestos Organotiofosforados/análisis , Compuestos Organotiofosforados/química , Peróxidos , AguaRESUMEN
C-reactive protein (CRP) can increase up to 1000-fold in blood and form complexes with very low density lipoproteins (VLDL). These complexes are associated with worse outcomes for septic patients, and this suggests a potential pathological role in sepsis. Complex formation is heightened when CRP is over 200 mg/l and levels are associated with the severity of sepsis and blood bacterial culture positivity. Using a mouse bacteremia model, blood bacterial clearance can be delayed by i.v. injection of CRP-VLDL complexes. Complexes are more efficiently taken up by activated U937 cells in vitro and Kupffer cells in vivo than VLDL alone. Both in vitro-generated and naturally occurring CRP-VLDL complexes reduce phagocytosis of bacteria by activated U937 cells. Fcγ and scavenger receptors are involved and a competitive mechanism for clearance of CRP-VLDL complexes and bacteria is demonstrated. Interaction of phosphocholine groups on VLDL with CRP is the major driver for complex formation and phosphocholine can disrupt the complexes to reverse their inhibitory effects on phagocytosis and bacterial clearance. Increased formation of CRP-VLDL complexes is therefore harmful and could be a novel target for therapy in sepsis.
Asunto(s)
Bacteriemia/metabolismo , Proteína C-Reactiva/metabolismo , Macrófagos del Hígado/fisiología , Lipoproteínas VLDL/metabolismo , Sepsis/metabolismo , Anciano , Animales , Proteína C-Reactiva/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Mutación/genética , Fagocitosis , Fosforilcolina/metabolismo , Unión Proteica , Receptores de IgG/metabolismo , Células U937RESUMEN
BACKGROUND: Neutrophil extracellular traps (NETs) facilitate bacterial clearance but also promote thrombosis and organ injury in sepsis. We quantified ex vivo NET induction in septic humans and murine models of sepsis to identify signalling pathways that may be modulated to improve outcome in human sepsis. METHODS: NET formation in human donor neutrophils was quantified after incubation with plasma obtained from patients with sepsis or systemic inflammation (double-blinded assessment of extracellular DNA using immunofluorescence microscopy). NET formation (% neutrophils forming NETs) was correlated with plasma cytokine levels (MultiPlex assay). Experimental sepsis (caecal ligation and puncture or intraperitoneal injection of Escherichia coli) was assessed in C57/BL6 male mice. The effect of pharmacological inhibition of CXCR1/2 signalling (reparixin) on NET formation, organ injury (hepatic, renal, and cardiac biomarkers), and survival in septic mice was examined. RESULTS: NET formation was higher after incubation with plasma from septic patients (median NETs=25% [10.5-46.5%]), compared with plasma obtained from patients with systemic inflammation (14% [4.0-23.3%]; P=0.02). Similar results were observed after incubation of plasma from mice with neutrophils from septic non-septic mice. Circulating CXCR1/2 ligands correlated with NETosis in patients (interleukin-8; r=0.643) and mice (macrophage inflammatory protein-2; r=0.902). In experimental sepsis, NETs were primarily observed in the lungs, correlating with fibrin deposition (r=0.702) and lung injury (r=0.692). Inhibition of CXCR1/2 using reparixin in septic mice reduced NET formation, multi-organ injury, and mortality, without impairing bacterial clearance. CONCLUSION: CXCR1/2 signalling-induced NET formation is a therapeutic target in sepsis, which may be guided by ex vivo NET assays.
Asunto(s)
Trampas Extracelulares/metabolismo , Inflamación/complicaciones , Sepsis/complicaciones , Sulfonamidas/farmacología , Trombosis/prevención & control , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación/tratamiento farmacológico , Lesión Pulmonar/etiología , Lesión Pulmonar/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Receptores de Interleucina-8A/antagonistas & inhibidores , Receptores de Interleucina-8B/antagonistas & inhibidores , Estudios Retrospectivos , Sepsis/tratamiento farmacológico , Sepsis/mortalidad , Trombosis/etiologíaRESUMEN
Chloramine-T, especially its solution in weak acidity, is one of the decontaminants for chemical warfare agents (CWAs), HD, and VX. A high CWAs recovery from decontamination (decon) sample via pretreatment was essential for evaluating decontamination effects. This paper performed experiments to optimize pretreatment methods to extract residual CWAs from chloramine-T decon samples before GC analysis. Effects of two neutralization methods, destroying decon activity by 15% Na2SO3 or decreasing decon activity by 3% NH3·H2O or 4% NaOH, were studied. Results showed they were all suitable for the HD decon sample, but only 4% NaOH was ideal for the VX decon sample. As for extractant, compared with dichloromethane, petroleum ether was more suitable for recovering CWAs from decon samples. A high recovery above 80% could be obtained for HD and VX samples ranging from 10 mg/L to 10,000 mg/L when optimized neutralization and extraction methods were simultaneously carried out.
Asunto(s)
Sustancias para la Guerra Química , Compuestos Organotiofosforados , Descontaminación/métodos , Hidróxido de Sodio , Sustancias para la Guerra Química/análisis , Compuestos Organotiofosforados/análisisRESUMEN
Active chlorine decontaminants like hypochlorite are used to destroy chemical warfare agents (CWAs) such as HD, VX and GD due to the former's strong oxidation capacity and high nucleophilicity. In this paper, experiments were performed to identify the main factors affecting agent recovery from decon water. Based on the results, a method to recover residual CWAs from hypochlorite decon water before quantitative determination by GC was developed. The results showed that the extraction solvent was a critical determinant of high CWA recovery. Dichloromethane was more suitable than petroleum either, especially for samples containing GD or low residual CWAs. For VX-containing samples, the use of an alkali solution improved VX recovery. Neutralization was also important for a high CWA recovery, especially for samples with low CWA concentrations and/or strong decontaminant reactivity. The use of 15% sodium sulfite as the neutralization solution gave the best results for hypochlorite decon water. When the optimized conditions of simultaneous sodium sulfite neutralization and dichloromethane extraction were used, the recovery of HD, VX and GD in hypochlorite decon water was greater than 85% at a concentration range of 20 mg/L to 10,000 mg/L.
Asunto(s)
Sustancias para la Guerra Química , Ácido Hipocloroso , Sustancias para la Guerra Química/análisis , Descontaminación , Compuestos Organotiofosforados , AguaRESUMEN
The pathological roles of bacterial DNA have been documented many decades ago. Bacterial DNAs are different from mammalian DNAs; the latter are heavily methylated. Mammalian cells have sensors such as TLR-9 to sense the DNAs with nonmethylated CpGs and distinguish them from host DNAs with methylated CpGs. Further investigation has identified many other types of DNA sensors distributed in a variety of cellular compartments. These sensors not only sense foreign DNAs, including bacterial and viral DNAs, but also sense damaged DNAs from the host cells. The major downstream signalling pathways includeTLR-9-MyD88-IKKa-IRF-7/NF-κB pathways to increase IFN/proinflammatory cytokine production, STING-TBK1-IRF3 pathway to increase IFN-beta, and AIM2-ASC-caspas-1 pathway to release IL-1beta. The major outcome is to activate host immune response by inducing cytokine production. In this review, we focus on the roles and potential mechanisms of DNA sensors and downstream pathways in sepsis. Although bacterial DNAs play important roles in sepsis development, bacterial DNAs alone are unable to cause severe disease nor lead to death. Priming animals with bacterial DNAs facilitate other pathological factors, such as LPS and other virulent factors, to induce severe disease and lethality. We also discuss compartmental distribution of DNA sensors and pathological significance as well as the transport of extracellular DNAs into cells. Understanding the roles of DNA sensors and signal pathways will pave the way for novel therapeutic strategies in many diseases, particularly in sepsis.
Asunto(s)
ADN Bacteriano/metabolismo , FN-kappa B/metabolismo , Sepsis/metabolismo , Animales , Humanos , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVES: Multiple organ dysfunction syndrome is characterized by simultaneous multiple organ failure, which is the leading cause of death in acute critically ill patients. However, what mediates multiple organ dysfunction syndrome is not fully understood. The discovery of toxic effects by extracellular histones on different individual organs strongly suggests their involvement in multiple organ dysfunction syndrome. In this study, we investigate whether circulating histones are major mediators of multiple organ dysfunction syndrome in acute critical illnesses. DESIGN: Combination of retrospective clinical studies and animal models with intervention. SETTING: ICU in a tertiary hospital and research laboratories. PATIENTS: Four hundred and twenty ICU patients, including sepsis (140), severe trauma (63), severe pancreatitis (89), and other admission diagnoses (128). LABORATORY INVESTIGATION: Cells from major organs are treated with calf thymus histones or histone-containing sera. Animal models for sepsis, trauma, and acute pancreatitis are treated with antihistone reagents. INTERVENTION: Antihistone reagents in in vitro, ex vivo, and animal models. MEASUREMENT AND MAIN RESULTS: Retrospective analysis of a prospectively recruited ICU cohort demonstrated a strong correlation between circulating histones and organ injury markers and Sequential Organ Failure Assessment scores. Ex vivo experiments showed that patient sera containing high histone levels were toxic to cultured cells from different origins, suggesting their universal toxicity to multiple organs. Animal models of sepsis, trauma, and pancreatitis further demonstrated a temporal correlation between histone levels and disease severity and multiple organ injury. Importantly, antihistone reagents, that is, antihistone single-chain variable fragment and nonanticoagulant heparin, could dramatically reduce multiple organ injury, particularly of the heart and lungs, and improve survival in mouse models. CONCLUSIONS: High levels of circulating histones are major mediators of multiple organ dysfunction syndrome. Our results indicate that monitoring upon ICU admission could inform on disease severity and developing antihistone therapy holds great potential of reducing multiple organ dysfunction syndrome and improving survival of critically ill patients.
Asunto(s)
Enfermedad Crítica , Histonas/sangre , Insuficiencia Multiorgánica/sangre , Sepsis/sangre , Biomarcadores/sangre , Humanos , Unidades de Cuidados Intensivos , Puntuaciones en la Disfunción de Órganos , Estudios RetrospectivosRESUMEN
Background and Objective: Chronic unexpected mild stress (CUMS) destroys synaptic plasticity of hippocampal regenerated neurons that may be involved in the occurrence of poststroke depression. Astrocytes uptake glutamate at the synapse and provide metabolic support for neighboring neurons. Currently, we aim to investigate whether CUMS inhibits synaptic formation of regenerated neurons through a glutamate transporter, GLT-1, of astrocytes in the ischemic stroke rats. Method: We exposed the ischemic stroke rats to ceftriaxone, during the CUMS intervention period to determine the effects of GLT-1 on glutamate circulation by immunofluorescence and mass spectrometry and its influences to synaptic plasticity by western blot and transmission electron microscopy. Result: CUMS evidently reduced the level of astroglial GLT-1 in the hippocampus of the ischemic rats (p < 0.05), resulting in smaller amount of glutamate being transported into astrocytes surrounding synapses (p < 0.05), and then expression of synaptophysin was suppressed (p < 0.05) in hippocampal dentate gyrus. The ultrastructures of synapses in dentate gyrus were adversely influenced including decreased proportion of smile synapses, shortened thickness of postsynaptic density, reduced number of vesicles, and widened average distance of the synaptic cleft (all p < 0.05). Moreover, ceftriaxone can promote glutamate circulation and synaptic plasticity (all p < 0.05) by raising astroglial GLT-1 (p < 0.05) and then improve depressive behaviors of the CUMS-induced model rats (p < 0.05). Conclusion: Our study shows that CUMS destroys synaptic plasticity of regenerated neurons in the hippocampus through a glutamate transporter, GLT-1, of astrocytes in the ischemic stroke rats. This may indicate one potential pathogenesis of poststroke depression.
Asunto(s)
Astrocitos/fisiología , Isquemia Encefálica/fisiopatología , Transportador 2 de Aminoácidos Excitadores/metabolismo , Hipocampo/fisiopatología , Plasticidad Neuronal , Neuronas/fisiología , Estrés Psicológico/fisiopatología , Accidente Cerebrovascular/fisiopatología , Animales , Isquemia Encefálica/complicaciones , Ácido Glutámico/metabolismo , Masculino , Ratas Sprague-Dawley , Estrés Psicológico/complicaciones , Accidente Cerebrovascular/complicacionesRESUMEN
OBJECTIVE: To investigate the impact of circulating histones on cardiac injury and dysfunction in a murine model and patients with sepsis. DESIGN: Prospective, observational clinical study with in vivo and ex vivo translational laboratory investigations. SETTING: General ICU and university research laboratory. SUBJECTS: Sixty-five septic patients and 27 healthy volunteers. Twelve-week-old male C57BL/6N mice. INTERVENTIONS: Serial blood samples from 65 patients with sepsis were analyzed, and left ventricular function was assessed by echocardiography. Patients' sera were incubated with cultured cardiomyocytes in the presence or absence of antihistone antibody, and cellular viability was assessed. Murine sepsis was initiated by intraperitoneal Escherichia coli injection (10(8) colony-forming unit/mouse) in 12-week-old male C57BL/6N mice, and the effect of antihistone antibody (10 mg/kg) was studied. Murine blood samples were collected serially, and left ventricular function was assessed by intraventricular catheters and electrocardiography. MEASUREMENTS AND MAIN RESULTS: Circulating histones and cardiac troponins in human and murine plasma were quantified. In 65 patients with sepsis, circulating histones were significantly elevated compared with healthy controls (n = 27) and linearly correlated with cardiac troponin T levels (rs = 0.650; p < 0.001), noradrenaline doses required to achieve hemodynamic stability (rs = 0.608; p < 0.001), Sequential Organ Failure Assessment scores (p = 0.028), and mortality (p = 0.008). In a subset of 36 septic patients without prior cardiac disease, high histone levels were significantly associated with new-onset left ventricular dysfunction (p = 0.001) and arrhythmias (p = 0.01). Left ventricular dysfunction only predicted adverse outcomes when combined with elevated histones or cardiac troponin levels. Furthermore, patients' sera directly induced histone-specific cardiomyocyte death ex vivo, which was abrogated by antihistone antibodies. In vivo studies on septic mice confirmed the cause-effect relationship between circulating histones and the development of cardiac injury, arrhythmias, and left ventricular dysfunction. CONCLUSION: Circulating histones are novel and important mediators of septic cardiomyopathy, which can potentially be utilized for prognostic and therapeutic purposes.
Asunto(s)
Cardiopatías/sangre , Cardiopatías/microbiología , Histonas/sangre , Sepsis/sangre , Sepsis/complicaciones , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
In this work, nanostructured copper oxide of varied morphologies and high surface area were prepared by calcination of copper oxalate precursors, and were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis and differential thermal analysis, and nitrogen adsorption-desorption measurements. The sphere-like CuO (specific surface area: 73 m(2) g(-1)) functionalized QCM resonators were fabricated and explored for HCN sensing. The sensitivity (10 s HCN exposure) of sphere-like CuO functionalized QCM resonators reached as high as 6.53 Hz µg(-1). The reproducibility and stability of sphere-like CuO functionalized QCM resonators was excellent, and the selectivity was very high with a converse response to examined common chemicals. The high surface area CuO functionalized QCM sensors may be applicable for HCN gas sensing.
RESUMEN
A highly sensitive and specific detection of 2,4,6-trinitrotoluene (TNT), a typical nitrated aromatic explosive, was demonstrated by a silicon nanowire (SiNW) array sensor. The SiNW array devices were self-assembled and functionalized with the anti-TNT peptide to obtain unique sensitivity toward TNT. Also, the effect of the biointerfacing linker's chemistry and Debye screening with varied ionic strength of phosphate buffer solution (PBS) on TNT binding response signals were investigated. The optimization of the peptide-functionalized SiNW array sensor showed high sensitivity for TNT with a detection limit of 0.2 fM, the highest sensitivity reported to date. These initial promising results may help accelerate the development of portable sensors for femtomolar level TNT detection.
RESUMEN
Most patients who die of cancer do so from its metastasis to other organs. The calcium-binding protein S100A4 can induce cell migration/invasion and metastasis in experimental animals and is overexpressed in most human metastatic cancers. Here, we report that a novel inhibitor of S100A4 can specifically block its increase in cell migration in rat (IC50, 46 µM) and human (56 µM) triple negative breast cancer (TNBC) cells without affecting Western-blotted levels of S100A4. The moderately-weak S100A4-inhibitory compound, US-10113 has been chemically attached to thalidomide to stimulate the proteasomal machinery of a cell. This proteolysis targeting chimera (PROTAC) RGC specifically eliminates S100A4 in the rat (IC50, 8 nM) and human TNBC (IC50, 3.2 nM) cell lines with a near 20,000-fold increase in efficiency over US-10113 at inhibiting cell migration (IC50, 1.6 nM and 3.5 nM, respectively). Knockdown of S100A4 in human TNBC cells abolishes this effect. When PROTAC RGC is injected with mouse TNBC cells into syngeneic Balb/c mice, the incidence of experimental lung metastases or local primary tumour invasion and spontaneous lung metastasis is reduced in the 10-100 nM concentration range (Fisher's Exact test, p ≤ 0.024). In conclusion, we have established proof of principle that destructive targeting of S100A4 provides the first realistic chemotherapeutic approach to selectively inhibiting metastasis.
Asunto(s)
Proteína de Unión al Calcio S100A4 , Neoplasias de la Mama Triple Negativas , Animales , Humanos , Ratones , Ratas , Línea Celular Tumoral , Movimiento Celular , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteína de Unión al Calcio S100A4/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Quimera Dirigida a la Proteólisis/metabolismo , Quimera Dirigida a la Proteólisis/farmacologíaRESUMEN
Degradation of sulfur mustard (HD), S-2-(di-isopropylamino)ethyl O-ethyl methylphosphonothioate (VX) and Soman (GD) in water by bleaching powder was investigated. The degradation products were comprehensively analyzed by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and ion chromatography. Degradation pathways were deduced based on the identified products. The product analysis results indicated that HD could be degraded through oxidation and chlorination reactions, and a small portion of sulfur atoms could be mineralized into SO(4)(2-) ion. Oxidative degradation of VX could finally generate O-ethyl methylphosphonate acid (EMPA), sulfonic acids, SO(4)(2-) and NO(3)(-) ions. GD would be converted into non-toxic pinacolyl methylphosphonate via nucleophilic substitution.
Asunto(s)
Sustancias para la Guerra Química/química , Aminopiridinas/química , Cromatografía de Gases y Espectrometría de Masas , Gas Mostaza/química , Compuestos Organotiofosforados/química , Oxidación-Reducción , Soman/química , Sulfonamidas/química , Ácidos Sulfónicos/químicaRESUMEN
Extracellular histones are cytotoxic to various cells and have been extensively proven a vital mediator of multiple organ injuries. However, the effect of extracellular histones on the intestine remains largely unknown. This study aimed to clarify the effect of extracellular histones on the intestine. IEC-6, a cell line of rat small intestinal epithelial crypt, and C57BL/6 or ICR mice were treated with histones. The IEC-6 cells treated with histones from 20 µg/mL to 200 µg/mL for 0-24 h displayed a decline of cell viability and an increase of cell death in a concentration- and time-dependent manner. Moreover, histones (100 µg/mL) induced IEC-6 apoptosis through activating caspase 3 and necroptosis through up-regulation of receptor-interacting serine/threonine protein kinase 1 and 3 (RIPK1 and RIPK3), phosphorylated mixed-lineage kinase domain-like protein (p-MLKL) along with the decrease of caspase-8. Histones treatment disturbed zonular occludens 1 (ZO-1) expression and increased permeability of IEC-6 cell monolayer. In vivo, histones 50 mg/kg injection caused mice intestinal edema, loss apex of villus, epithelial lifting down the sides of the villi, and increased neutrophil infiltration. Elevation of serum intestinal fatty acid binding protein (I-FABP), d-lactate, or Diamine oxidase (DAO) and loss of tight junction protein, ZO-1, at 3 h and 6 h after histones injection strongly indicated severe intestinal epithelium injury, which led to increased permeability of the intestine. In conclusion, extracellular histones cause intestinal epithelial damage via direct cytotoxicity. Consequently, intestinal epithelial tight junction and barrier integrity are disrupted, which may play pivotal roles in diverse diseases.
Asunto(s)
Histonas , Mucosa Intestinal , Animales , Histonas/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Necroptosis , RatasRESUMEN
BACKGROUND: Multiple organ injury and dysfunction often occurs in acute critical illness and adversely affects survival. However, in patients who survive, organ function usually recovers without permanent damage. It is, therefore, likely that there are reversible mechanisms, but this is poorly understood in the pathogenesis of multiple organ dysfunction syndrome (MODS). AIMS: Based on our knowledge of extracellular histones and pneumolysin, as endogenous and exogenous pore-forming toxins, respectively, here we clarify if the extent of cell membrane disruption and recovery is important in MODS. METHODS: This is a combination of retrospective clinical studies of a cohort of 98 patients from an intensive care unit (ICU) in a tertiary hospital, with interventional animal models and laboratory investigation. RESULTS: In patients without septic shock and/or disseminate intravascular coagulation (DIC), circulating histones also strongly correlated with sequential organ failure assessment (SOFA) scores, suggesting their pore-forming property might play an important role. In vivo, histones or pneumolysin infusion similarly caused significant elevation of cell damage markers and multiple organ injury. In trauma and sepsis models, circulating histones strongly correlated with these markers, and anti-histone reagents significantly reduced their release. Comparison of pneumolysin deletion and its parental strain-induced sepsis mouse model showed that pneumolysin was not essential for sepsis development, but enhanced multiple organ damage and reduced survival time. In vitro, histones and pneumolysin treatment disrupt cell membrane integrity, resulting in changes in whole-cell currents and elevated intracellular Ca2+ to lead to Ca2+ overload. Cell-specific damage markers, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and cardiac troponin I (cTnI), were released from damaged cells. Once toxins were removed, cell membrane damage could be rapidly repaired and cellular function recovered. CONCLUSION: This work has confirmed the importance of pore-forming toxins in the development of MODS and proposed a potential mechanism to explain the reversibility of MODS. This may form the foundation for the development of effective therapies.
RESUMEN
In this work, quartz crystal microbalance (QCM) sensors for detection of trace hydrogen cyanide (HCN) gas were developed based on nanostructural (flower-like, boat-like, ellipsoid-like, plate-like) CuO. Responses of all the sensors to HCN were found to be in an opposite direction as compared with other common volatile substances, offering excellent selectivity for HCN detection. The sensitivity of these sensors is dependent on the morphology of CuO nanostructures, among which the plate-like CuO has the highest sensitivity (2.26 Hz/µg). Comparison of the specific surface areas of CuO nanostructures shows that CuO of higher surface area (9.3 m(2)/g) is more sensitive than that of lower surface area (1.5 m(2)/g), indicating that the specific surface area of these CuO nanostructures plays an important role in the sensitivity of related sensors. On the basis of experimental results, a sensing mechanism was proposed in which a surface redox reaction occurs between CuO and Cu(2)O on the CuO nanostructures reversibly upon contact with HCN and air, respectively. The CuO-functionalized QCM sensors are considered to be a promising candidate for trace HCN gas detection in practical applications.
Asunto(s)
Contaminantes Atmosféricos/análisis , Cobre/química , Monitoreo del Ambiente/instrumentación , Cianuro de Hidrógeno/análisis , Nanoestructuras/química , Tecnicas de Microbalanza del Cristal de Cuarzo/instrumentación , Monitoreo del Ambiente/métodos , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Difracción de Rayos XRESUMEN
Sepsis was first described by the ancient Greek physicians over 2000 years ago. The pathophysiology of the disease, however, is still not fully understood and hence the mortality rate is still unacceptably high due to lack of specific therapies. In the last decade, great progress has been made by shifting the focus of research from systemic inflammatory response syndrome (SIRS) to multiple organ dysfunction syndrome (MODS). Sepsis has been re-defined as infection-induced MODS in 2016. How infection leads to MODS is not clear, but what mediates MODS becomes the major topic in understanding the molecular mechanisms and developing specific therapies. Recently, the mechanism of infection-induced extensive immune cell death which releases a large quantity of damage-associated molecular patterns (DAMPs) and their roles in the development of MODS as well as immunosuppression during sepsis have attracted much attention. Growing evidence supports the hypothesis that DAMPs, including high-mobility group box 1 protein (HMGB1), cell-free DNA (cfDNA) and histones as well as neutrophil extracellular traps (NETs), may directly or indirectly contribute significantly to the development of MODS. Here, we provide an overview of the mechanisms and consequences of infection-induced extensive immune cell death during the development of sepsis. We also propose a pivotal pathway from a local infection to eventual sepsis and a potential combined therapeutic strategy for targeting sepsis.
Asunto(s)
Apoptosis , Sistema Inmunológico/inmunología , Insuficiencia Multiorgánica/inmunología , Sepsis/inmunología , Alarminas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Coagulación Sanguínea , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Interacciones Huésped-Patógeno , Humanos , Sistema Inmunológico/metabolismo , Sistema Inmunológico/patología , Insuficiencia Multiorgánica/metabolismo , Insuficiencia Multiorgánica/patología , Sepsis/metabolismo , Sepsis/patología , Transducción de SeñalRESUMEN
BACKGROUND: Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated. AIM: To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis. METHODS: In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated. RESULTS: Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice. CONCLUSION: Extracellular histones potentially enhance fibrogenesis via the TLR4-MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.