RESUMEN
Neuroinflammation is a pathophysiological hallmark of multiple sclerosis and has a close mechanistic link to neurodegeneration. Although this link is potentially targetable, robust translatable models to reliably quantify and track neuroinflammation in both mice and humans are lacking. The choroid plexus (ChP) plays a pivotal role in regulating the trafficking of immune cells from the brain parenchyma into the cerebrospinal fluid (CSF) and has recently attracted attention as a key structure in the initiation of inflammatory brain responses. In a translational framework, we here address the integrity and multidimensional characteristics of the ChP under inflammatory conditions and question whether ChP volumes could act as an interspecies marker of neuroinflammation that closely interrelates with functional impairment. Therefore, we explore ChP characteristics in neuroinflammation in patients with multiple sclerosis and in two experimental mouse models, cuprizone diet-related demyelination and experimental autoimmune encephalomyelitis. We demonstrate that ChP enlargement-reconstructed from MRI-is highly associated with acute disease activity, both in the studied mouse models and in humans. A close dependency of ChP integrity and molecular signatures of neuroinflammation is shown in the performed transcriptomic analyses. Moreover, pharmacological modulation of the blood-CSF barrier with natalizumab prevents an increase of the ChP volume. ChP enlargement is strongly linked to emerging functional impairment as depicted in the mouse models and in multiple sclerosis patients. Our findings identify ChP characteristics as robust and translatable hallmarks of acute and ongoing neuroinflammatory activity in mice and humans that could serve as a promising interspecies marker for translational and reverse-translational approaches.
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Plexo Coroideo/diagnóstico por imagen , Esclerosis Múltiple/fisiopatología , Enfermedades Neuroinflamatorias/diagnóstico por imagen , Adulto , Animales , Barrera Hematoencefálica/fisiología , Encéfalo/fisiología , Plexo Coroideo/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/diagnóstico por imagen , Proteómica/métodosRESUMEN
Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.
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Canales de Potasio de Dominio Poro en Tándem , Humanos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Células Endoteliales/metabolismo , Enfermedades Neuroinflamatorias , Encéfalo/metabolismo , Moléculas de Adhesión Celular/metabolismoRESUMEN
Blood-brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.
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Glicina , Receptores de N-Metil-D-Aspartato , Animales , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Glicina/metabolismo , Glicina/farmacología , Ratones , N-Metilaspartato/farmacología , Receptores de Glicina , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
BACKGROUND: Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice. METHODS: In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo. RESULTS: Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient. CONCLUSIONS: Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood-brain barrier independently of its peripheral immunosuppressant action.
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Encefalomielitis Autoinmune Experimental , Encefalomielitis , Fármacos Neuroprotectores , Ratones , Animales , Encefalomielitis Autoinmune Experimental/patología , Cladribina/uso terapéutico , Fármacos Neuroprotectores/farmacología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Inmunosupresores/uso terapéuticoRESUMEN
The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.
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Artritis Reumatoide/tratamiento farmacológico , Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Calcio/inmunología , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Señalización del Calcio , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Moduladores del Transporte de Membrana/química , Ratones , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/inmunología , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/inmunologíaRESUMEN
Flow stagnation of peri-ischemic capillaries due to dynamic leukocyte stalls has been described to be a contributor to ongoing penumbral injury in transient brain ischemia, but has not been investigated in permanent experimental stroke so far. Moreover, it is discussed that obstructing neutrophils are involved in this process; however, their contribution has not yet been proven. Here, we characterize the dynamics of neutrophil granulocytes in two models of permanent stroke (photothrombosis and permanent middle cerebral artery occlusion) using intravital two-photon fluorescence microscopy. Different to previous studies on LysM-eGFP+ cells we additionally apply a transgenic mouse model with tdTomato-expressing neutrophils to avoid interference from additional immune cell subsets. We identify repetitively occurring capillary stalls of varying duration promoted by neutrophils in both models of permanent cerebral ischemia, validating the suitability of our new transgenic mouse model in determining neutrophil occlusion formation in vivo. Flow cytometric analysis of peripheral blood (PB) and brain tissue from mice subjected to photothrombosis reveal an increase in the total proportion of neutrophils, with selective upregulation of endothelial adherence markers in the PB. In conclusion, the dynamic microcirculatory stall phenomenon that is described after transient ischemia followed by reperfusion also occurs after permanent small- or large-vessel stroke and is clearly attributable to neutrophils.
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Isquemia Encefálica , Accidente Cerebrovascular , Animales , Capilares , Modelos Animales de Enfermedad , Granulocitos , Infarto de la Arteria Cerebral Media , Ratones , Ratones Endogámicos C57BL , Microcirculación , NeutrófilosRESUMEN
Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.
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Virus del Dengue/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Antivirales/farmacología , Línea Celular , Virus del Dengue/enzimología , VIH-1/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Replicación Viral/efectos de los fármacosRESUMEN
The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Bacillus cereus/metabolismo , Proteínas Bacterianas/inmunología , Enterotoxinas/inmunología , Animales , Bacillus cereus/inmunología , Línea Celular , Clonación Molecular , Enterotoxinas/metabolismo , Enterotoxinas/toxicidad , Humanos , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
TWIK-related spinal cord potassium (TRESK) and TWIK-related potassium (TREK) channels are both subfamilies of the two-pore domain potassium (K2P) channel group. Despite major structural, pharmacological, as well as biophysical differences, emerging data suggest that channels of these two subfamilies are functionally more closely related than previously assumed. Recent studies, for instance, indicate an assembling of TRESK and TREK subunits, leading to the formation of heterodimeric channels with different functional properties compared to homodimeric ones. Formation of tandems consisting of TRESK and TREK subunits might thus multiply the functional diversity of both TRESK and TREK activity. Based on the involvement of these channels in the pathophysiology of migraine, we here highlight the role as well as the impact of the interplay of TRESK and TREK subunits in the context of different disease settings. In this regard, we focus on their involvement in migraine and pain syndromes, as well as on their influence on (neuro-)inflammatory processes. Furthermore, we describe the potential implications for innovative therapeutic strategies that take advantage of TRESK and TREK modulation as well as obstacles encountered in the development of therapies related to the aforementioned diseases.
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Enfermedades Neuroinflamatorias/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio/metabolismo , Humanos , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/genética , Multimerización de ProteínaRESUMEN
Rag1-/- mice, lacking functional B and T cells, have been extensively used as an adoptive transfer model to evaluate neuroinflammation in stroke research. However, it remains unknown whether natural killer (NK) cell development and functions are altered in Rag1-/- mice as well. This connection has been rarely discussed in previous studies but might have important implications for data interpretation. In contrast, the NOD-Rag1nullIL2rgnull (NRG) mouse model is devoid of NK cells and might therefore eliminate this potential shortcoming. Here, we compare immune-cell frequencies as well as phenotype and effector functions of NK cells in Rag1-/- and wildtype (WT) mice using flow cytometry and functional in vitro assays. Further, we investigate the effect of Rag1-/- NK cells in the transient middle cerebral artery occlusion (tMCAO) model using antibody-mediated depletion of NK cells and adoptive transfer to NRG mice in vivo. NK cells in Rag1-/- were comparable in number and function to those in WT mice. Rag1-/- mice treated with an anti-NK1.1 antibody developed significantly smaller infarctions and improved behavioral scores. Correspondingly, NRG mice supplemented with NK cells were more susceptible to tMCAO, developing infarctions and neurological deficits similar to Rag1-/- controls. Our results indicate that NK cells from Rag1-/- mice are fully functional and should therefore be considered in the interpretation of immune-cell transfer models in experimental stroke. Fortunately, we identified the NRG mice, as a potentially better-suited transfer model to characterize individual cell subset-mediated neuroinflammation in stroke.
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Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Infarto de la Arteria Cerebral Media , Células Asesinas Naturales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones NoqueadosRESUMEN
It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.
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Canales de Potasio , Receptores de Antígenos de Linfocitos T , Linfocitos T Reguladores , Animales , Diferenciación Celular , Factores de Transcripción Forkhead , Humanos , Ratones , FN-kappa B , Timocitos , TimoRESUMEN
OBJECTIVE: The objective of this study is to report a 15-month follow-up with the Endurant Stent Graft System in patients with challenging aortic anatomies. METHODS: At three German clinics, a consecutive series of 50 patients underwent endovascular abdominal aortic repair (EVAR) for challenging abdominal aortic aneurysm with the Endurant stent graft between November 2008 and May 2009. EVAR was elective in 48 cases and emergent in two. Patients had short (≤15 mm) aortic necks, severe suprarenal/infrarenal angulation, and/or small (<8 mm), calcified, severely angulated, or tortuous iliac or femoral access vessels. Additionally, a cohort of 40 patients without challenging anatomies were retrospectively analysed to clarify differences concerning technical success, mortality, and morbidity between these groups. RESULTS: The primary technical success rate was 92% (46/50). The 30-day mortality rate was 2% (1/50), the death due to multiorgan failure. Intraoperative angiograms revealed three type I endoleaks (2 proximal and 1 distal), and one of those was persisting at 30 days (30-day rate, 2%). Postoperative imaging discovered no further type I or type III endoleaks. The 30-day rate of the type II endoleak was 6% (3/50). There were two cases of graft limb occlusion, both requiring reintervention within 30 days. Follow-up was available in all of the 50 patients (100%) over a median of 15 months (1-25). During this time, seven patients died (overall mortality, 16%; 8/50), besides the above-described patient, all of them unrelated to the procedure. Compared to the 30-day results with the Endurant stent graft in non-challenging anatomies (no type I endoleak; no graft limb occlusion; all-cause mortality, 0%), procedure-related complications in challenging anatomies are increasing. CONCLUSION: Early and 15-month results with the Endurant stent graft in patients with challenging aortic anatomies are encouraging.
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Aneurisma de la Aorta Abdominal/cirugía , Implantación de Prótesis Vascular/métodos , Prótesis Vascular , Stents , Anciano , Anciano de 80 o más Años , Angiografía , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/mortalidad , Femenino , Estudios de Seguimiento , Alemania , Humanos , Interpretación de Imagen Asistida por Computador , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tasa de Supervivencia , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Regulatory T cells (Tregs) are the major determinant of peripheral immune tolerance. Many Treg subsets have been described, however thymus-derived and peripherally induced Tregs remain the most important subpopulations. In multiple sclerosis, a prototypical autoimmune disorder of the central nervous system, Treg dysfunction is a pathogenic hallmark. In contrast, induction of Treg proliferation and enhancement of their function are central immune evasion mechanisms of infectious pathogens. In accordance, Treg expansion is compartmentalized to tissues with high viral replication and prolonged in chronic infections. In friend retrovirus infection, Treg expansion is mainly based on excessive interleukin-2 production by infected effector T cells. Moreover, pathogens seem also to enhance Treg functions as shown in human immunodeficiency virus infection, where Tregs express higher levels of effector molecules such as cytotoxic T-lymphocyte-associated protein 4, CD39 and cAMP and show increased suppressive capacity. Thus, insights into the molecular mechanisms by which intracellular pathogens alter Treg functions might aid to find new therapeutic approaches to target central nervous system autoimmunity. In this review, we summarize the current knowledge of the role of pathogens for Treg function in the context of autoimmune neuroinflammation. We discuss the mechanistic implications for future therapies and provide an outlook for new research directions.
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Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/microbiología , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/microbiología , Linfocitos T Reguladores/inmunología , Animales , Humanos , Infección Persistente/inmunologíaRESUMEN
In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, the role of each central nervous system (CNS)-resident cell type during inflammation, neurodegeneration, and remission has been frequently addressed. Although protocols for the isolation of different individual CNS-resident cell types exist, none can harvest all of them within a single experiment. In addition, isolation of individual cells is more demanding in adult mice and even more so from the inflamed CNS. Here, we present a protocol for the simultaneous purification of viable single-cell suspensions of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) from adult mice-applicable in healthy mice as well as in EAE. After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes and, neurons were isolated via magnetic-activated cell sorting (MACS). Validations comprised flow cytometry, immunocytochemistry, as well as functional analyses (immunoassay and Sholl analysis). The purity of each cell isolation averaged 90%. All cells displayed cell-type-specific morphologies and expressed specific surface markers. In conclusion, this new protocol for the simultaneous isolation of all major CNS-resident cell types from one CNS offers a sophisticated and comprehensive way to investigate complex cellular networks ex vivo and simultaneously reduce mice numbers to be sacrificed.
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Encéfalo/citología , Separación Celular , Microglía/citología , Oligodendroglía/citología , Médula Espinal/citología , Animales , Encéfalo/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Inflamación/metabolismo , Ratones , Microglía/metabolismo , Esclerosis Múltiple/metabolismo , Oligodendroglía/metabolismo , Médula Espinal/metabolismoRESUMEN
This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.
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Bacillus cereus/patogenicidad , Enterotoxinas/toxicidad , Animales , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Enterotoxinas/química , Enterotoxinas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Células VeroRESUMEN
Autoimmune diseases of the central nervous system (CNS) like multiple sclerosis (MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease, whereas progressive MS (PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with relapsing MS have limited success in PMS. BAF312 (siponimod) is a novel sphingosine-1-phosphate receptor modulator shown to delay progression in PMS. Besides reducing inflammation by sequestering lymphocytes in lymphoid tissues, BAF312 crosses the blood-brain barrier and binds its receptors on neurons, astrocytes and oligodendrocytes. To evaluate potential direct neuroprotective effects, BAF312 was systemically or locally administered in the CNS of experimental autoimmune encephalomyelitis mice with distinct grey- and white-matter lesions (focal experimental autoimmune encephalomyelitis using an osmotic mini-pump). Ex-vivo flow cytometry revealed that systemic but not local BAF312 administration lowered immune cell infiltration in animals with both grey and white matter lesions. Ex-vivo voltage-sensitive dye imaging of acute brain slices revealed an altered spatio-temporal pattern of activation in the lesioned cortex compared to controls in response to electrical stimulation of incoming white-matter fiber tracts. Here, BAF312 administration showed partial restore of cortical neuronal circuit function. The data suggest that BAF312 exerts a neuroprotective effect after crossing the blood-brain barrier independently of peripheral effects on immune cells. Experiments were carried out in accordance with German and EU animal protection law and approved by local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen; 87-51.04.2010.A331) on December 28, 2010.
RESUMEN
Sodium chloride promotes vascular fibrosis, arterial hypertension, pro-inflammatory immune cell polarization and endothelial dysfunction, all of which might influence outcomes following stroke. But despite enormous translational relevance, the functional importance of sodium chloride in the pathophysiology of acute ischemic stroke is still unclear. In the current study, we show that high-salt diet leads to significantly worse functional outcomes, increased infarct volumes, and a loss of astrocytes and cortical neurons in acute ischemic stroke. While analyzing the underlying pathologic processes, we identified the migrasome as a novel, sodium chloride-driven pathomechanism in acute ischemic stroke. The migrasome was previously described in vitro as a migrating organelle, which incorporates and dispatches cytosol of surrounding cells and plays a role in intercellular signaling, whereas a pathophysiological meaning has not been elaborated. We here confirm previously reported characteristics of the migrasome in vivo. Immunohistochemistry, electron microscopy and proteomic analyses further demonstrate that the migrasome incorporates and dispatches cytosol of surrounding neurons following stroke. The clinical relevance of these findings is emphasized by neuropathological examinations, which detected migrasome formation in infarcted brain parenchyma of human stroke patients. In summary, we demonstrate that high-salt diet aggravates stroke outcomes, and we characterize the migrasome as a novel mechanism in acute stroke pathophysiology.
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Lesiones Encefálicas , Isquemia Encefálica , Corteza Cerebral , Orgánulos , Cloruro de Sodio Dietético/efectos adversos , Accidente Cerebrovascular , Animales , Astrocitos/metabolismo , Astrocitos/patología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Citosol/metabolismo , Citosol/patología , Masculino , Ratones , Neuronas/metabolismo , Neuronas/patología , Orgánulos/metabolismo , Orgánulos/patología , Cloruro de Sodio Dietético/farmacología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatologíaRESUMEN
A comparative analysis on the relevance of the Bacillus cereus enterotoxins Nhe (nonhemolytic enterotoxin), HBL (haemolysin BL) and CytK (cytotoxin K) was accomplished, concerning their toxic activity towards different target cell lines. Overall, among the components secreted by the reference strains for Nhe and HBL, the enterotoxin complexes accounted for over 90% of the total toxicity. Vero and primary endothelial cells (HUVEC) were highly susceptible to Nhe, whereas Hep-G2, Vero and A549 reacted most sensitive to Nhe plus HBL. For CytK the highest toxicity was observed on CaCo-2 cells. As HBL positive strains always produce Nhe in parallel, the specific contribution of both enterotoxin complexes to the overall observed cytotoxic effects was determined by consecutively removing their single components. While in most cell lines Nhe and HBL contributed more or less equally (40-60%) to cytotoxicity, the relative activity of Nhe was approximately 90% in HUVEC, and that of HBL 75% in A549 cells. With U937, a nearly Nhe resistant cell line was identified for the first time. This distinct susceptibility of cell lines was confirmed by investigating a set of 37 B. cereus strains. Interestingly, whereas Nhe is the enterotoxin mainly responsible for cell death as determined by WST-1 bioassays, more rapid pore formation was observed when HBL was present, pointing to a different mode of action of the two enterotoxin complexes. Furthermore, correlation was observed between cytotoxicity of solely Nhe producing strains and NheB. Cytotoxicity of Nhe/HBL producing isolates correlated with the expression of HBL L1, NheB and HBL B. In conclusion, the observed susceptibilities of target cell lines of different histological origin underline that B. cereus enterotoxins represent major virulence factors and that toxicity is not restricted to gastrointestinal infections. The varying contribution of Nhe and HBL to total cytotoxicity strongly indicates that Nhe as well as HBL specific B. cereus enterotoxin receptors exist.
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Bacillus cereus/química , Proteínas Bacterianas/toxicidad , Citotoxinas/toxicidad , Enterotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Factores de Virulencia/toxicidad , Animales , Células CACO-2 , Muerte Celular/efectos de los fármacos , Chlorocebus aethiops , Cromatografía de Afinidad , Células Endoteliales de la Vena Umbilical Humana , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa/metabolismo , Receptores de Péptidos/metabolismo , Células VeroRESUMEN
CONTEXT: Adrenal tumors are highly prevalent and represent a wide range of different pathological entities. Conventional imaging often provides only limited information on the origin of these lesions. Novel specific imaging methods are, therefore, of great clinical interest. OBJECTIVE: We evaluated [(123)I]iodometomidate ([(123)I]IMTO) imaging for noninvasive characterization of adrenal masses. DESIGN/SETTING: This was a prospective monocentric diagnostic study in a tertiary care center. PATIENTS AND INTERVENTION: A total of 51 patients with an adrenal lesion underwent [(123)I]IMTO imaging after injection of 185 MBq of [(123)I]IMTO. Sequential planar whole-body scans until 24 hours postinjection and single photon emission computed tomography (SPECT)/computed tomography imaging 4 to 6 hours postinjection were performed. MAIN OUTCOME MEASURE: Sensitivity and specificity of [(123)I]IMTO imaging for the noninvasive characterization of adrenal lesions were measured. RESULTS: Adrenocortical tissue showed high and specific tracer uptake with a short investigation time and low radiation exposure. Qualitative analysis of SPECT/computed tomography data resulted in a sensitivity of 89% and a specificity of 85% for differentiating adrenocortical tumors from lesions of nonadrenocortical origin. Receiver-operating characteristic analysis of semiquantitative data revealed a sensitivity of 83% and a specificity of 86% for identification of adrenocortical lesions at a cutoff value of tumor to liver ratio of 1.3. CONCLUSIONS: [(123)I]IMTO is a highly specific radiotracer for imaging of adrenocortical tissue with a short investigation time and low radiation exposure. Because of the general availability of SPECT technology, [(123)I]IMTO scintigraphy has the potential to become a widely used tool to noninvasively characterize the biology of adrenal lesions.
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Neoplasias de las Glándulas Suprarrenales/diagnóstico por imagen , Carcinoma/diagnóstico por imagen , Etomidato/análogos & derivados , Radioisótopos de Yodo , Imagen Multimodal/métodos , Tomografía de Emisión de Positrones , Tomografía Computarizada por Rayos X , Adolescente , Neoplasias de las Glándulas Suprarrenales/epidemiología , Neoplasias de las Glándulas Suprarrenales/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/epidemiología , Carcinoma/fisiopatología , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Etomidato/efectos adversos , Femenino , Humanos , Radioisótopos de Yodo/efectos adversos , Masculino , Persona de Mediana Edad , Imagen Multimodal/efectos adversos , Sensibilidad y Especificidad , Imagen de Cuerpo Entero , Adulto JovenRESUMEN
CONTEXT: Imaging with [¹²³I]iodometomidate ([¹²³I]IMTO) has been shown to diagnose adrenocortical lesions with high sensitivity and specificity. OBJECTIVE: Our objective was to evaluate the clinical utility of [¹²³I]IMTO imaging in adrenocortical carcinoma (ACC). DESIGN: We conducted a prospective monocentric diagnostic study and a prospective case series at a single tertiary referral center. PATIENTS AND INTERVENTIONS: Fifty-eight patients with histologically confirmed ACC, all European Network for the Study of Adrenal Tumors stage IV (with distant metastases), received 185 MBq [¹²³I]IMTO. Sequential planar whole-body scans until 24 hours post injection and single photon emission computed tomography/computed tomography (SPECT/CT) hybrid imaging 4 to 6 hours post injection were performed. MAIN OUTCOME MEASURES: Outcome measures included uptake of [¹²³I]IMTO in ACC lesions, sensitivity and specificity of [¹²³I]IMTO imaging compared with conventional imaging, and number of patients eligible for [¹³¹I]IMTO therapy. RESULTS: Of 430 lesions detected by conventional imaging, 30% showed strong, 8% moderate, and 62% no tracer accumulation. [¹²³I]IMTO detected both primary and metastatic lesions of ACC. However, a substantial percentage of lesions failed to show [¹²³I]IMTO uptake. The overall sensitivity and specificity values were 38% and 100%, respectively. Thirty-four patients (59%) had at least 1 [¹²³I]IMTO-positive lesion. Cortisol and aldosterone secretion by ACC was positively correlated to [¹²³I]IMTO uptake (P = .01); cytotoxic chemotherapy and mitotane treatment presumably did not influence tracer uptake. Twenty-one patients (36.2%) had radiotracer uptake in all lesions ≥ 2 cm and therefore were potential candidates for targeted systemic radiotherapy with [¹³¹I]IMTO. CONCLUSION: About one-third of patients with ACC show specific retention of [¹²³I]IMTO in metastatic lesions. This study provides support for the conduct of a prospective trial to determine whether the first molecular informed therapy using [¹³¹I]IMTO will be of value to patients with metastatic ACC.