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1.
J Neuroinflammation ; 20(1): 24, 2023 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-36739434

RESUMEN

BACKGROUND: Previous reports have indicated that disrupting the Wnt/ß-catenin pathway in dendritic cells (DCs) may affect the progression of autoimmune inflammation; however, the factors and timing that regulate Wnt/ß-catenin signaling have not been clearly understood. METHODS: Experimental autoimmune uveitis (EAU) mice and Vogt-Koyanagi-Harada disease (VKH) patient samples were used to detect the expression of Wnt/ß-catenin pathway genes. Western blot, real-time PCR, flow cytometry, and ELISA were performed to examine the expression of components of the Wnt/ß-catenin pathway and inflammatory factors. DC-specific ß-catenin knockout mice and 6-bromoindirubin-3'-oxime (BIO) administered mice were used to observe the effect of disrupting the Wnt pathway on EAU pathogenesis. RESULTS: Wnt/ß-catenin signaling was inhibited in DCs during the induction phase of EAU. The inhibition was mediated by pertussis toxin (PTX), which promoted DC maturation, in turn promoting pathogenic T cell proliferation and differentiation. In vivo experiments confirmed that deleting ß-catenin in DCs enhanced EAU severity, and pre-injection of PTX advanced EAU onset. Administration of a Wnt activator (BIO) limited the effects of PTX, in turn ameliorating EAU. CONCLUSIONS: Our results demonstrate that PTX plays a key role as a virulence factor in initiating autoimmune inflammation via DCs by inhibiting Wnt/ß-catenin signaling in EAU, and highlight the potential mechanism by which infection can trigger apparent autoimmunity.


Asunto(s)
Enfermedades Autoinmunes , Uveítis , Ratones , Animales , Toxina del Pertussis/toxicidad , Autoinmunidad , Vía de Señalización Wnt , beta Catenina/metabolismo , Uveítis/inducido químicamente , Uveítis/tratamiento farmacológico , Inflamación/metabolismo , Células Dendríticas
2.
J Infect Dis ; 225(1): 172-176, 2022 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-34145457

RESUMEN

Pertussis, caused by Bordetella pertussis, is a reemerging disease that can produce severe disease manifestations in infants, including pulmonary hypertension (PH). B. pertussis-induced PH is a major risk factor for infection-induced death, but the molecular mechanisms promoting PH are unknown and there is no effective treatment. We examined B. pertussis-induced PH in infant and adult mouse models of pertussis by Fulton index, right heart catheterization, or Doppler echocardiogram. Our results demonstrate that B. pertussis-induced PH is age related and dependent on the expression of pertussis toxin by the bacterium. Hence, pertussis toxin-targeting treatments may ameliorate PH and fatal infant infection.


Asunto(s)
Infecciones por Bordetella , Bordetella pertussis , Hipertensión Pulmonar/inducido químicamente , Toxina del Pertussis/toxicidad , Animales , Modelos Animales de Enfermedad , Ratones , Factores de Virulencia de Bordetella , Tos Ferina
3.
Toxins (Basel) ; 13(8)2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34437436

RESUMEN

One of the main virulence factors produced by Bordetella pertussis is pertussis toxin (PTx) which, in its inactivated form, is the major component of all marketed acellular pertussis vaccines. PTx ADP ribosylates Gαi proteins, thereby affecting the inhibition of adenylate cyclases and resulting in the accumulation of cAMP. Apart from this classical model, PTx also activates some receptors and can affect various ADP ribosylation- and adenylate cyclase-independent signalling pathways. Due to its potent ADP-ribosylation properties, PTx has been used in many research areas. Initially the research primarily focussed on the in vivo effects of the toxin, including histamine sensitization, insulin secretion and leukocytosis. Nowadays, PTx is also used in toxicology research, cell signalling, research involving the blood-brain barrier, and testing of neutralizing antibodies. However, the most important area of use is testing of acellular pertussis vaccines for the presence of residual PTx. In vivo models and in vitro assays for PTx often reflect one of the toxin's properties or details of its mechanism. Here, the established and novel in vivo and in vitro methods used to evaluate PTx are reviewed, their mechanisms, characteristics and limitations are described, and their application for regulatory and research purposes are considered.


Asunto(s)
Bioensayo , Modelos Biológicos , Toxina del Pertussis/toxicidad , Animales , Humanos , Toxina del Pertussis/química
4.
Toxins (Basel) ; 13(8)2021 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-34437445

RESUMEN

Production and secretion of pertussis toxin (PT) is essential for the virulence of Bordetella pertussis. Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order to maintain pathogenicity. During the secretion process, PT undergoes a two-step transport process. The first step involves transport of the individual polypeptide chains of PT across the inner membrane utilizing a generalized secretion pathway, most likely the bacterial Sec system. The second step involves the use of a specialized apparatus to transport the toxin across the outer membrane of the bacterial cell. This apparatus, which has been termed the Ptl transporter and which is unique to the PT secretion pathway, is a member of the type IV family of bacterial transporters. Here, the current understanding of the PT secretion process is reviewed including a description of the Ptl proteins that assemble to form the transporter, the general structure of type IV transporters, the known similarities and differences between canonical type IV substrate transport and Ptl-mediated transport of PT, as well as the known sequence of events in the assembly and secretion of PT.


Asunto(s)
Transporte Biológico/fisiología , Bordetella pertussis/química , Bordetella pertussis/metabolismo , Proteínas de Transporte de Membrana/fisiología , Toxina del Pertussis/biosíntesis , Toxina del Pertussis/toxicidad , Factores de Virulencia de Bordetella/biosíntesis , Factores de Virulencia de Bordetella/toxicidad
5.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34417310

RESUMEN

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.


Asunto(s)
Encefalomielitis Autoinmune Experimental/inducido químicamente , Glicoproteína Mielina-Oligodendrócito/farmacología , Oligodendroglía/efectos de los fármacos , Células Th17/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Adyuvante de Freund , Inflamación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oligodendroglía/metabolismo , Toxina del Pertussis/toxicidad
6.
Pharmeur Bio Sci Notes ; 2021: 69-87, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33934749

RESUMEN

Recently, the Chinese hamster ovary (CHO) cell-based clustering assay replaced the in vivo Histamine Sensitisation Test (HIST) in mice in European Pharmacopoeia (Ph. Eur.) general chapter 2.6.33. 'Residual pertussis toxin' as the recommended method to test for residual pertussis toxin in acellular pertussis vaccine intermediates. To support the standardised CHO clustering assay, availability of a reference standard is critical. Ph. Eur. pertussis toxin Biological Reference Preparation (BRP) batch 1 was first calibrated in International Units in 2008 for the HIST and subsequently also calibrated for the CHO clustering assay in 2017. However, its stocks were dwindling and needed to be replaced. In an effort to maintain adequate supply, a project (BSP141) was initiated by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to establish a second pertussis toxin BRP (BRP2). Candidate material was manufactured ad hoc by an acellular pertussis vaccine manufacturer and an optimal formulation for long-term stability was defined. Exhaustive in-process and post-production controls demonstrated that the material was fit for its intended purpose and therefore a collaborative study for calibration and stability assessment of the candidate material was organised, which included 10 laboratories worldwide. As a result of the study, the candidate material was established as Ph. Eur. Pertussis toxin BRP batch 2 with a potency of 130 IU/vial for the CHO clustering assay. Unopened vials must be stored at −20°C. The BRP may be used for up to two weeks after reconstitution if appropriately handled and stored at 2­8°C.


Asunto(s)
Cooperación Internacional , Animales , Células CHO , Análisis por Conglomerados , Cricetinae , Cricetulus , Europa (Continente) , Ratones , Toxina del Pertussis/toxicidad , Estándares de Referencia
7.
Sci Rep ; 11(1): 5429, 2021 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-33686161

RESUMEN

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.


Asunto(s)
Bordetella pertussis/enzimología , Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Leucocitosis , Chaperonas Moleculares , Toxina del Pertussis/toxicidad , Animales , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidad , Células CHO , Cricetulus , Células Epiteliales/microbiología , Células HEK293 , Humanos , Leucocitosis/inducido químicamente , Leucocitosis/tratamiento farmacológico , Leucocitosis/metabolismo , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo
8.
Sci Rep ; 10(1): 14160, 2020 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-32843685

RESUMEN

Immediate hypersensitivity reaction (IHR) can be divided into allergic- and non-allergic-mediated, while "anaphylaxis" is reserved for severe IHR. Clinically, true penicillin allergy is rare and most reported penicillin allergy is "spurious". Penicillin-initiated anaphylaxis is possible to occur in skin test- and specific IgE-negative patients. The contact system is a plasma protease cascade initiated by activation of factor XII (FXII). Many agents with negative ion surface can activate FXII to drive contact system. Our data showed that penicillin significantly induced hypothermia in propranolol- or pertussis toxin-pretreated mice. It also caused a rapid and reversible drop in rat blood pressure, which did not overlap with IgE-mediated hypotension. These effects could be countered by a bradykinin-B2 receptor antagonist icatibant, and consistently, penicillin indeed increased rat plasma bradykinin. Moreover, penicillin not only directly activated contact system FXII-dependently, but also promoted bradykinin release in plasma incubated-human umbilical vein endothelial cells. In fact, besides penicillin, other beta-lactams also activated the contact system in vitro. Since the autoactivation of FXII can be affected by multiple-factors, plasma from different healthy individuals showed vastly different amidolytic activity in response to penicillin, suggesting the necessity of determining the potency of penicillin to induce individual plasma FXII activation. These results clarify that penicillin-initiated non-allergic anaphylaxis is attributed to contact system activation, which might bring more effective diagnosis options for predicting penicillin-induced fatal risk and avoiding costly and inappropriate treatment clinically.


Asunto(s)
Anafilaxia/inducido químicamente , Coagulación Sanguínea/efectos de los fármacos , Factor XIIa/metabolismo , Sistema Calicreína-Quinina/efectos de los fármacos , Penicilina G/toxicidad , Anafilaxia/inmunología , Animales , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Bradiquinina/fisiología , Antagonistas de los Receptores de Bradiquinina/farmacología , Permeabilidad Capilar/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hipotermia/inducido químicamente , Masculino , Ratones , Ratones Endogámicos BALB C , Penicilina G/efectos adversos , Toxina del Pertussis/toxicidad , Propranolol/toxicidad , Ratas , Ratas Sprague-Dawley , Receptor de Bradiquinina B2/efectos de los fármacos , Receptor de Bradiquinina B2/fisiología , beta-Lactamas/toxicidad
9.
Mol Cell Biochem ; 469(1-2): 89-95, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32301060

RESUMEN

Lysophosphatidic acid (LPA) signaling through LPA receptors (LPA1 to LPA6) regulates a variety of malignant properties in cancer cells. Recently, we show that LPA2 expression is elevated by long-term cisplatin (CDDP) treatment in melanoma A375 cells. In the present study, we investigated whether LPA2-mediated signaling is involved in the modulation of chemoresistance in A375 cells. In cell survival assay, cells were treated with CDDP and dacarbazine (DTIC) every 24 h for 2 days. The cell survival rates to CDDP and DTIC were markedly increased by an LPA2 agonist, GRI-977143. To validate the effects of LPA2 on cell survival, LPA2 knockdown cells were generated from A375 cells. The cell survival rates elevated by GRI-977143 were suppressed by LPA2 knockdown. To evaluate the roles of LPA2-mediated signaling in cell survival, cells were pretreated with a Gi protein inhibitor, pertussis toxin (PTX). In the presence of GRI-977143, the cell survival rates to CDDP and DTIC were significantly lower in PTX-treated cells than in untreated cells. In addition, pretreatment of an adenylyl cyclase inhibitor, SQ22536, increased the cell survival of A375 cells treated with CDDP and DTIC. These results suggest that LPA2-mediated signaling plays an important role in the enhancement of chemoresistance of A375 cells treated with anticancer drugs.


Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Dacarbazina/farmacología , Resistencia a Antineoplásicos/genética , Lisofosfolípidos/metabolismo , Melanoma/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Lisofosfolípidos/agonistas , Lisofosfolípidos/genética , Melanoma/genética , Toxina del Pertussis/toxicidad , Receptores del Ácido Lisofosfatídico/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
10.
J Med Microbiol ; 69(1): 111-119, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31778110

RESUMEN

Introduction. Differences between the genomic and virulence profile of Bordetella pertussis circulating strains and vaccine strains are considered as one of the important reasons for the resurgence of whooping cough (pertussis) in the world. Genetically inactivated B. pertussis is one of the new strategies to generate live-attenuated vaccines against whooping cough.Aim. The aim of this study was to construct a B. pertussis strain based on a predominant profile of circulating Iranian isolates that produces inactivated pertussis toxin (PTX).Methodology. The B. pertussis strain BPIP91 with predominant genomic and virulence pattern was selected from the biobank of the Pasteur Institute of Iran. A BPIP91 derivative with R9K and E129G alterations in the S1 subunit of PTX (S1mBPIP91) was constructed by the site-directed mutagenesis and homologous recombination. Genetic stability and antigen expression of S1mBPIP91 were tested by serially in vitro passages and immunoblot analyses, respectively. The reduction in toxicity of S1mBPIP91 was determined by Chinese hamster ovary (CHO) cell clustering.Results. All constructs and S1mBPIP91 were confirmed via restriction enzyme analysis and DNA sequencing. The engineered mutations in S1mBPIP91 were stable after 20 serial in vitro passages. The production of virulence factors was also confirmed in S1mBPIP91. The CHO cell-clustering test demonstrated the reduction in PTX toxicity in S1mBPIP91.Conclusion. A B. pertussis of the predominant genomic and virulence lineage in Iran was successfully engineered to produce inactive PTX. This attenuated strain will be useful to further studies to develop both whole cell and acellular pertussis vaccines.


Asunto(s)
Antígenos Bacterianos/genética , Bordetella pertussis/genética , Bordetella pertussis/inmunología , Proteínas Mutantes/genética , Toxina del Pertussis/genética , Vacuna contra la Tos Ferina/genética , Animales , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/toxicidad , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetulus , Irán , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidad , Toxina del Pertussis/metabolismo , Toxina del Pertussis/toxicidad , Vacuna contra la Tos Ferina/efectos adversos , Ingeniería de Proteínas , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética
11.
Adv Exp Med Biol ; 1183: 35-51, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31376138

RESUMEN

Bordetella pertussis produces several toxins that affect host-pathogen interactions. Of these, the major toxins that contribute to pertussis infection and disease are pertussis toxin, adenylate cyclase toxin-hemolysin and tracheal cytotoxin. Pertussis toxin is a multi-subunit protein toxin that inhibits host G protein-coupled receptor signaling, causing a wide array of effects on the host. Adenylate cyclase toxin-hemolysin is a single polypeptide, containing an adenylate cyclase enzymatic domain coupled to a hemolysin domain, that primarily targets phagocytic cells to inhibit their antibacterial activities. Tracheal cytotoxin is a fragment of peptidoglycan released by B. pertussis that elicits damaging inflammatory responses in host cells. This chapter describes these three virulence factors of B. pertussis, summarizing background information and focusing on the role of each toxin in infection and disease pathogenesis, as well as their role in pertussis vaccination.


Asunto(s)
Toxina de Adenilato Ciclasa/toxicidad , Bordetella pertussis/patogenicidad , Toxina del Pertussis/toxicidad , Factores de Virulencia de Bordetella/toxicidad , Adenilil Ciclasas/fisiología , Toxinas Bacterianas , Bordetella pertussis/enzimología , Bordetella pertussis/genética , Proteínas Hemolisinas/fisiología , Humanos , Factores de Virulencia , Tos Ferina/microbiología , Tos Ferina/prevención & control
12.
Toxins (Basel) ; 11(7)2019 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-31319496

RESUMEN

Whooping cough is caused by the bacterium Bordetella pertussis. There are currently two types of vaccines that can prevent the disease; whole cell vaccines (WCV) and acellular vaccines (ACV). The main virulence factor produced by the organism is pertussis toxin (PTx). This toxin is responsible for many physiological effects on the host, but it is also immunogenic and in its detoxified form is the main component of all ACVs. In producing toxoid for vaccines, it is vital to achieve a balance between sufficiently detoxifying PTx to render it safe while maintaining enough molecular structure that it retains its protective immunogenicity. To ensure that the first part of this balancing act has been successfully achieved, assays are required to accurately measure residual PTx activity in ACV products accurately. Quality control assays are also required to ensure that the detoxification procedures are robust and stable. This manuscript reviews the methods that have been used to achieve this aim, or may have the potential to replace them, and highlights their continuing requirement as vaccines that induce a longer lasting immunity are developed to prevent the re-occurrence of outbreaks that have been observed recently.


Asunto(s)
Toxina del Pertussis/análisis , Vacuna contra la Tos Ferina/análisis , Animales , Bioensayo , Humanos , Toxina del Pertussis/toxicidad
13.
Toxins (Basel) ; 11(7)2019 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-31252532

RESUMEN

Pertussis, caused by respiratory tract infection with the bacterial pathogen Bordetella pertussis, has long been considered to be a toxin-mediated disease. Bacteria adhere and multiply extracellularly in the airways and release several toxins, which have a variety of effects on the host, both local and systemic. Predominant among these toxins is pertussis toxin (PT), a multi-subunit protein toxin that inhibits signaling through a subset of G protein-coupled receptors in mammalian cells. PT activity has been linked with severe and lethal pertussis disease in young infants and a detoxified version of PT is a common component of all licensed acellular pertussis vaccines. The role of PT in typical pertussis disease in other individuals is less clear, but significant evidence supporting its contribution to pathogenesis has been accumulated from animal model studies. In this review we discuss the evidence indicating a role for PT in pertussis disease, focusing on its contribution to severe pertussis in infants, modulation of immune and inflammatory responses to infection, and the characteristic paroxysmal cough of pertussis.


Asunto(s)
Toxina del Pertussis/toxicidad , Tos Ferina/etiología , Animales , Humanos , Tos Ferina/inmunología
14.
Pain ; 160(5): 1037-1049, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30649100

RESUMEN

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease with many known structural and functional changes in the central nervous system. A well-recognized, but poorly understood, complication of MS is chronic pain. Little is known regarding the influence of sex on the development and maintenance of MS-related pain. This is important to consider, as MS is a predominantly female disease. Using the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we demonstrate sex differences in measures of spinal cord inflammation and plasticity that accompany tactile hypersensitivity. Although we observed substantial inflammatory activity in both sexes, only male EAE mice exhibit robust staining of axonal injury markers and increased dendritic arborisation in morphology of deep dorsal horn neurons. We propose that tactile hypersensitivity in female EAE mice may be more immune-driven, whereas pain in male mice with EAE may rely more heavily on neurodegenerative and plasticity-related mechanisms. Morphological and inflammatory differences in the spinal cord associated with pain early in EAE progression supports the idea of differentially regulated pain pathways between the sexes. Results from this study may indicate future sex-specific targets that are worth investigating for their functional role in pain circuitry.


Asunto(s)
Sistema Nervioso Central/fisiopatología , Encefalomielitis Autoinmune Experimental/complicaciones , Plasticidad Neuronal/fisiología , Dolor/etiología , Dolor/patología , Animales , Axones/patología , Axones/ultraestructura , Proteínas de Unión al Calcio/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/ultraestructura , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Ciclo Estral/fisiología , Femenino , Adyuvante de Freund/toxicidad , Hiperalgesia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Actividad Motora/fisiología , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Umbral del Dolor/fisiología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Toxina del Pertussis/toxicidad , Estimulación Física/efectos adversos , Factores Sexuales
15.
J Neuroinflammation ; 15(1): 270, 2018 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-30231889

RESUMEN

BACKGROUND: Axonal degeneration and neuronal loss have been described as the major causes of irreversible clinical disability in multiple sclerosis (MS). The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) protein has been associated with neuroprotection in models of ischemia and neuronal responses to stressors. METHODS: To characterize its potential to influence inflammatory neurodegeneration, we examined ARNT2 expression in the experimental autoimmune encephalomyelitis (EAE) model of MS and characterized mediators that influence ARNT2 expression as well as plausible partners and targets. RESULTS: Arnt2 message and protein levels dropped significantly in EAE spinal cords as disease developed and were lowest at peak disability. ARNT2 expression is prominent in neuronal cell bodies within the gray matter with some staining in glial fibrillary acidic protein (GFAP)+ astrocytes in healthy animals. At peak disease, ARNT2 expression is reduced by 20-50% in gray matter neurons compared to healthy controls. ARNT2 intensity in neurons throughout the EAE spinal cord correlated inversely with the degree of immune cell infiltration (r = - 0.5085, p < 0.01) and axonal damage identified with SMI32 staining (r = - 0.376, p = 0.032). To understand the relationship between ARNT2 expression and neuronal health, we exposed enriched cortical cultures of neurons to hydrogen peroxide (H2O2) to mimic oxidative stress. H2O2 at lower doses rapidly increased ARNT2 protein levels which returned to baseline within 3-4 h. Exposure to higher doses of H2O2) dropped ARNT2 levels below baseline, preceding cytotoxicity measured by morphological changes and lactate dehydrogenase release from cells. Decreases in ARNT2 secondary to staurosporine and H2O2 preceded increases in cleaved caspase 3 and associated apoptosis. We also examined expression of neuronal pas 4 (Npas4), whose heterodimerization with ARNT2 drives expression of the neurotrophic factor brain-derived neurotrophic factor (Bdnf). Like ARNT2, Npas4 levels also decline at the onset of EAE and are linked to decreases in Bdnf. In vitro, H2O2 exposure drives Npas4 expression that is tied to increases in Bdnf. CONCLUSION: Our data support ARNT2 as a neuronal transcription factor whose sustained expression is linked to neuronal and axonal health, protection that may primarily be driven through its partnering with Npas4 to influence BDNF expression.


Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica/fisiología , Esclerosis Múltiple/complicaciones , Neuronas/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Axones/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Embrión de Mamíferos , Femenino , Adyuvante de Freund/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Toxina del Pertussis/toxicidad
16.
Cell Microbiol ; 20(12): e12948, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30152075

RESUMEN

Pertussis toxin (PTx) is a major protective antigen produced by Bordetella pertussis that is included in all current acellular vaccines. Of several well-characterized monoclonal antibodies binding this toxin, the humanised hu1B7 and hu11E6 antibodies are highly protective in multiple in vitro and in vivo assays. In this study, we determine the molecular mechanisms of protection mediated by these antibodies. Neither antibody directly binds the B. pertussis bacterium nor supports antibody-dependent complement cytotoxicity. Both antibodies, either individually or as a cocktail, form multivalent complexes with soluble PTx that bind the FcγRIIb receptor more tightly than antibody alone, suggesting that the antibodies may accelerate PTx clearance via immune complex formation. However, a receptor binding assay and cellular imaging indicate that the main mechanism used by hu11E6 is competitive inhibition of PTx binding to its cellular receptor. In contrast, the main hu1B7 neutralising mechanism appears to be inhibition of PTx internalisation and retrograde trafficking. We assessed the effects of hu1B7 on PTx retrograde trafficking in CHO-K1 cells using quantitative immunofluorescence microscopy. In the absence of hu1B7 or after incubation with an isotype control antibody, PTx colocalizes to organelles in a manner consistent with retrograde transport. However, after preincubation with hu1B7, PTx appears restricted to the membrane surface with colocalization to organelles associated with retrograde transport significantly reduced. Together, these data support a model whereby hu11E6 and hu1B7 interfere with PTx receptor binding and PTx retrograde trafficking, respectively.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Bordetella pertussis/efectos de los fármacos , Toxina del Pertussis/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Bordetella pertussis/inmunología , Bordetella pertussis/metabolismo , Células CHO , Cricetulus , Endocitosis/efectos de los fármacos , Humanos , Toxina del Pertussis/toxicidad , Transporte de Proteínas/efectos de los fármacos , Receptores de IgG/metabolismo
17.
Toxins (Basel) ; 10(5)2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29723951

RESUMEN

The Bordetella pertussis toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.


Asunto(s)
Ciclofilinas/antagonistas & inhibidores , Ciclosporina/farmacología , Toxina del Pertussis/toxicidad , Animales , Bordetella pertussis , Células CHO , Cricetulus , Ciclofilinas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismo
18.
Sci Rep ; 8(1): 4649, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29545630

RESUMEN

The placenta has emerged as an attractive source of mesenchymal stem cells (MSCs) because of the absence of ethical issues, non-invasive access, and abundant yield. However, inflammatory cell invasion into grafts negatively impacts the survival and efficacy of transplanted cells. Previous studies have shown that synthetic C16 peptide can competitively block the transmigration of leukocytes into the central nerve system, while angiopoietin-1 (Ang-1) can inhibit inflammation-induced blood vessel leakage and inflammatory cell infiltration in rats with experimental allergic encephalomyelitis (EAE). In this study, we investigated the effects of intravenous administration of C16 and Ang-1 on the efficacy of placenta-derived MSC (PMSC) transplantation in a rat model of EAE. We found that, compared with PMSCs alone, treatment with PMSCs along with intravenously administered C16 and Ang-1 was more effective at ameliorating demyelination/neuronal loss and neurological dysfunction, reducing inflammatory cell infiltration, perivascular edema, and reactive astrogliosis (p < 0.05). Mechanistic studies revealed that intravenous C16 and Ang-1 increased PMSC engraftment in the central nervous system and promoted expression of the neurotropic proteins brain-derived neurotrophic factor, growth-associated protein 43, and p75 neurotrophin receptor as well as the neuronal-glial lineage markers neurofilament protein 200 and myelin basic protein in the engrafted PMSCs.


Asunto(s)
Angiopoyetina 1/administración & dosificación , Enfermedades del Sistema Nervioso Central/prevención & control , Encefalomielitis Autoinmune Experimental/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Fragmentos de Péptidos/administración & dosificación , Placenta/citología , Administración Intravenosa , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Integrina alfaVbeta3/metabolismo , Masculino , Toxina del Pertussis/toxicidad , Embarazo , Ratas , Ratas Endogámicas Lew , Receptores de Vitronectina/metabolismo
19.
Methods Mol Biol ; 1727: 353-360, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29222794

RESUMEN

The protocol in this chapter presents a method to actively induce experimental autoimmune encephalomyelitis (EAE), one of the most widely used animal models to study efficacy of potential drugs for treatment of multiple sclerosis. Multiple sclerosis is an inflammatory, demyelinating disease of the central nervous system and the most common cause of chronic neurological impairment in young people. In this model EAE is induced in female C57BL/6 mice by immunization with an emulsion of myelin oligodendrocyte glycoprotein (fragment 35-55) in complete Freund's adjuvant, followed by administration of pertussis toxin in phosphate-buffered saline. EAE is evidenced by ascending flaccid paralysis with inflammation targeting the spinal cord.


Asunto(s)
Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Glicoproteína Mielina-Oligodendrócito/inmunología , Toxina del Pertussis/toxicidad , Animales , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Adyuvante de Freund , Inmunización , Ratones , Ratones Endogámicos C57BL
20.
Neuron ; 96(6): 1290-1302.e6, 2017 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-29268096

RESUMEN

Brain aging and neurodegeneration are associated with prominent microglial reactivity and activation of innate immune response pathways, commonly referred to as neuroinflammation. One such pathway, the type I interferon response, recognizes viral or mitochondrial DNA in the cytoplasm via activation of the recently discovered cyclic dinucleotide synthetase cGAS and the cyclic dinucleotide receptor STING. Here we show that the FDA-approved antiviral drug ganciclovir (GCV) induces a type I interferon response independent of its canonical thymidine kinase target. Inhibition of components of the STING pathway, including STING, IRF3, Tbk1, extracellular IFNß, and the Jak-Stat pathway resulted in reduced activity of GCV and its derivatives. Importantly, functional STING was necessary for GCV to inhibit inflammation in cultured myeloid cells and in a mouse model of multiple sclerosis. Collectively, our findings uncover an unexpected new activity of GCV and identify the STING pathway as a regulator of microglial reactivity and neuroinflammation.


Asunto(s)
Encefalomielitis Autoinmune Experimental/patología , Regulación de la Expresión Génica/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Microglía/metabolismo , Animales , Animales Recién Nacidos , Antivirales/uso terapéutico , Células Cultivadas , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Femenino , Adyuvante de Freund/toxicidad , Ganciclovir/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Monocitos/efectos de los fármacos , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/inmunología , Toxina del Pertussis/toxicidad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
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