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Introduction: Clobazam is a benzodiazepine drug, used to treat Lennox-Gastaut syndrome in patients aged 2 years and older. Objective: To support patient care, our laboratory developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma or serum samples. Methods: The chromatographic separation was achieved with an Agilent Zorbax Eclipse Plus C-18 RRHD column with mobile phase consisting of 0.05% formic acid in 5 mM ammonium formate, pH 3.0 and 0.1% formic acid in acetonitrile at a flow rate of 600 µL/minute and an injection volume of 5 µL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor precursor-to-product ion transitions in positive electrospray ionization mode. Results: The method was validated over a concentration range of 20-2000 ng/mL for CLB and 200-10,000 ng/mL for N-CLB. The lower limit of quantification was 20 ng/mL for CLB and 200 ng/mL for N-CLB with good accuracy and precision. The method performance was successfully evaluated by comparison with two different external laboratories. Retrospective data analysis was performed to evaluate the positivity rate and metabolic patterns for clobazam from our patient population, as a reference laboratory. Among the positive samples, both parent and metabolite were detected in 96.4% of the samples. Conclusion: The method was developed to support therapeutic drug monitoring and the data generated from retrospective analysis could be useful for result interpretation in conjunction with clinical patient information.
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Mutant superoxide dismutase 1 (SOD1) can be constitutively released from motor neurons and transmitted to naïve motor neurons to promote the progression of amyotrophic lateral sclerosis (ALS). However, the biological impacts of this process and the precise mechanisms of SOD1 release remain to be fully resolved. Using biochemical and fluorescent techniques, this study aimed to determine if P2X7 receptor activation could induce mutant SOD1 release from motor neurons and whether this released SOD1 could be transmitted to motor neurons or microglia to mediate effects associated with neurodegeneration in ALS. Aggregated SOD1G93A, released from murine NSC-34 motor neurons transiently transfected with SOD1G93A, could be transmitted to naïve NSC-34 cells and murine EOC13 microglia to induce endoplasmic reticulum (ER) stress and tumour necrosis factor-alpha (TNFα) release, respectively. Immunoblotting revealed NSC-34 cells expressed P2X7. Extracellular ATP induced cation dye uptake into these cells, which was blocked by the P2X7 antagonist AZ10606120, demonstrating these cells express functional P2X7. Moreover, ATP induced the rapid release of aggregated SOD1G93A from NSC-34 cells transiently transfected with SOD1G93A, a process blocked by AZ10606120 and revealing a role for P2X7 in this process. ATP-induced SOD1G93A release coincided with membrane blebbing. Finally, aggregated SOD1G93A released via P2X7 activation could also be transmitted to NSC-34 and EOC13 cells to induce ER stress and TNFα release, respectively. Collectively, these results identify a novel role for P2X7 in the prion-like propagation of SOD1 in ALS and provide a possible explanation for the therapeutic benefits of P2X7 antagonism previously observed in ALS SOD1G93A mice.
Assuntos
Esclerose Lateral Amiotrófica , Receptores Purinérgicos P2X7 , Superóxido Dismutase-1 , Animais , Camundongos , Trifosfato de Adenosina/farmacologia , Esclerose Lateral Amiotrófica/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Neurônios Motores/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Superóxido Dismutase-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.
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Esclerose Lateral Amiotrófica/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Compostos de Cobre Orgânico , Superóxido Dismutase-1/metabolismo , Animais , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos de Cobre Orgânico/administração & dosagem , Compostos de Cobre Orgânico/efeitos adversos , Compostos de Cobre Orgânico/farmacologiaRESUMO
The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins.
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Exotoxinas/metabolismo , Prófagos/genética , Streptococcus pyogenes/patogenicidade , Streptococcus pyogenes/virologia , Animais , Proteínas de Bactérias/farmacologia , Linhagem Celular , Eritrócitos/efeitos dos fármacos , Exotoxinas/genética , Feminino , Glutationa/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Faringe/citologia , Escarlatina/epidemiologia , Escarlatina/microbiologia , Streptococcus pyogenes/genética , Estreptolisinas/farmacologia , Superantígenos/genética , Superantígenos/metabolismoRESUMO
CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.
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5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Separação Celular/métodos , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Criopreservação , Citometria de Fluxo , HumanosRESUMO
BACKGROUND & AIMS: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. METHODS: C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. RESULTS: hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. CONCLUSIONS: We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.
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Clostridioides difficile/efeitos dos fármacos , Enterocolite Pseudomembranosa/etiologia , Enterocolite Pseudomembranosa/patologia , Plasminogênio/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Humanos , Intestino Delgado , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNß and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1G93A mice.
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Esclerose Lateral Amiotrófica/patologia , Niacinamida/análogos & derivados , Piperazinas/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Animais , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Niacinamida/farmacologia , Superóxido Dismutase-1/genéticaRESUMO
BACKGROUND: Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. METHODS: NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry; gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology. RESULTS: Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling. CONCLUSION: Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.
Assuntos
Trato Gastrointestinal/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Pele/metabolismo , Adulto , Animais , Antígenos CD/metabolismo , Modelos Animais de Doenças , Orelha/patologia , Feminino , Doença Enxerto-Hospedeiro/sangue , Humanos , Interferon gama/sangue , Leucócitos Mononucleares/metabolismo , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Proteínas Associadas a Pancreatite/metabolismo , Baço/imunologia , Linfócitos T/imunologia , Regulação para Cima/genética , Adulto JovemRESUMO
Invasive infections due to group A Streptococcus (GAS) advance rapidly causing tissue degradation and unregulated inflammation. Neutrophils are the primary immune cells that respond to GAS. The neutrophil response to GAS was characterised in response to two M1T1 isolates; 5448 and animal passaged variant 5448AP. Co-incubation of neutrophils with 5448AP resulted in proliferation of GAS and lowered the production of reactive oxygen species when compared with 5448. Infection with both strains invoked neutrophil death, however apoptosis was reduced in response to 5448AP. Both strains induced neutrophil caspase-1 and caspase-4 expression in vitro, with inflammatory caspase activation detected in vitro and in vivo. GAS infections involving strains such as 5448AP that promote an inflammatory neutrophil phenotype may contribute to increased inflammation yet ineffective bacterial eradication, contributing to the severity of invasive GAS infections.
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Infecções Estreptocócicas , Streptococcus pyogenes , Animais , Caspases/genética , Neutrófilos , FenótipoRESUMO
Plasmin(ogen) acquisition is critical for invasive disease initiation by Streptococcus pyogenes (GAS). Host urokinase plasminogen activator (uPA) plays a role in mediating plasminogen activation for GAS dissemination, however the contribution of tissue-type plasminogen activator (tPA) to GAS virulence is unknown. Using novel tPA-deficient ALBPLG1 mice, our study revealed no difference in mouse survival, bacterial dissemination or the pathology of GAS infection in the absence of tPA in AlbPLG1/tPA-/- mice compared to AlbPLG1 mice. This study suggests that tPA has a limited role in this humanized model of GAS infection, further highlighting the importance of its counterpart uPA in GAS disease.
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Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Carga Bacteriana , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Viabilidade Microbiana , Mutação , Infecções Estreptocócicas/patologia , Ativador de Plasminogênio Tecidual/genética , VirulênciaRESUMO
Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289.
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Proteínas de Bactérias/metabolismo , Plasminogênio/metabolismo , Infecções Estreptocócicas/enzimologia , Streptococcus pyogenes/metabolismo , Aminocaproatos/química , Aminocaproatos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Ativação Enzimática , Glicosilação , Humanos , Kringles , Plasminogênio/química , Plasminogênio/genética , Ligação Proteica , Conformação Proteica , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
Polymorphonuclear leukocyte (PMN) cell death strongly influences the resolution of inflammatory episodes, and may exacerbate adverse pathologies in response to infection. We investigated PMN cell death mechanisms following infection by virulent group A Streptococcus (GAS). Human PMNs were infected in vitro with a clinical, virulent GAS isolate and an avirulent derivative strain, and compared for phagocytosis, the production of reactive oxygen species (ROS), mitochondrial membrane depolarization and apoptotic markers. C57BL/6J mice were then infected, in order to observe the effects on murine PMNs in vivo. Human PMNs phagocytosed virulent GAS less efficiently, produced less ROS and underwent reduced mitochondrial membrane depolarization compared with phagocytosis of avirulent GAS. Morphological and biochemical analyses revealed that PMNs infected with avirulent GAS exhibited nuclear fragmentation and caspase-3 activation consistent with an anti-inflammatory apoptotic phenotype. Conversely, virulent GAS induced PMN vacuolization and plasma membrane permeabilization, leading to a necrotic form of cell death. Infection of the mice with virulent GAS engendered significantly higher systemic pro-inflammatory cytokine release and localized infiltration of murine PMNs, with cells associated with virulent GAS infection exhibiting reduced apoptotic potential. Avirulent GAS infection was associated with lower levels of proinflammatory cytokines and tissue PMN apoptosis. We propose that the differences in PMN cell death mechanisms influence the inflammatory responses to infection by GAS.
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Apoptose/imunologia , Caspase 3/imunologia , Neutrófilos/imunologia , Fagocitose , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Feminino , Humanos , Masculino , Camundongos , Membranas Mitocondriais/imunologia , Membranas Mitocondriais/patologia , Necrose , Espécies Reativas de Oxigênio/imunologia , Infecções Estreptocócicas/patologiaRESUMO
The globally significant human pathogen group A Streptococcus (GAS) sequesters the host protease plasmin to the cell surface during invasive disease initiation. Recent evidence has shown that localized plasmin activity prevents opsonization of several bacterial species by key components of the innate immune system in vitro. Here we demonstrate that plasmin at the GAS cell surface resulted in degradation of complement factor C3b, and that plasminogen acquisition is associated with a decrease in C3b opsonization and neutrophil-mediated killing in vitro. Furthermore, the ability to acquire cell surface plasmin(ogen) correlates directly with a decrease in C3b opsonization, neutrophil phagocytosis, and increased bacterial survival in a humanized plasminogen mouse model of infection. These findings demonstrate that localized plasmin(ogen) plays an important role in facilitating GAS escape from the host innate immune response and increases bacterial virulence in the early stages of infection.
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Complemento C3b/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Plasminogênio/imunologia , Streptococcus pyogenes/imunologia , Animais , Western Blotting , Complemento C3b/metabolismo , Feminino , Fibrinolisina/imunologia , Fibrinolisina/metabolismo , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Masculino , Camundongos Transgênicos , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Plasminogênio/genética , Plasminogênio/metabolismo , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/fisiologia , Estreptoquinase/imunologia , Estreptoquinase/metabolismoRESUMO
Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA-/- mice. The observed decrease in virulence in uPA-/-mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.
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Resistência à Doença , Plasminogênio/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Cruzamentos Genéticos , Suscetibilidade a Doenças , Precursores Enzimáticos/sangue , Precursores Enzimáticos/metabolismo , Fibrinolisina/metabolismo , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasminogênio/genética , Proteólise , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/patogenicidade , Estreptoquinase/metabolismo , Propriedades de Superfície , Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/genética , VirulênciaRESUMO
The cystic fibrosis (CF)-causing mutant, deltaF508-CFTR, is misfolded and fails to traffic out of the endoplasmic reticulum (ER) to the cell surface. Introduction of second site mutations that disrupt a diarginine (RXR)-based ER retention motif in the first nucleotide binding domain rescues the trafficking defect of deltaF508-CFTR, supporting a role for these motifs in mediating ER retention of the major mutant. To determine if these RXR motifs mediate retention of the native deltaF508-CFTR protein in situ, we generated peptides that mimic these motifs and should antagonize mistrafficking mediated via their aberrant exposure. Here we show robust rescue of deltaF508-CFTR in cell lines and in respiratory epithelial tissues by transduction of RXR motif-mimetics, showing that abnormal accessibility of this motif is a key determinant of mistrafficking of the major CF-causing mutant.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Peptídeos/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Mucosa Respiratória/metabolismoRESUMO
Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed approximately 450 unique proteins fused to the Cl(-)-sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI(-)/l(-) exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trk-fused gene, Caveolin 2, PAP/REG3alpha, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of high-content analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block correction of F508del-CFTR, small molecule, and peptide screens.