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BACKGROUND: Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD. METHODS: This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182. FINDINGS: At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes. INTERPRETATION: Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy. FUNDING: Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.
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Cerebrosídeo Sulfatase/genética , Transplante de Células-Tronco Hematopoéticas , Lentivirus/genética , Leucodistrofia Metacromática , Idade de Início , Criança , Pré-Escolar , Feminino , Terapia Genética , Vetores Genéticos , Humanos , Itália , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/terapia , Masculino , Estudos Prospectivos , Resultado do TratamentoRESUMO
Mycoplasma pneumoniae is an atypical bacterial respiratory pathogen known to cause a range of airway inflammation and lung and extrapulmonary pathologies. We recently reported that an M. pneumoniae-derived ADP-ribosylating and vacuolating toxin called community-acquired respiratory distress syndrome (CARDS) toxin is capable of triggering NLRP3 (NLR-family, leucine-rich repeat protein 3) inflammasome activation and interleukin-1ß (IL-1ß) secretion in macrophages. However, it is unclear whether the NLRP3 inflammasome is important for the immune response during M. pneumoniae acute infection. In the current study, we utilized in vitro and in vivo models of M. pneumoniae infection to characterize the role of the NLRP3 inflammasome during acute infection. M. pneumoniae-infected macrophages deficient for inflammasome components NLRP3, ASC (apoptosis speck-like protein containing a caspase activation and recruitment domain), or caspase-1 failed to process and secrete IL-1ß. The MyD88/NF-κB signaling pathway was found to be critical for proinflammatory gene expression in macrophages infected with M. pneumoniae C57BL/6 mice deficient for NLRP3 expression were unable to produce IL-1ß in the airways during acute infection, and lack of this inflammatory response led to deficient immune cell activation and delayed bacterial clearance. These findings are the first to report the importance of the NLRP3 inflammasome in regulating the inflammatory response and influencing the progression of M. pneumoniae during acute infection.
Assuntos
Imunidade Inata/imunologia , Inflamação/metabolismo , Mycoplasma pneumoniae/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/metabolismo , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/imunologia , Caspase 1/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Pneumonia por Mycoplasma/microbiologia , Transdução de Sinais/imunologiaRESUMO
Recognition of viral dsRNA by endosomal TLR3 activates innate immune response during virus infection. Trafficking of TLR3 to the endolysosomal compartment arising from fusion of late endosome (LE) with lysosome is required for recognition and detection of pathogen associated molecular patterns, which results in activation of the TLR3-dependent signaling cascade. Existing knowledge about the mechanism(s) and cellular factor(s) governing TLR3 trafficking is limited. In the current study, we identified intracellular S100A9 protein as a critical regulator of TLR3 trafficking. S100A9 was required for maturation of TLR3 containing early endosome (EE) into LE, the compartment that fuses with lysosome to form the endolysosomal compartment. A drastic reduction in cytokine production was observed in S100A9-knockout (KO) primary macrophages following RNA virus infection and treatment of cells with polyinosinic-polycytidylic acid (polyIC; a dsRNA mimetic that acts as a TLR3 agonist). Mechanistic studies revealed colocalization and interaction of S100A9 with TLR3 following polyIC treatment. S100A9-TLR3 interaction was critical for maturation of TLR3 containing EE into LE because TLR3 could not be detected in the LE of polyIC-treated S100A9-KO macrophages. Subsequently, TLR3 failed to colocalize with its agonist (i.e., biotin-labeled polyIC) in S100A9-deficient macrophages. The in vivo physiological role of S100A9 was evident from loss of cytokine production in polyIC-treated S100A9-KO mice. Thus, we identified intracellular S100A9 as a regulator of TLR3 signaling and demonstrated that S100A9 functions during pre-TLR3 activation stages by facilitating maturation of TLR3 containing EE into LE.
Assuntos
Calgranulina B/imunologia , Macrófagos/imunologia , Vírus de RNA/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Western Blotting , Calgranulina B/genética , Calgranulina B/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Poli I-C/imunologia , Poli I-C/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Interferência de RNA , Vírus de RNA/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/metabolismoRESUMO
Pathogen-associated molecular patterns (PAMPs) trigger host immune response by activating pattern recognition receptors like toll-like receptors (TLRs). However, the mechanism whereby several pathogens, including viruses, activate TLRs via a non-PAMP mechanism is unclear. Endogenous "inflammatory mediators" called damage-associated molecular patterns (DAMPs) have been implicated in regulating immune response and inflammation. However, the role of DAMPs in inflammation/immunity during virus infection has not been studied. We have identified a DAMP molecule, S100A9 (also known as Calgranulin B or MRP-14), as an endogenous non-PAMP activator of TLR signaling during influenza A virus (IAV) infection. S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response, cell-death and virus pathogenesis. Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection. Furthermore, the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studies have thus, underscored the role of a DAMP molecule (i.e. extracellular S100A9) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Calgranulina B/imunologia , RNA Helicases DEAD-box/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Infecções por Orthomyxoviridae/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Calgranulina B/genética , RNA Helicases DEAD-box/genética , Cães , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Transdução de Sinais/genética , Receptor 4 Toll-Like/genéticaRESUMO
How intestinal epithelial cells (IECs) recognize pathogens and activate inflammasomes at intestinal surfaces is poorly understood. We hypothesized that IECs use integrin receptors to recognize pathogens and initiate inflammation within the intestinal tract. We find that IECs infected with Yersinia enterocolitica, an enteric pathogen, use ß1 integrins as pathogen recognition receptors detecting the bacterial adhesin invasin (Inv). The Inv-integrin interaction provides the first signal for NLRP3 inflammasome activation with the type three secretion system translocon providing the second signal for inflammasome activation, resulting in release of IL-18. During infection, Yersinia employs two virulence factors, YopE and YopH, to counteract Inv-mediated integrin-dependent inflammasome activation. Furthermore, NLRP3 inflammasome activation in epithelial cells requires components of the focal adhesion complex signaling pathway, focal adhesion kinase, and rac1. The binding of Inv to ß1 integrins rapidly induces IL-18 mRNA expression, suggesting integrins provide a first signal for NLRP3 inflammasome activation. These data suggest integrins function as pathogen recognition receptors on IECs to rapidly induce inflammasome-derived IL-18-mediated responses.
Assuntos
Células Epiteliais/imunologia , Inflamassomos/imunologia , Inflamassomos/metabolismo , Integrina alfa5beta1/fisiologia , Mucosa Intestinal/imunologia , Transdução de Sinais/imunologia , Yersinia enterocolitica/imunologia , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/farmacologia , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ligação Proteica/imunologia , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Fatores de Virulência/fisiologia , Yersinia enterocolitica/genéticaRESUMO
Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. NO is a cellular mediator involved in tissue damage through its apoptosis of target cells and resulting enhancement of local inflammation. Inducible NO synthase (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism, including the role of NO in apoptosis during infection, has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of the host transactivator(s) required for iNOS gene expression. In the current study, we identified Kruppel-like factor 6 (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We also underscored the requirement for iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in the drastic loss of NO production, iNOS promoter-specific luciferase activity, and expression of iNOS mRNA following flu infection. Chromatin immunoprecipitation assay revealed a direct interaction of KLF6 with iNOS promoter during in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. A significant reduction in flu-mediated apoptosis was noted in KLF6-silenced cells, cells treated with iNOS inhibitor, and primary murine macrophages derived from iNOS knockout mice. A similar reduction in apoptosis was noted in the lungs following intratracheal flu infection of iNOS knockout mice.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Infecções por Orthomyxoviridae/imunologia , Proteínas Proto-Oncogênicas/fisiologia , Ativação Transcricional/imunologia , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Inativação Gênica/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/deficiência , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Regiões Promotoras Genéticas/imunologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Ativação Transcricional/genéticaRESUMO
Background/Motivation: This article presents a systematic review aimed at examining the utilization of learning analytics (LA) to enhance teachers' professional capital. Aim: The study focuses on three primary research questions: (1) exploring the characteristics and approaches of LA in professional capital, (2) investigating suggestions from LA for assessing and improving professional capital, and (3) examining variables studied in enhancing the most intricate dimension of professional capital using LA. Methodology: To address the research objectives, a systematic review was conducted focusing on the key concepts "learning analytics" and "professional capital." Following the procedures outlined encompassed in four stages: identification, screening, inclusion, and adequacy. The PRISMA 2009 protocol guided the systematic review process. Principal findings: The findings of the study underscore the efficacy of LA as a catalyst for improving professional capital, particularly through collaborative learning and the utilization of tools like forums and online learning platforms. Social capital emerges as a pivotal component in integrating diverse types of professional capital, fostering opportunities for knowledge creation and social networking. Conclusion/Significance: In conclusion, the study highlights the paramount significance of addressing teachers' professional capital development through collaborative approaches and leveraging technology, particularly in primary education. The article concludes by emphasizing the imperative for more research and knowledge dissemination in this field, aiming to ensure equity in learning and address the challenges posed by the COVID-19 pandemic.
RESUMO
Human respiratory syncytial virus (RSV) is a highly pathogenic lung-tropic virus that causes severe respiratory diseases. Enzymatic activity of inducible nitric oxide (iNOS) is required for NO generation. Although NO contributes to exaggerated lung disease during RSV infection, the role of NO in apoptosis during infection is not known. In addition, host trans-activator(s) required for iNOS gene expression during RSV infection is unknown. In the current study we have uncovered the mechanism of iNOS gene induction by identifying kruppel-like factor 6 (KLF6) as a critical transcription factor required for iNOS gene expression during RSV infection. Furthermore, we have also uncovered the role of iNOS as a critical host factor regulating apoptosis during RSV infection.
Assuntos
Apoptose/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Animais , Imunoprecipitação da Cromatina , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Proteínas Proto-Oncogênicas/genética , Infecções por Vírus Respiratório Sincicial/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
Hybrid designs with both randomized arms and an external control cohort preserve key features of randomization and utilize external information to augment clinical trials. In this study, we propose to leverage high-quality, patient-level concurrent registries to enhance clinical trials and illustrate the impact on trial design for amyotrophic lateral sclerosis. The proposed methodology was evaluated in a randomized, placebo-controlled clinical trial. We used patient-level information from a well-defined, population-based registry, that was running parallel to the randomized clinical trial, to identify concurrently nonparticipating, eligible patients who could be matched with trial participants, and integrate them into the statistical analysis. We assessed the impact of the addition of the external controls on the treatment effect estimate, precision, and time to reach a conclusion. During the runtime of the trial, a total of 1,141 registry patients were alive; 473 (41.5%) of them fulfilled the eligibility criteria and 133 (11.7%) were enrolled in the study. A matched control population could be identified among the nonparticipating patients. Augmenting the randomized controls with matched external controls could have avoided unnecessary randomization of 17 patients (-12.8%) as well as reducing the study duration from 30.1 months to 22.6 months (-25.0%). Matching eligible external controls from a different calendar period led to bias in the treatment effect estimate. Hybrid trial designs utilizing a concurrent registry with rigorous matching can minimize bias due to a mismatch in calendar time and differences in standard of care, and may accelerate the development of new treatments.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Estudos de Viabilidade , Projetos de PesquisaRESUMO
BACKGROUND: Human respiratory syncytial virus (RSV) infection is associated with airway remodeling and subsequent asthma development. Transforming growth factor-beta (TGF) plays a crucial role in asthma development. The mechanism regulating TGF gene expression during RSV infection is not known. Kruppel-like factor family of transcription factors are critical regulators of cellular/tissue homeostasis. Previous studies have shown that Kruppel-like factor 6 (KLF6) could function as a trans-activator of TGF gene; however, whether KLF members play a role during infection is unknown. In the current study we have evaluated the role of KLF6 during TGF expression in RSV infected cells. FINDINGS: Silencing KLF6 expression by shRNA led to drastic inhibition in TGF production during RSV infection, as assessed by ELISA analysis of medium supernatants. RT-PCR analysis revealed loss of TGF expression in KLF6 silenced cells. Chromatin-immunoprecipitation assay conducted with RSV infected cells showed binding of KLF6 protein to the TGF promoter during RSV infection. We further observed reduced RSV infectivity in KLF6 silenced cells and in cells incubated with TGF neutralizing antibody. In contrast, enhanced RSV infection was noted in cells incubated with purified TGF. CONCLUSION: We have identified KLF6 as a key transcription factor required for trans-activation of TGF gene during RSV infection. Moreover, TGF production is required for efficient RSV infection and thus, KLF6 is also required for efficient RSV infection by virtue of KLF6 dependent TGF production during infection.
Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vírus Sincicial Respiratório Humano/patogenicidade , Fator de Crescimento Transformador beta/biossíntese , Linhagem Celular , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Hidroxicolesteróis/metabolismo , Imunidade Inata/imunologia , Integrina alfa5beta1/imunologia , Integrina alfaVbeta3/imunologia , Macrófagos/imunologia , Animais , Adesões Focais , Inflamação , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Interleucina-6/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/imunologiaRESUMO
PURPOSE: Phosphodiesterase 4 (PDE4) inhibition in the brain has been reported to improve cognitive function in animal models. Therefore, PDE4 inhibitors are one of key targets potential for drug development. Investigation of brain PDE4 occupancy would help to understand the effects of PDE4 inhibition to cognitive functions. Roflumilast is a selective phosphodiesterase type 4 (PDE4) inhibitor used clinically for severe chronic obstructive pulmonary disease, but the effects to the brain have not been well investigated. In this study, we aimed to investigate whether roflumilast entered the brain and occupied PDE4 in nonhuman primates. PROCEDURES: Positron emission tomography (PET) measurements with (R)-[11C]rolipram were performed at baseline and after intravenous (i.v.) administration of roflumilast (3.6 to 200 µg/kg) in three female rhesus monkeys. Arterial blood samples were taken to obtain the input function. Protein binding was measured to obtain the free fraction (fp) of the radioligand. Total distribution volume (VT) and VT/fp were calculated as outcome measures from two tissue compartment model. Lassen plot approach was taken to estimate the target occupancy. RESULTS: The brain uptake of (R)-[11C]rolipram decreased after roflumilast administration. PDE 4 occupancy by roflumilast showed dose- and plasma concentration-dependent increase, although PDE4 occupancy did not reach 50 % even after the administration of up to 200 µg/kg of roflumilast, regardless of outcome measures, VT or VT/fp. CONCLUSIONS: This PET study showed that the brain PDE4 binding was blocked to a certain extent after i.v. administration of clinical relevant doses of roflumilast in nonhuman primates. Further clinical PET evaluation is needed to understand the relationship between PDE4 inhibition and potential improvement of cognitive function in human subjects.
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Aminopiridinas/farmacologia , Benzamidas/farmacologia , Encéfalo/enzimologia , Radioisótopos de Carbono/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Tomografia por Emissão de Pósitrons , Rolipram/farmacologia , Aminopiridinas/sangue , Aminopiridinas/química , Aminopiridinas/farmacocinética , Animais , Benzamidas/sangue , Benzamidas/química , Benzamidas/farmacocinética , Ciclopropanos/sangue , Ciclopropanos/química , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , Feminino , Humanos , Macaca mulatta , Rolipram/sangue , Rolipram/química , Rolipram/farmacocinéticaRESUMO
In dentistry and oral and maxillofacial surgery, the development of implantable biomaterials and the understanding of their molecular, cellular and pharmaceutical aspects are currently major fields of research and education, with a considerable impact on the daily clinical practice and the evolution of therapeutic strategies. In the era of globalized economy of knowledge and science, this scientific domain needs the development of global cooperation and a paradigm evolution in the organizational culture of the dental sciences and related dental industry. Despite political pressure and theoretical efforts, the internationalization of higher education and research today in dentistry and biomaterials remains in general quite superficial and mostly dependent on the efforts of a few leaders of internationalization working through their personal networks, as it was assessed through the FAST scores (Fast Assessment Screening Test) calculated in various dental schools and groups worldwide through the ISAIAS program (Intercultural Sensitivity Academic Index &Advanced Standards). Cooperation in a multipolar multicultural community requires the development of strong intercultural competences, and this process remains limited in most institutions. These limits of international scientific cooperation can be observed through different markers, particularly the difficult and limited production of ISO standards (International Organization for Standardization) and the relatively low SCIENTI scores (Scientific Cooperation Internationalization Effort &Network Test &Index) of the specialized dental literature, particularly in comparison to the most significant medical literature. However, as an analytical tool to assess the scientific international cooperation effort between fields and periods, the SCIENTI screening system also highlighted a significant increase of the internationalization effort in the last years in the best dental biomaterials publications. Finally, an internationalization of higher education and research perspective is a very important approach to assess the evolution of the dental biomaterial science and highlights very clearly the future endeavors of this field, particularly the impact and interferences of private entities and companies in the development of this corpus of knowledge. It also reveals that the concept of independent not-for-profit Cooperation Internationalization Effort Literature (CIEL), in the various informal models that can be found worldwide around diverse leaderships, is the best perspective for a better science and understanding of molecular, cellular and pharmaceutical aspects of biomaterials in dentistry and oral and maxillofacial surgery.
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Materiais Biocompatíveis , Pesquisa Biomédica/tendências , Odontologia/tendências , Educação em Odontologia/tendências , Intercâmbio Educacional Internacional , Cirurgia Bucal/tendências , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Biotecnologia/tendências , HumanosRESUMO
Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP1-734. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP1-734 in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence.
Assuntos
Proteínas de Bactérias/genética , Deleção de Genes , Streptococcus pneumoniae/genética , Fatores de Virulência/genética , Células A549 , Animais , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Adesão Celular , Células Epiteliais/microbiologia , Feminino , Genes Bacterianos , Ilhas Genômicas/genética , Humanos , Pulmão/citologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Óperon/genética , Streptococcus pneumoniae/patogenicidadeRESUMO
Caspase-1 is activated by the inflammasome complex to process cytokines like interleukin-1ß (IL-1ß). Pro-caspase-1 consists of three domains, CARD, p20, and p10. Association of pro-caspase-1 with the inflammasome results in initiation of its autocatalytic activity, culminating in self-cleavage that generates catalytically active subunits (p10 and p20). In the current study, we show that Nedd8 is required for efficient self-cleavage of pro-caspase-1 to generate its catalytically active subunits. Nedd8 silencing or treating cells with the neddylation inhibitor MLN4924 led to diminished caspase-1 processing and reduced IL-1ß maturation following inflammasome activation. Coimmunoprecipitation and mass spectrometric analysis of 293 cells overexpressing pro-caspase-1 (and CARD) and Nedd8 suggested possible neddylation of caspase-1 CARD. Following inflammasome activation in primary macrophages, we observed colocalization of endogenous Nedd8 with caspase-1. Similarly, interaction of endogenous Nedd8 with caspase-1 CARD was detected in inflammasome-activated macrophages. Furthermore, enhanced autocatalytic activity of pro-caspase-1 was observed following Nedd8 overexpression in 293 cells, and such activity in inflammasome-activated macrophages was drastically diminished upon treatment of cells with MLN4924. Thus, our studies demonstrate a role of Nedd8 in regulating caspase-1 activation following inflammasome activation, presumably via augmenting autoprocessing/cleavage of pro-caspase-1 into its corresponding catalytically active subunits.
Assuntos
Caspase 1/metabolismo , Inflamassomos/metabolismo , Vírus da Influenza A/isolamento & purificação , Ubiquitinas/metabolismo , Animais , Proteínas de Transporte , Ativação Enzimática , Humanos , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Proteína NEDD8RESUMO
UNLABELLED: The inflammasome is a major regulator of inflammation through its activation of procaspase-1, which cleaves prointerleukin-1ß (pro-IL-1ß) into its mature form. IL-1ß is a critical proinflammatory cytokine that dictates the severity of inflammation associated with a wide spectrum of inflammatory diseases. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the current study, we uncovered a yet unknown mechanism of NLRP3 inflammasome activation by a pathogen-derived factor. We show that the unique bacterial ADP-ribosylating and vacuolating toxin produced by Mycoplasma pneumoniae and designated community-acquired respiratory distress syndrome (CARDS) toxin activates the NLRP3 inflammasome by colocalizing with the NLRP3 inflammasome and catalyzing the ADP-ribosylation of NLRP3. Mutant full-length CARDS toxin lacking ADP-ribosyltransferase (ADPRT) activity and truncated CARDS toxins unable to bind to macrophages and be internalized failed to activate the NLRP3 inflammasome. These studies demonstrate that CARDS toxin-mediated ADP-ribosylation constitutes an important posttranslational modification of NLRP3, that ADPRT activity of CARDS toxin is essential for NLRP3 inflammasome activation, and that posttranslational ADPRT-mediated modification of the inflammasome is a newly discovered mechanism for inflammasome activation with subsequent release of IL-1ß and associated pathologies. IMPORTANCE: Inflammation is a fundamental innate immune response to environmental factors, including infections. The inflammasome represents a multiprotein complex that regulates inflammation via its ability to activate specific proinflammatory cytokines, resulting in an effective host protective response. However, excessive release of proinflammatory cytokines can occur following infection that skews the host response to "hyperinflammation" with exaggerated tissue damage. Mycoplasma pneumoniae, a common bacterial airway pathogen, possesses a unique protein toxin with ADP-ribosyltransferase and vacuolating properties capable of reproducing the robust inflammation and cytopathology associated with mycoplasma infection. Here, we show that the toxin uniquely activates the NLRP3 inflammasome by colocalizing with and ADP-ribosylating NLRP3, possibly leading to "hyperinflammation" and thus uncovering a novel target for therapeutic intervention.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Transporte/metabolismo , Interações Hospedeiro-Patógeno , Inflamassomos/metabolismo , Mycoplasma pneumoniae/fisiologia , Processamento de Proteína Pós-Traducional , Animais , Células Cultivadas , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Mycoplasma pneumoniae/patogenicidade , Proteína 3 que Contém Domínio de Pirina da Família NLRRESUMO
Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in the life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.
Assuntos
Colesterol/fisiologia , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Microdomínios da Membrana/fisiologia , Vírus Sincicial Respiratório Humano/fisiologia , Liberação de Vírus/fisiologia , Linhagem Celular , Células Epiteliais/virologia , Fibroblastos/virologia , Humanos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1ß (IL-1ß) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1ß is synthesized as an immature pro-IL-1ß form. It is cleaved by activated caspase-1 to yield mature IL-1ß that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1ß release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1ß secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1ß production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1ß and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1ß release during RSV infection.