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One of the hallmarks of type 1 but also type 2 diabetes is pancreatic islet inflammation, associated with altered pancreatic islet function and structure, if unresolved. IL-1ß is a proinflammatory cytokine which detrimentally affects ß-cell function. In the course of diabetes, complement components, including the central complement protein C3, are deregulated. Previously, we reported high C3 expression in human pancreatic islets, with upregulation after IL-1ß treatment. In the current investigation, using primary human and rodent material and CRISPR/Cas9 gene-edited ß-cells deficient in C3, or producing only cytosolic C3 from a noncanonical in-frame start codon, we report a protective effect of C3 against IL-1ß-induced ß-cell death, that is attributed to the cytosolic fraction of C3. Further investigation revealed that intracellular C3 alleviates IL-1ß-induced ß-cell death, by interaction with and inhibition of Fyn-related kinase (FRK), which is involved in the response of ß-cells to cytokines. Furthermore, these data were supported by increased ß-cell death in vivo in a ß-cell-specific C3 knockout mouse. Our data indicate that a functional, cytoprotective association exists between FRK and cytosolic C3.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Camundongos , Animais , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Morte Celular , Citocinas/metabolismo , Camundongos KnockoutRESUMO
The term "intracellular complement" has been introduced recently as an umbrella term to distinguish functions of complement proteins that take place intracellularly, rather than in the extracellular environment. However, this rather undefined term leaves some confusion as to the classification of what intracellular complement really is, and as to which intracellular compartment(s) it should refer to. In this review, we will describe the evidence for both canonical and non-canonical functions of intracellular complement proteins, as well as the current controversies and unanswered questions as to the nature of the intracellular complement. We also suggest new terms to facilitate the accurate description and discussion of specific forms of intracellular complement and call for future experiments that will be required to provide more definitive evidence and a better understanding of the mechanisms of intracellular complement activity.
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Proteínas do Sistema Complemento , Humanos , Proteínas do Sistema Complemento/metabolismoRESUMO
NXP900 is a selective and potent SRC family kinase (SFK) inhibitor, currently being dosed in a phase 1 clinical trial, that locks SRC in the "closed" conformation, thereby inhibiting both kinase-dependent catalytic activity and kinase-independent functions. In contrast, several multi-targeted kinase inhibitors that inhibit SRC, including dasatinib and bosutinib, bind their target in the active "open" conformation, allowing SRC and other SFKs to act as a scaffold to promote tumorigenesis through non-catalytic functions. NXP900 exhibits a unique target selectivity profile with sub-nanomolar activity against SFK members over other kinases. This results in highly potent and specific SFK pathway inhibition. Here, we demonstrate that esophageal squamous cell carcinomas (ESCC) and head and neck squamous cell carcinomas (HNSCC) are exquisitely sensitive to NXP900 treatment in cell culture and in vivo, and we identify a patient population that could benefit from treatment with NXP900.
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The complement system is a proteolytic cascade triggered by pathogen and danger-associated molecular patterns, with resultant outcomes of inflammation, cellular activation, and opsonization of material for removal by phagocytosis. While first discovered as an activity in serum, it is now recognized that complement components play important roles at local and individual cell-intrinsic levels. In particular, apart from the extracellular serum activities of complement, it is now believed that complement also acts intracellularly, as part of a cellular signal transduction cascade that can stimulate cellular survival and activation, and individual immune cell phenotypes, via effects on cellular metabolism. This review will describe what is currently known about how complement functions in intracellular signal transduction, and outline the functional advantages of a compartmentalized and intracellular complement system.
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Ativação do Complemento , Proteínas do Sistema Complemento , Transdução de Sinais , Humanos , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Animais , Transdução de Sinais/imunologia , Ativação do Complemento/imunologia , Fagocitose/imunologia , Inflamação/imunologia , Inflamação/metabolismoRESUMO
Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of ß-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse ß-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed nonGPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.
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Processamento Alternativo , Diabetes Mellitus , Antígenos CD59/genética , Antígenos CD59/metabolismo , Diabetes Mellitus/genética , Humanos , Secreção de Insulina , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Kidney disease disproportionately impacts people with low socioeconomic status, and low socioeconomic status is associated with worse outcomes for people with kidney disease. Unstable housing, which includes housing insecurity and homelessness, is increasing due to rising housing costs. There is mounting evidence that unstable housing and other health-related social needs are partially driving worse outcomes for people with low socioeconomic status. In this perspective, we consider the challenges to addressing housing for people with kidney disease, such as difficulty with identification of those with unstable housing, strict eligibility criteria for housing support, inadequate supply of affordable housing, and flaws in communities' prioritization of affordable housing. We discuss ways to tailor management for people experiencing unstable housing with kidney disease, and the importance of addressing safety, trauma, and emotional concerns as a part of care. We identify opportunities for the nephrology community to surmount challenges through increased screening, investment in workforce dedicated to community resource navigation, advocacy for investment in affordable housing, restructuring of communities' prioritization of affordable housing, and conducting needed research. Identifying and addressing housing needs among people with kidney disease is critical to eliminating kidney health disparities.
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Habitação , Humanos , Pessoas Mal Alojadas , Nefropatias/terapiaRESUMO
OBJECTIVE: Popular musicians are exposed to many occupational stressors, including unpredictable work schedules, touring and economic precarity, that have been associated in prior studies with psychological ill health. This study sought to identify occupational stressors most strongly associated with depression, anxiety, and alcohol misuse in popular musicians. METHODS: An online survey was completed by 317 popular musicians that included the Musician Occupational Stress Scale (MOSS), a validated measure of occupational stress in popular musicians. An exploratory principle-factor analysis (EFA) investigated the dimensions of response pat¬terns in the items comprising the MOSS. RESULTS: Four factors were identified that predicted 50% of musician occupational stress: Work Insecurity Stress, Tour Stress, Performance Stress, and Professional Relationship Stress. In addition to financial distress, each factor was significantly associated with depression and anxiety and Tour Stress also was associated with alcohol misuse. After adjusting for all other factors and financial distress, only Work Insecurity Stress remained associated with depression (odds ratio [OR]= 5.42; 95% confidence interval [CI] = 3.23-9.09) and anxiety (OR=5.95; 95% CI = 3.51-10.11). Tour Stress became inversely associated with depression (OR= 0.59; 95% CI = 0.40-0.89) and anxiety (OR=0.60; 95% CI = 0.40-0.89). After adjustment, alcohol misuse was associated with Professional Relationship Stress (OR=1.66; 95% CI = 1.04-2.65) but inversely correlated with Performance Stress (OR=0.60; 95% CI = 0.40-0.91). CONCLUSIONS: The four-factor model was shown to reliably simplify driving associations of occupational stressors that negatively impact psychological functioning in popular musicians. Dissemination of these findings could have practical implications in developing effective outreach messaging to promote psychological resilience and guide psychotherapeutic intervention strategies for this high-risk occupational group.
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Ansiedade , Depressão , Música , Estresse Ocupacional , Humanos , Masculino , Estresse Ocupacional/psicologia , Estresse Ocupacional/epidemiologia , Feminino , Adulto , Análise Fatorial , Música/psicologia , Pessoa de Meia-Idade , Depressão/epidemiologia , Depressão/psicologia , Ansiedade/epidemiologia , Ansiedade/psicologia , Inquéritos e Questionários , Doenças Profissionais/psicologia , Doenças Profissionais/epidemiologia , Alcoolismo/psicologia , Alcoolismo/epidemiologia , Estresse Psicológico/psicologia , Estresse Psicológico/epidemiologia , Adulto JovemRESUMO
Complement C3 was originally regarded as a serum effector protein, although recent data has emerged suggesting that intracellular C3 can also regulate basic cellular processes. Despite the growing interest in intracellular C3 functions, the mechanism behind its generation has not been demonstrated. In this study we show that C3 can be expressed from an alternative translational start site, resulting in C3 lacking the signal peptide, which is therefore translated in the cytosol. In contrast to the secreted form, alternatively translated cytosolic C3 is not glycosylated, is present mainly in a reduced state, and is turned over by the ubiquitin-proteasome system. C3 can also be retrotranslocated from the endoplasmic reticulum into the cytosol, structurally resembling secreted C3. Finally, we demonstrate that intracellular cytosolic C3 can opsonize invasive Staphylococcus aureus within epithelial cell, slowing vacuolar escape as well as impacting bacterial survival on subsequent exposure to phagocytes. Our work therefore reveals the existence and origin of intracellular, cytosolic C3, and demonstrates functions for cytosolic C3 in intracellular detection of cytoinvasive pathogens.
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Complemento C3 , Complexo de Endopeptidases do Proteassoma , Bactérias/metabolismo , Complemento C3/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
The future quantum internet will leverage existing communication infrastructures, including deployed optical fibre networks, to enable novel applications that outperform current information technology. In this scenario, we perform a feasibility study of quantum communications over an industrial 224 km submarine optical fibre link deployed between Southport in the United Kingdom (UK) and Portrane in the Republic of Ireland (IE). With a characterisation of phase drift, polarisation stability and the arrival time of entangled photons, we demonstrate the suitability of the link to enable international UK-IE quantum communications for the first time.
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Health care is a human right. Achieving universal health insurance coverage for all US residents requires significant system-wide reform. The most equitable and cost-effective health care system is a public, single-payer (SP) system. The rapid growth in national health expenditures can be addressed through a system that yields net savings over projected trends by eliminating profit and waste. With universal health insurance coverage through SP financing, providers can focus on optimizing delivery of services, rather than working within a system covered by payers who have incentives to limit costs regardless of benefit. Rather, with a SP, the people act as their own insurer through a partnership with provider organizations where tax dollars work for everyone. Consumer choice is then based on the best care to meet need with no out-of-pocket payments. SP financing is the best option to ensure equity, fairness, and public health priorities align with medical needs, providing incentives for wellness. Consumer choice will drive market forces, not provider network profits or insurer restrictions. This approach benefits public health, as everyone will have universal access to needed care, with treatment plans developed by providers based on what works best for the patient. In 2021, the American Public Health Association adopted a policy statement calling for comprehensive reforms to implement a SP system. The proposed action steps in this policy will help build a healthier nation, saving lives and reducing wasted health care expenditures while addressing inequities rooted in social, demographic, mental health, economic, and political determinants.
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American Public Health Association , Sistema de Fonte Pagadora Única , Atenção à Saúde , Reforma dos Serviços de Saúde , Humanos , Seguradoras , Cobertura Universal do Seguro de SaúdeRESUMO
This introduction describes the impetus and context for this special issue on multimorbidity in homeless populations. The guest editors begin the introduction by describing the problem of homelessness which has been exacerbated by the coronavirus disease 2019 (COVID-19) pandemic. The editors then describe the content of this special issue, which includes original research examining special populations such as homeless youth, aging populations, and Veterans as well as medical and behavioral health conditions such as tuberculosis, HIV, and opioid use disorder. Two editorials are also included in this special issue that comment on the history of homelessness and the link between homelessness and suicide. The editors acknowledge the different stakeholders that helped support this special issue and highlight the need for continued research and innovative solutions to improve the health, housing, and well-being of homeless populations.
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Pessoas Mal Alojadas , Multimorbidade , Adolescente , Adulto , Idoso , COVID-19 , Infecções por HIV , Habitação , Humanos , Transtornos Relacionados ao Uso de Opioides , SARS-CoV-2 , Suicídio , Tuberculose , VeteranosRESUMO
Protein kinase RNA-activated (PKR) is a cytoplasmic receptor for dsRNA, and as such is involved in detection of viral infection. On binding dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates its substrate, eukaryotic translation initiation factor 2 subunit α (eIF2α), causing inhibition of mRNA translation and shutdown of viral protein production. However, active PKR has also been found to be involved in the NF-κB signaling pathway by inducing phosphorylation of IκBα. PKR is regulated by the noncoding RNA nc886, which has altered expression in cancer. We have found that expression of nc886 is highly upregulated during activation of human CD4+ T cells. As has been described in other cell types, nc886 bound to PKR in human T cell lysates, preventing PKR phosphorylation by polyinosinic:polycytidylic acid or HIV trans-activation response element RNA in lysates of T cell lines or primary human CD4+ T cells. Using clonal human T cell lines, we found that nc886 expression was strictly required for IFN-γ and IL-2 expression and secretion after T cell activation but did not affect proliferation or activation-induced cell death. In stimulated human PBMCs, nc886 expression strongly correlated with IFN-γ expression. Although nc886 inhibited PKR activation by dsRNA, it was required for PKR phosphorylation during T cell stimulation, with subsequent NF-κB signaling and CREB phosphorylation. nc886 also regulated PKR phosphorylation during human monocyte-derived macrophage activation. We have therefore identified nc886 as a noncoding RNA marker of T cell activation and regulator of PKR-dependent signaling.
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Linfócitos T CD4-Positivos/imunologia , RNA Longo não Codificante/genética , eIF-2 Quinase/metabolismo , Linhagem Celular , Células Clonais , Citocinas/metabolismo , Dimerização , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/genética , Ativação de Macrófagos/genética , NF-kappa B/metabolismo , Fosforilação , RNA Viral/imunologia , Transdução de SinaisRESUMO
CD59 is a glycosylphosphatidylinositol (GPI)-anchored cell surface inhibitor of the complement membrane attack complex (MAC). We showed previously that CD59 is highly expressed in pancreatic islets but is down-regulated in rodent models of diabetes. CD59 knockdown but not enzymatic removal of cell surface CD59 led to a loss of glucose-stimulated insulin secretion (GSIS), suggesting that an intracellular pool of CD59 is required. In this current paper, we now report that non-GPI-anchored CD59 is present in the cytoplasm, colocalizes with exocytotic protein vesicle-associated membrane protein 2, and completely rescues GSIS in cells lacking endogenous CD59 expression. The involvement of cytosolic non-GPI-anchored CD59 in GSIS is supported in phosphatidylinositol glycan class A knockout GPI anchor-deficient ß-cells, in which GSIS is still CD59 dependent. Furthermore, site-directed mutagenesis demonstrated different structural requirements of CD59 for its 2 functions, MAC inhibition and GSIS. Our results suggest that CD59 is retrotranslocated from the endoplasmic reticulum to the cytosol, a process mediated by recognition of trimmed N-linked oligosaccharides, supported by the partial glycosylation of non-GPI-anchored cytosolic CD59 as well as the failure of N-linked glycosylation site mutant CD59 to reach the cytosol or rescue GSIS. This study thus proposes the previously undescribed existence of non-GPI-anchored cytosolic CD59, which is required for insulin secretion.-Golec, E., Rosberg, R., Zhang, E., Renström, E., Blom, A. M., King, B. C. A cryptic non-GPI-anchored cytosolic isoform of CD59 controls insulin exocytosis in pancreatic ß-cells by interaction with SNARE proteins.
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Antígenos CD59/metabolismo , Citosol/metabolismo , Exocitose , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas SNARE/metabolismo , Animais , Antígenos CD59/genética , Células CHO , Cricetulus , Insulina/genética , Células Secretoras de Insulina/citologia , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Ratos , Proteínas SNARE/genéticaRESUMO
BACKGROUND: Neutrophil extracellular traps (NETs) are known to play an important role in the pathophysiology of acute pancreatitis (AP). Activation of the complement cascade has been shown to occur in AP. The aim of this study was to examine whether complement component 3 is involved in the generation of NETs in AP. METHODS: AP was induced in wild-type and C3-deficient mice by retrograde infusion of taurocholate into the pancreatic duct. Blood, lung, and pancreas tissue were collected and MPO activity was determined in lung and pancreas tissue. Histological examination of the inflamed pancreas was performed. Plasma levels of CXCL2, MMP-9, IL-6, and DNA-histone complexes as well as pancreatic levels of CXCL1 and CXCL2 were determined by use of enzyme-linked immunosorbent assay. NETs were detected in the pancreas by electron microscopy. The amount of MPO and citrullinated histone 3 in neutrophils isolated from bone marrow was examined using flow cytometry. RESULTS: In C3-deficient mice, challenge with taurocholate yielded much fewer NETs in the pancreatic tissue compared with wild-type controls. Taurocholate-induced blood levels of amylase, tissue injury, and neutrophil recruitment in the pancreas were markedly reduced in the mice lacking C3. Furthermore, MPO levels in the lung, and plasma levels of IL-6, MMP-9, and CXCL2 were significantly lower in the C3-deficient mice compared to wild-type mice after the induction of AP. In vitro studies revealed that neutrophils from C3-deficient mice had normal NET-forming ability and recombinant C3a was not capable of directly inducing NETs formation in the wild-type neutrophils. CONCLUSION: C3 plays an important role in the pathophysiology of AP as it is necessary for the recruitment of neutrophils into the pancreas and ensuring NETs formation. Targeting C3 could hence be a potential strategy to ameliorate local damage as well as remote organ dysfunction in AP.
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Complemento C3/fisiologia , Armadilhas Extracelulares/metabolismo , Infiltração de Neutrófilos , Neutrófilos/fisiologia , Pancreatite/imunologia , Animais , Modelos Animais de Doenças , Pulmão/imunologia , Camundongos Endogâmicos C57BL , Pâncreas/imunologia , Pâncreas/metabolismo , Pancreatite/sangueRESUMO
CD46 is a cell surface complement inhibitor widely expressed in human tissues, in contrast to mice, where expression is limited to the testes. In humans, it has been identified as an important T cell costimulatory receptor, and patients deficient in CD46 or its endogenous ligands are unable to mount effective Th1 T cell responses. Stimulation of human CD4(+) T cells with CD3 and CD46 also leads to the differentiation of a "switched" Th1 population, which shuts down IFN-γ secretion and upregulates IL-10 and is thought to be important for negative feedback regulation of the Th1 response. In the present study, we show that CD46 costimulation leads to amplified microRNA (miR) expression changes in human CD4(+) T cells, with associated increases in activation more potent than those mediated by the "classic" costimulator CD28. Blockade of cell surface CD46 inhibited CD28-mediated costimulation, identifying autocrine CD46 signaling as downstream of CD28. We also identify a downregulation of miR-150 in CD46-costimulated T cells and identify the glucose transporter 1 encoding transcript SLC2A1 as a target of miR-150 regulation, connecting miR-150 with modulation of glucose uptake. We also investigated microRNA expression profiles of CD46-induced switched IL-10-secreting Th1 T cells and found increased expression of miR-150, compared with IFN-γ-secreting Th1 cells. Knockdown of miR-150 led to a reduction in IL-10 but not IFN-γ. CD46 therefore controls both Th1 activation and regulation via a miR-150-dependent mechanism.
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Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Transportador de Glucose Tipo 1/biossíntese , Ativação Linfocitária/genética , Proteína Cofatora de Membrana/imunologia , MicroRNAs/imunologia , Células Th1/imunologia , Separação Celular , Citocinas/metabolismo , Humanos , Immunoblotting , Ativação Linfocitária/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
Periodontal disease is one of the most common inflammatory infectious diseases worldwide and it is associated with other syndromes, such as cardiovascular disease or rheumatoid arthritis. Recent advances in sequencing allowed for identification of novel periodontopathogens such as Gram-positive Filifactor alocis, but its virulence mechanisms remain largely unknown. We confirmed that F. alocis is a prevalent species in periodontitis patients, and we also observed strong correlation of this bacterium with clinical parameters, highlighting its role in the pathogenesis of the disease. Further, we found that preincubation of human serum with F. alocis resulted in abolished bactericidal activity and that F. alocis was surviving readily in full blood. We demonstrated that one of the key contributors to F. alocis complement resistance is a unique protein, FACIN (F. alocis complement inhibitor), which binds to C3, resulting in suppression of all complement pathways. Interestingly, FACIN is a nonclassical cell surface protein, a cytosolic enzyme acetylornithine transaminase, for which we now identified a moonlighting function. FACIN binds to C3 alone, but more importantly it also captures activated complement factor 3 within the complex with factor B, thereby locking in the convertase in an inactive state. Because of the indispensable role of alternative pathway convertase in amplifying complement cascades, its inhibition by FACIN results in a very potent downregulation of activated complement factor 3 opsonization on the pathogen surface, accompanied by reduction of downstream C5 cleavage.
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Bactérias Anaeróbias/enzimologia , Bactérias Anaeróbias/imunologia , Complemento C3/antagonistas & inibidores , Complemento C3/metabolismo , Transaminases/metabolismo , Ativação do Complemento , Complemento C3/imunologia , HumanosRESUMO
OBJECTIVE: The objective of this study was to determine if a rapid albuterol delivery pathway with a breath-enhanced nebulizer can reduce emergency department (ED) length of stay (LOS), while maintaining admission rates and side effects, when compared to a traditional asthma pathway with a standard jet nebulizer. METHODS: Children aged 3-18 presenting to a large urban pediatric ED for asthma were enrolled if they were determined by pediatric asthma score to have a moderate to severe exacerbation. Subjects were randomized to either a standard treatment arm where they received up to 2 continuous albuterol nebulizations, or a rapid albuterol arm where they received up to 4 rapid albuterol treatments with a breath-enhanced nebulizer, depending on severity scoring. The primary endpoint was ED LOS from enrollment until disposition decision. Asthma scores, albuterol dose, side effects, and return visits were also recorded. RESULTS: A total of 50 subjects were enrolled (25 in each arm). The study LOS was shorter in the rapid albuterol group (118 vs. 163 minutes, p = 0.0002). When total ED LOS was analyzed, the difference was no longer statistically significant (192 vs. 203 minutes, p = 0.65). There were no statistically significant differences with respect to admission rates, asthma score changes, side effects, or return visits. CONCLUSION: A rapid albuterol treatment pathway that utilizes a breath-enhanced nebulizer is an effective alternative to traditional pathways that utilize continuous nebulizations for children with moderate to severe asthma exacerbations in the ED.
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Albuterol/administração & dosagem , Asma/tratamento farmacológico , Broncodilatadores/administração & dosagem , Serviço Hospitalar de Emergência/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Nebulizadores e Vaporizadores , Doença Aguda , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , MasculinoRESUMO
AIMS/HYPOTHESIS: Inflammasome activation and subsequent IL-1ß production is a driver of islet pathology in type 2 diabetes. Oligomers, but not mature amyloid fibrils, of human islet amyloid polypeptide (IAPP), which is co-secreted with insulin, trigger NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome activation. C4b-binding protein (C4BP), present in serum, binds to IAPP and affects transition of IAPP monomers and oligomers to amyloid fibrils. We therefore hypothesised that C4BP inhibits IAPP-mediated inflammasome activation and IL-1ß production. METHODS: Macrophages were exposed to IAPP in the presence or absence of plasma-purified human C4BP, and inflammasome activation was assessed by IL-1ß secretion as detected by ELISA and reporter cell lines. IAPP fibrillation was assessed by thioflavin T assay. Uptake of IAPP-C4BP complexes and their effects on phagolysosomal stability were assessed by flow cytometry and confocal microscopy. The effect of C4BP regulation of IAPP-mediated inflammasome activation on beta cell function was assessed using a clonal rat beta cell line. Immunohistochemistry was used to examine the association of IAPP amyloid deposits and macrophage infiltration in isolated human and mouse pancreatic islets, and expression of C4BP from isolated human pancreatic islets was assessed by quantitative PCR, immunohistochemistry and western blot. RESULTS: C4BP significantly inhibited IAPP-mediated IL-1ß secretion from primed macrophages at physiological concentrations in a dose-dependent manner. C4BP bound to and was internalised together with IAPP. C4BP did not affect IAPP uptake into phagolysosomal compartments, although it did inhibit its formation into amyloid fibrils. The loss of macrophage phagolysosomal integrity induced by IAPP incubation was inhibited by co-incubation with C4BP. Supernatant fractions from macrophages activated with IAPP inhibited both insulin secretion and viability of clonal beta cells in an IL-1ß-dependent manner but the presence of C4BP during macrophage IAPP incubation rescued beta cell function and viability. In human and mouse islets, the presence of amyloid deposits correlated with higher numbers of infiltrating macrophages. Isolated human islets expressed and secreted C4BP, which increased with addition of IL-1ß. CONCLUSIONS/INTERPRETATION: IAPP deposition is associated with inflammatory cell infiltrates in pancreatic islets. C4BP blocks IAPP-induced inflammasome activation by preventing the loss of macrophage phagolysosomal integrity required for NLRP3 activation. The consequence of this is the preservation of beta cell function and viability. C4BP is secreted directly from human pancreatic islets and this increases in response to inflammatory cytokines. We therefore propose that C4BP acts as an extracellular chaperone protein that limits the proinflammatory effects of IAPP.
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Proteína de Ligação ao Complemento C4b/metabolismo , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Idoso , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Insulina/metabolismo , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , RatosRESUMO
C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.
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Colesterol/biossíntese , Proteína de Ligação ao Complemento C4b/metabolismo , Regulação da Expressão Gênica/fisiologia , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/biossíntese , Animais , Linhagem Celular Tumoral , Colesterol Oxidase/metabolismo , Humanos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos WistarRESUMO
Surface electromyography (sEMG) is used as an adjuvant to dysphagia therapy to demonstrate the activity of submental muscles during swallowing exercises. Mechanomyography (MMG) has been suggested as a potential superior alternative to sEMG; however, this advantage is not confirmed for signal acquired from submental muscles. This study compared the signal-to-noise ratio (SNR) obtained from sEMG and MMG sensors during swallowing tasks, in healthy participants and those with a history of head and neck cancer (HNC), a population with altered anatomy and a high incidence of dysphagia. Twenty-two healthy adults and 10 adults with a history of HNC participated in this study. sEMG and MMG signals were acquired during dry, thin liquid, effortful, and Mendelsohn maneuver swallows. SNR was compared between the two sensors using repeated measures ANOVAs and subsequent planned pairwise comparisons. Test-retest measures were collected on 20 % of participants. In healthy participants, MMG SNR was higher than that of sEMG for dry [t(21) = -3.02, p = 0.007] and thin liquid swallows [t(21) = -4.24, p < 0.001]. Although a significant difference for sensor was found in HNC participants F(1,9) = 5.54, p = 0.043, planned pairwise comparisons by task revealed no statistically significant difference between the two sensors. sEMG also showed much better test-retest reliability than MMG. Biofeedback provided as an adjuvant to dysphagia therapy in patients with HNC should employ sEMG technology, as this sensor type yielded better SNR and overall test-retest reliability. Poor MMG test-retest reliability was noted in both healthy and HNC participants and may have been related to differences in sensor application.