RESUMO
Plasma gelsolin (pGSN) is the secreted isoform of an intracellular actin remodeling protein found in high concentrations in human plasma. Clinical studies demonstrate reduced pGSN concentrations in several disease states, including severe trauma, burns, and sepsis. Markedly decreased pGSN concentrations in these conditions precede and predict adverse clinical outcomes. In this study, we measured pGSN in patients with chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and dysregulated inflammation. pGSN was quantified using a sandwich ELISA in plasma from healthy volunteers, clinically stable CGD patients, and X-linked CGD carriers and in sera from 12 CGD patients undergoing bone marrow transplantation. pGSN was also quantified in healthy volunteers challenged with intravenous endotoxin. pGSN concentrations were lower in CGD patients without active infection or systemic inflammation compared with healthy control subjects. In CGD patients undergoing bone marrow transplantation, pGSN concentrations increased significantly following successful transplant. X-linked carriers of CGD had normal pGSN. Despite reduction of pGSN in CGD patients, we did not detect significant changes in pGSN over 24 h following challenge of healthy volunteers with intravenous endotoxin (4 ng/kg) that elicited a febrile response. We describe, for the first time, significantly lower pGSN in clinically stable patients with CGD compared with age- and sex-matched healthy volunteers. Low pGSN levels in CGD patients significantly increased following bone marrow transplantation. X-linked carriers of CGD had normal pGSN. In healthy volunteers challenged with intravenous endotoxin, pGSN is not an acute phase reactant.
Assuntos
Gelsolina/sangue , Doença Granulomatosa Crônica/sangue , Doença Granulomatosa Crônica/diagnóstico , Adolescente , Adulto , Biomarcadores/sangue , Transplante de Medula Óssea/métodos , Estudos de Coortes , Endotoxinas/toxicidade , Feminino , Febre/sangue , Febre/induzido quimicamente , Febre/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Plasma gelsolin (pGSN) is an abundant circulating protein quickly consumed by extensive tissue damage. Marked depletion is associated with later poor outcomes in diverse clinical circumstances. Repletion with recombinant human (rhu)-pGSN in animal models of inflammation lessens mortality and morbidity. METHODS: Neutropenic mice were treated with different meropenem doses ±12 mg of rhu-pGSN commencing 1 day before an intratracheal challenge with multidrug-resistant Pseudomonas aeruginosa. Survival, bacterial counts, and pulmonary pathology were compared between corresponding meropenem groups with and without rhu-pGSN. RESULTS: Overall survival was 35/64 (55%) and 46/64 (72%) in mice given meropenem without and with rhu-pGSN, respectively (Δâ =â 17%; 95% CI, 1-34). In control mice receiving meropenem 1250 mg/kg/d where the majority died, the addition of rhu-pGSN increased survival from 5/16 (31%) to 12/16 (75%) (Δâ =â 44%; 95% CI, 13-75). Survival with minor lung injury was found in 26/64 (41%) mice receiving only meropenem, vs 38/64 (59%) in mice given meropenem plus rhu-pGSN (Δâ =â 19%; 95% CI, 2-36). CONCLUSIONS: In a series of dose-ranging experiments, both mortality and lung injury were reduced by the addition of rhu-pGSN to meropenem against carbapenem-resistant P. aeruginosa. Rhu-pGSN offers a novel candidate therapy for antibiotic-resistant pneumonia.
RESUMO
BACKGROUND: Plasma gelsolin (pGSN) is an abundant circulating protein that neutralizes actin exposed by damaged cells, modulates inflammatory responses, and enhances alveolar macrophage antimicrobial activity. We investigated whether adults with low pGSN at hospital admission for community-acquired pneumonia (CAP) were at high risk for severe outcomes. METHODS: Admission pGSN concentrations in 455 adults hospitalized with CAP were measured using enzyme-linked immunosorbent assay. Patients were grouped into the following 4 hierarchical, mutually exclusive categories based on maximum clinical severity experienced during their hospitalization: general floor care without intensive care unit (ICU) admission, invasive respiratory or vasopressor support (IRVS), or death; ICU care without IRVS or death; IRVS without death; or death. Admission pGSN concentrations were compared across these discrete outcome categories. Additionally, outcomes among patients in the lowest quartile of pGSN concentration were compared to those in the upper 3 quartiles. RESULTS: Overall, median (interquartile range) pGSN concentration was 38.1 (32.1, 45.7) µg/mL. Patients with more severe outcomes had lower pGSN concentrations (P = .0001); median values were 40.3 µg/mL for floor patients, 36.7 µg/mL for ICU patients, 36.5 µg/mL for patients receiving IRVS, and 25.7 µg/mL for patients who died. Compared to patients with higher pGSN concentrations, patients in the lowest quartile (pGSN ≤ 32.1 µg/mL) more often required IRVS (21.2% vs 11.7%, P = .0114) and died (8.8% vs 0.9%, P < .0001). CONCLUSIONS: Among adults hospitalized with CAP, lower pGSN concentrations were associated with more severe clinical outcomes. Future studies are planned to investigate possible therapeutic benefits of recombinant human pGSN in this population.
Assuntos
Infecções Comunitárias Adquiridas/sangue , Infecções Comunitárias Adquiridas/epidemiologia , Gelsolina/sangue , Admissão do Paciente , Pneumonia/sangue , Pneumonia/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Infecções Comunitárias Adquiridas/tratamento farmacológico , Infecções Comunitárias Adquiridas/etiologia , Resistência Microbiana a Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados da Assistência ao Paciente , Pneumonia/tratamento farmacológico , Pneumonia/etiologia , Prognóstico , Adulto JovemRESUMO
Thomas P. Stossel is an American Cancer Society Professor of Medicine Emeritus at Harvard Medical School, Honorary Physician in the Hematology Division of Brigham & Women's Hospital, founding scientist of BioAegis Therapeutics, Visiting Scholar of The American Enterprise Institute and cofounder of Options for Children in Zambia.
Assuntos
Indústria Farmacêutica/normas , Guias como Assunto , Invenções/normas , Centros Médicos Acadêmicos , Pesquisa Biomédica , Difusão de Inovações , Humanos , Narração , Transtornos Fóbicos , Estados UnidosRESUMO
BACKGROUND: Preeclampsia, a pregnancy-specific inflammatory disorder, is characterized by high levels of anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), in the maternal circulation. sFlt1 producing molecular machinery is present in syncytiotrophoblast extracellular vesicles that are released by the placenta into maternal plasma during normal pregnancy, a process greatly accelerated in preeclampsia. We hypothesized that syncytiotrophoblast extracellular vesicles exposes cytoplasmic actin to plasma resulting in depletion of plasma gelsolin (pGSN), an abundant plasma protein that scavenges circulating actin and other pro-inflammatory mediators. OBJECTIVE: To test whether pGSN levels would be lower in preeclampsia and to assess whether recombinant human plasma gelsolin (rhpGSN) may promote placental health by decreasing shedding of syncytiotrophoblast extracellular vesicles. METHODS: We tested pGSN levels in third trimester plasma samples from women with preeclampsia and non-hypertensive pregnancies. We then assessed whether rhpGSN may act as a negative regulator of syncytial shedding in placental explant culture and dynamic mechanical stretch studies. RESULTS: pGSN levels fall in late pregnancy and decline further in preeclampsia patients. Recombinant human pGSN (rhpGSN) at 100µg/ml limits spontaneous syncytiotrophoblast vesicle release and sFlt1 protein dissemination by normal placental explants. Higher rhpGSN doses (500µg/ml) also limit syncytiotrophoblast vesicle and sFlt1 dissemination from preeclamptic placental explants. rhpGSN also mitigates syncytiotrophoblast vesicle during dynamic mechanical stretch. CONCLUSIONS: 1) pGSN, an anti-inflammatory factor of maternal origin is reduced in preeclampsia and may contribute to disease progression and 2) exogenous rhpGSN supplementation can limit the dissemination of toxic syncytiotrophoblast vesicle that characterizes the disease state.
Assuntos
Vesículas Extracelulares , Gelsolina/sangue , Gelsolina/farmacologia , Pré-Eclâmpsia/sangue , Proteínas Recombinantes/farmacologia , Trofoblastos/efeitos dos fármacos , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Gravidez , Terceiro Trimestre da Gravidez , Técnicas de Cultura de Tecidos , Trofoblastos/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
PURPOSE: This study surveyed pediatric dentists' knowledge of the Physician Payments Sunshine Act (PPSA) and their impressions of its effect on their future interactions with the dental products industry and on their clinical practice. METHODS: Seven hundred seventy four practicing dentists responded to a survey, with 13 responding to a follow-up survey. RESULTS: Most respondents were unfamiliar (43 percent) or only vaguely familiar (33 percent) with the PPSA. In response to the required PPSA disclosures, 62 percent said they would see company representatives less often (37 percent indicated no change); 50 percent said they would attend fewer industry-sponsored continuing education events (49 percent indicated no change); and 57 percent indicated they would attend fewer promotional speaking events (43 percent indicated no change). Respondents indicated that self-banning detailing (68 percent), samples (72 percent), industry-sponsored continuing education events (81 percent), and promotional speaking (82 percent) would negatively impact the quality of care they are able to provide their patients. CONCLUSION: Pediatric dentists anticipate reducing or severing their existing ties to pharmaceutical, biotechnology, and device manufacturers in response to the Physician Payments Sunshine Act, despite believing that interactions with the dental products' industry enhance the quality of patient care they are able to provide their patients.
Assuntos
Gastos em Saúde , Atitude do Pessoal de Saúde , Criança , Humanos , Assistência ao Paciente , Inquéritos e QuestionáriosRESUMO
Filamin A (FLNA) is an actin filament crosslinking protein with multiple intracellular binding partners. Mechanical force exposes cryptic FLNA binding sites for some of these ligands. To identify new force-dependent binding interactions, we used a fusion construct composed of two FLNA domains, one of which was previously identified as containing a force-dependent binding site as a bait in a yeast two-hybrid system and identified the Rho dissociation inhibitor 2 (RhoGDI2) as a potential interacting partner. A RhoGDI2 truncate with 81 N-terminal amino acid residues and a phosphomimetic mutant, RhoGDI(Tyr153Glu) interacted with the FLNA construct. However, neither wild-type or full-length RhoGDI2 phosphorylated at Y153 interacted with FLNA. Our interpretation of these contradictions is that truncation and/or mutation of RhoGDI2 perturbs its conformation to expose a site that adventitiously binds FLNA and is not a bona-fide interaction. Therefore, previous studies reporting that a RhoGDI(Y153E) mutant suppresses the metastasis of human bladder cancer cells must be reinvestigated in light of artificial interaction of this point mutant with FLNA.
Assuntos
Filaminas/química , Filaminas/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/química , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Fosforilação , Ligação ProteicaRESUMO
Gelsolin is the fourth most abundant protein in the body and its depletion in the blood has been found in multiple sclerosis (MS) patients. How gelsolin affects the MS brain has not been studied. We found that while the secreted form of gelsolin (pGSN) decreased in the blood of experimental autoimmune encephalomyelitis (EAE) mice, pGSN concentration increased in the EAE brain. Recombinant human pGSN (rhp-GSN) decreased extracellular actin and myeloperoxidase activity in the brain, resulting in reduced disease activity and less severe clinical disease, suggesting that gelsolin could be a potential therapeutic target for MS.
Assuntos
Actinas/toxicidade , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Gelsolina/metabolismo , Gelsolina/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Actinas/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Adjuvante de Freund/toxicidade , Glioma/patologia , Humanos , Camundongos , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Mycobacterium tuberculosis/imunologia , Proteína Proteolipídica de Mielina/toxicidade , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fragmentos de Peptídeos/toxicidade , Peroxidase/metabolismo , Fatores de TempoRESUMO
Plasma gelsolin (pGSN) functions as part of the "extracellular actin-scavenging system," but its potential to improve host defense against infection has not been studied. In a mouse model of primary pneumococcal pneumonia, recombinant human pGSN (rhu-pGSN) caused enhanced bacterial clearance, reduced acute inflammation, and improved survival. In vitro, rhu-pGSN rapidly improved lung macrophage uptake and killing of bacteria (Streptococcus pneumoniae, Escherichia coli, and Francisella tularensis). pGSN triggers activating phosphorylation (Ser(1177)) of macrophage nitric oxide synthase type III (NOS3), an enzyme with important bactericidal functions in lung macrophages. rhu-pGSN failed to enhance bacterial killing by NOS3(-/-) macrophages in vitro or bacterial clearance in NOS3(-/-) mice in vivo. Prophylaxis with immunomodulators may be especially relevant for patients at risk for secondary bacterial pneumonia, e.g., after influenza. Treatment of mice with pGSN challenged with pneumococci on postinfluenza day 7 (the peak of enhanced susceptibility to secondary infection) caused a â¼15-fold improvement in bacterial clearance, reduced acute neutrophilic inflammation, and markedly improved survival, even without antibiotic therapy. pGSN is a potential immunomodulator for improving lung host defense against primary and secondary bacterial pneumonia.
Assuntos
Gelsolina/farmacologia , Pulmão/microbiologia , Macrófagos Alveolares/imunologia , Óxido Nítrico Sintase Tipo III/imunologia , Pneumonia Pneumocócica/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Escherichia coli/imunologia , Francisella tularensis/imunologia , Gelsolina/sangue , Inflamação/tratamento farmacológico , Inflamação/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Macrófagos Alveolares/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Infecções por Orthomyxoviridae/imunologia , Fagocitose/imunologia , Pneumonia Pneumocócica/mortalidade , Pneumonia Pneumocócica/prevenção & controle , Proteínas Recombinantes/farmacologia , Streptococcus pneumoniae/imunologiaRESUMO
Despite the growth of research in universities on point-of-care (POC) diagnostics for global health, most devices never leave the laboratory. The processes that move diagnostic technology from the laboratory to the field--the processes intended to evaluate operation and performance under realistic conditions--are more complicated than they might seem. Two case studies illustrate this process: the development of a paper-based device to measure liver function, and the development of a device to identify sickle cell disease based on aqueous multiphase systems (AMPS) and differences in the densities of normal and sickled cells. Details of developing these devices provide strategies for forming partnerships, prototyping devices, designing studies, and evaluating POC diagnostics. Technical and procedural lessons drawn from these experiences may be useful to those designing diagnostic tests for developing countries, and more generally, technologies for use in resource-limited environments.
Assuntos
Anemia Falciforme/diagnóstico , Anemia Falciforme/economia , Testes de Função Hepática/economia , Sistemas Automatizados de Assistência Junto ao Leito/economia , HumanosRESUMO
Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare congenital immunodeficiency often caused by mutations in the last 10 to 19 C-terminal amino acids of CXCR4. These mutations impair CXCR4 internalization and increase responsiveness to CXCL12. The CXCR4 C-terminal domain (C-tail) also has a binding site for the actin-binding protein filamin A (FLNA); it is not known whether FLNA binds to WHIM CXCR4 mutants or whether this interaction is implicated in the hyperfunction of these receptors. Here we show that, in addition to interacting with the CXCR4 C-tail, FLNA interacted with a region in the receptor third intracellular loop (ICL3) spanning amino acids 238 to 246. This interaction involved specific FLNA repeats and was sensitive to Rho kinase inhibition. Deletion of the 238-246 motif accelerated CXCL12-induced wild-type (WT) receptor endocytosis but enabled CXCL12-mediated endocytosis and normalized signaling by the WHIM-associated receptor CXCR4(R334X). CXCL12 stimulation triggered CXCR4(R334X) internalization in FLNA-deficient M2 cells but not in the FLNA-expressing M2 subclone A7; this suggests a role for FLNA in stabilization of WHIM-like CXCR4 at the cell surface. FLNA increased ß-arrestin2 binding to CXCR4(R334X) in vivo, which provides a molecular basis for FLNA-mediated hyperactivation of WHIM receptor signaling. We propose that FLNA interaction with ICL3 is central for endocytosis and signaling of WT and WHIM-like CXCR4 receptors.
Assuntos
Endocitose/genética , Filaminas/metabolismo , Síndromes de Imunodeficiência/genética , Receptores CXCR4/metabolismo , Verrugas/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Linhagem Celular Tumoral , Filaminas/química , Células HEK293 , Humanos , Síndromes de Imunodeficiência/metabolismo , Dados de Sequência Molecular , Doenças da Imunodeficiência Primária , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Receptores CXCR4/química , Receptores CXCR4/genética , Transdução de Sinais/genética , Verrugas/metabolismoRESUMO
Although simple and low-cost interventions for sickle cell disease (SCD) exist in many developing countries, child mortality associated with SCD remains high, in part, because of the lack of access to diagnostic tests for SCD. A density-based test using aqueous multiphase systems (SCD-AMPS) is a candidate for a low-cost, point-of-care diagnostic for SCD. In this paper, the field evaluation of SCD-AMPS in a large (nâ=â505) case-control study in Zambia is described. Of the two variations of the SCD-AMPS used, the best system (SCD-AMPS-2) demonstrated a sensitivity of 86% (82-90%) and a specificity of 60% (53-67%). Subsequent analysis identified potential sources of false positives that include clotting, variation between batches of SCD-AMPS, and shipping conditions. Importantly, SCD-AMPS-2 was 84% (62-94%) sensitive in detecting SCD in children between 6 months and 1 year old. In addition to an evaluation of performance, an assessment of end-user operability was done with health workers in rural clinics in Zambia. These health workers rated the SCD-AMPS tests to be as simple to use as lateral flow tests for malaria and HIV.
Assuntos
Anemia Falciforme/diagnóstico , Testes Diagnósticos de Rotina/métodos , Eritrócitos/patologia , Adolescente , Contagem de Células , Criança , Pré-Escolar , Coleta de Dados , Testes Diagnósticos de Rotina/economia , Feminino , Pessoal de Saúde , Humanos , Lactente , Masculino , Serviços de Saúde Rural , Manejo de Espécimes , Fatores de Tempo , Volatilização , ZâmbiaRESUMO
Endogenously and externally generated mechanical forces influence diverse cellular activities, a phenomenon defined as mechanotransduction. Deformation of protein domains by application of stress, previously documented to alter macromolecular interactions in vitro, could mediate these effects. We engineered a photon-emitting system responsive to unfolding of two repeat domains of the actin filament (F-actin) crosslinker protein filamin A (FLNA) that binds multiple partners involved in cell signalling reactions and validated the system using F-actin networks subjected to myosin-based contraction. Expressed in cultured cells, the sensor-containing FLNA construct reproducibly reported FLNA domain unfolding strikingly localized to dynamic, actively protruding, leading cell edges. The unfolding signal depends upon coherence of F-actin-FLNA networks and is enhanced by stimulating cell contractility. The results establish protein domain distortion as a bona fide mechanism for mechanotransduction in vivo.
Assuntos
Movimento Celular/fisiologia , Filaminas/química , Filaminas/fisiologia , Rim/fisiologia , Mecanotransdução Celular/fisiologia , Actinas/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas In Vitro , Rim/citologia , Óptica e Fotônica/métodosRESUMO
The actin crosslinking protein filamin A (FLNa) mediates mechanotransduction, a conversion of mechanical forces into cellular biochemical signals to regulate cell growth and survival. To provide more quantitative insight into this process, we report results using magnetic tweezers that relate mechanical force to conformational changes of FLNa immunoglobulin-like repeats (IgFLNa) 20-21, previously identified as a mechanosensing domain. We determined the force magnitudes required to unfold previously identified structural organizations of the ß-strands in the two domains: IgFLNa 20 unfolds at ~15â pN and IgFLNa 21 unfolding requires significantly larger forces. Unfolded domain IgFLNa 20 can exist in two different conformational states, which lead to different refolding kinetics of the IgFLNa 20 and imply a significant impact on the reformation of the domain pair at reduced force values. We discuss the relevance of the findings to force bearing and mechanosensing functions of FLNa.
Assuntos
Filaminas/química , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Actinas/química , Actinas/metabolismo , Filaminas/metabolismo , Cinética , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Redobramento de Proteína , Desdobramento de ProteínaRESUMO
Collaborations between physicians, particularly those in academic medicine, and industries that develop pharmaceutical products, medical devices, and diagnostic tests have led to substantial advances in patient care. At the same time, there is a strong awareness that these relationships, however beneficial they may be, should conform to established principles of ethical professional practice. Through a writing committee drawn from diverse disciplines across several institutions, the Association of Clinical Researchers and Educators (ACRE) has written a code of conduct to provide guidance to physicians in observing these principles. Our recommendations are not intended to be prescriptive or inflexible, but rather to be of assistance to physicians in making their own personal decisions on whether, or how, to be involved in research, education, or other collaborations with industry.
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Ética Profissional , Setor de Assistência à Saúde/ética , Relações Interprofissionais/ética , Médicos/ética , Códigos de Ética , Educação Médica Continuada/ética , Humanos , Editoração/ética , Pesquisadores/éticaAssuntos
Ensaios Clínicos como Assunto/métodos , Indústria Farmacêutica/organização & administração , Publicações Periódicas como Assunto/normas , Publicações/normas , Apoio à Pesquisa como Assunto/organização & administração , Estatística como Assunto/educação , Estatística como Assunto/organização & administração , HumanosRESUMO
Overwhelming evidence that relationships between universities, physicians and the medical products industry benefit patients explains the ubiquitous calls to encourage such relationships. Yet accumulating 'conflict of interest' regulations in academic health centers, government and industry have had the opposite effect. Justifications underlying the regulations lack quantitative rigor, and the rules they enforce impose costly bureaucratic requirements of dubious benefit. Evidence shows that they have diminished the collaborations deemed beneficial to health enhancement.