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1.
Biochem Pharmacol ; 168: 204-213, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295463

RESUMO

Impulsivity is a multifaceted behavioral manifestation with implications in several neuropsychiatric disorders. Glutamate neurotransmission through the N-methyl-D-aspartate receptors (NMDARs) in the medial prefrontal cortex (mPFC), an important brain region in decision-making and goal-directed behaviors, plays a key role in motor impulsivity. We discovered that inherent motor impulsivity predicted responsiveness to D-cycloserine (DCS), a partial NMDAR agonist, which prompted the hypothesis that inherent motor impulsivity is associated with the pattern of expression of cortical NMDAR subunits (GluN1, GluN2A, GluN2B), specifically the protein levels and synaptosomal trafficking of the NMDAR subunits. Outbred male Sprague-Dawley rats were identified as high (HI) or low (LI) impulsive using the one-choice serial reaction time task. Following phenotypic identification, mPFC synaptosomal protein was extracted from HI and LI rats to assess the expression pattern of the NMDAR subunits. Synaptosomal trafficking and stabilization for the GluN2 subunits were investigated by co-immunoprecipitation for postsynaptic density 95 (PSD95) and synapse associated protein 102 (SAP102). HI rats had lower mPFC GluN1 and GluN2A, but higher GluN2B and pGluN2B synaptosomal protein expression versus LI rats. Further, higher GluN2B:PSD95 and GluN2B:SAP102 protein:protein interactions were detected in HI versus LI rats. Thus, the mPFC NMDAR subunit expression pattern and/or synaptosomal trafficking associates with high inherent motor impulsivity. Increased understanding of the complex regulation of NMDAR balance within the mPFC as it relates to inherent motor impulsivity may lead to a better understanding of risk factors for impulse-control disorders.


Assuntos
Comportamento Impulsivo/fisiologia , Córtex Pré-Frontal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Ciclosserina/farmacologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Masculino , Neuropeptídeos/metabolismo , Fenótipo , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistas
2.
Mediators Inflamm ; 2019: 3481430, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182931

RESUMO

Macrophages (Mφ) play a central role in coordinating host response to pathogens, cellular injury, and environmental stimuli. Herein, we report multidimensional, nuclear proteomic analyses of protein expression and posttranslational modifications (PTMs) that control biological processes during Mφ activation. For this, Mφ were incubated with IFN-γ/LPS and IL-4, and their differentiation to proinflammatory (M1) and anti-inflammatory (M2a, referred as M2 for simplicity throughtout the manuscript) phenotypes was confirmed by detection of CD64 and CD206 surface markers and TNF-α, arginase I, and iNOS-dependent nitrite levels. We used a sequential method of organellar enrichment and labeling of nuclear fractions with BODIPY FL-maleimide fluorescence dye followed by two-dimensional electrophoresis (2DE) to capture quantitative changes in abundance and S-nitrosylated (SNO) proteome signatures. Exact same gels were then labeled with Pro-Q Diamond to detect protein phosphorylation. MALDI-TOF/TOF MS analysis of the protein spots with fold change of ≥|1.5| in any of the groups yielded 229 identifications. We found that 145, 78, and 173 protein spots in M1 Mφ and 105, 81, and 164 protein spots in M2 Mφ were changed in abundance, S-nitrosylation, and phosphorylation, respectively, with respect to M0 controls (fold change: ≥|1.5|, p ≤ 0.05). Targeted analysis by immunoprecipitation and Western blotting was performed to verify the differential abundance and phosphorylation levels of two of the proteins in M1 and M2 (vs. M0) Mφ. Ingenuity Pathway Analysis of the nuclear proteome datasets showed that the abundance and posttranslational (SNO and Phosphor) modifications of the proteins predicted to be involved in cytoskeletal organization/cell movement, phagocytosis/endocytosis, and cell proliferation/cell death were differentially regulated with proinflammatory and anti-inflammatory activation of Mφ.


Assuntos
Macrófagos/metabolismo , Animais , Western Blotting , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Imunoprecipitação , Espectrometria de Massas , Camundongos , Óxido Nítrico/metabolismo , Fosforilação , Análise de Componente Principal , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
J Immunol ; 201(9): 2753-2766, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275049

RESUMO

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein that translocates to the cytoplasm and is subsequently released to the extracellular space during infection and injury. Once released, it acts as a damage-associated molecular pattern and regulates immune and inflammatory responses. Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants and elderly, for which no effective treatment or vaccine is currently available. This study investigated the effects of HMGB1 on cytokine secretion, as well as the involvement of NF-κB and TLR4 pathways in RSV-induced HMGB1 release in human airway epithelial cells (AECs) and its proinflammatory effects on several human primary immune cells. Purified HMGB1 was incubated with AECs (A549 and small alveolar epithelial cells) and various immune cells and measured the release of proinflammatory mediators and the activation of NF-κB and P38 MAPK. HMGB1 treatment significantly increased the phosphorylation of NF-κB and P38 MAPK but did not induce the release of cytokines/chemokines from AECs. However, addition of HMGB1 to immune cells did significantly induce the release of cytokines/chemokines and activated the NF-κB and P38 MAPK pathways. We found that activation of NF-κB accounted for RSV-induced HMGB1 secretion in AECs in a TLR4-dependent manner. These results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis. Therefore, blocking the proinflammatory function of HMGB1 may be an effective approach for developing novel therapeutics.


Assuntos
Proteína HMGB1/imunologia , Leucócitos Mononucleares/imunologia , Mucosa Respiratória/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Humanos , Imunidade Inata/imunologia , Vírus Sincicial Respiratório Humano/imunologia
4.
Surgery ; 164(4): 887-894, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30093278

RESUMO

Historically, thyroidectomies have been performed as inpatient operations due to concerns of postoperative bleeding and symptomatic hypocalcemia. We aim to demonstrate that outpatient thyroidectomy can be performed safely. METHODS: This report outlines a 7-year retrospective analysis (2009-2016) of outpatient vs inpatient thyroidectomies, with outcomes including hematoma, blood loss, recurrent laryngeal nerve injury, symptomatic hypocalcemia, and postoperative emergency room (ER) visits. RESULTS: A total of 1460 thyroidectomies were performed: 1272 (87%) outpatient and 188 (13%) inpatient. Five outpatients: 4 total thyroidectomies (TT), 1 TT with a central lymph node dissection (CLND), and 1 partial thyroidectomy (PT) developed postoperative hematomas (0.34%) at post-discharge hour 3, 9, 10, 13, and 42. Average time to discharge was 2 hours and 37 minutes. Hematomas were evacuated successfully in the operating room under local anesthesia with a 2-day average hospital stay. There were no differences between TT, thyroid lobectomy (TL), and PT procedures for postoperative hematoma (p=0.17). Outpatient compared to inpatient thyroidectomy was more likely to have been performed in patients with lower American Society of Anesthesia scores (2.3 vs 2.9, p<0.0001), less mean blood loss (74 vs 227 ml, p<0.0001), lesser age (52 vs 56 years, p=0.0012), less extensive dissection (p<0.0001), and fewer RLN injuries (2.4% vs 8.5%, p<0.0001). There was no difference between outpatient and inpatient symptomatic hypocalcemia (6.3% vs 9.6%, p=0.09), 30-day postoperative ER visits (8.8% vs 9.6%, p=0.73), and postoperative hematoma (0.39% vs 0%, p=0.39). There was one inpatient mortality from stroke. CONCLUSION: Postoperative hematomas can be managed safely without life-threatening complications suggesting outpatient thyroidectomy can be performed safely by an experienced surgeon, and adverse sequelae dealt with in a safe and effective manner.


Assuntos
Procedimentos Cirúrgicos Ambulatórios/estatística & dados numéricos , Doenças da Glândula Tireoide/cirurgia , Tireoidectomia/estatística & dados numéricos , Humanos , Tempo de Internação/estatística & dados numéricos , Alta do Paciente/estatística & dados numéricos , Estudos Retrospectivos , Tireoidectomia/efeitos adversos , Tireoidectomia/métodos
5.
Clin J Oncol Nurs ; 21(5): 633-636, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28945726

RESUMO

Clinical research nurses are essential in the coordination of clinical trials and the management of research participants. Without a stable, knowledgeable research nurse workforce, the conduct of research is affected. A research nurse residency is a novel approach to preparing new graduate nurses for the oncology research nurse role. This article will describe the development and content of the research nurse residency and how this approach is being used to address a need for clinical research nurses to support burgeoning clinical trials at a National Cancer Institute-designated comprehensive cancer center.
.


Assuntos
Internato não Médico , Enfermeiras Clínicas , Pesquisa em Enfermagem , Desenvolvimento de Programas , Currículo , Papel do Profissional de Enfermagem , Enfermagem Oncológica , Estados Unidos
6.
J Proteome Res ; 16(8): 2663-2679, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28679203

RESUMO

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.


Assuntos
Citocinas/farmacologia , Eosinófilos/efeitos dos fármacos , Fosfoproteínas/análise , Proteínas/análise , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Asma/sangue , Células Cultivadas , Citocinas/metabolismo , Eletroforese em Gel Bidimensional , Eosinófilos/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Interleucina-3/farmacologia , Interleucina-5/farmacologia , Lectinas Tipo C/análise , Masculino , Proteômica/métodos , Espectrometria de Massas em Tandem , Fatores de Tempo
7.
J Immunol ; 197(10): 3782-3791, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27742828

RESUMO

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.


Assuntos
Citocinas/imunologia , Eosinófilos/imunologia , Erros Inatos do Metabolismo/imunologia , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/metabolismo , Apoptose/efeitos dos fármacos , Asma/complicações , Asma/imunologia , Dexametasona/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Hipersensibilidade/metabolismo , Interleucina-2/farmacologia , Interleucina-3/farmacologia , Interleucina-4/farmacologia , Interleucina-5/farmacologia , Lipoxinas/farmacologia , Erros Inatos do Metabolismo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteômica , RNA Interferente Pequeno/farmacologia , Receptores de Glucocorticoides/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
8.
Int J Proteomics ; 2016: 1384523, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27635260

RESUMO

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

9.
PLoS Negl Trop Dis ; 10(2): e0004490, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26919708

RESUMO

Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Leucócitos Mononucleares/química , Proteínas/química , Proteoma/química , Cardiomiopatia Chagásica/parasitologia , Doença Crônica , Eletroforese em Gel Bidimensional , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , Trypanosoma cruzi/fisiologia
10.
Apoptosis ; 21(4): 421-31, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26880402

RESUMO

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.


Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Dexametasona/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Interleucina-5/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Asma/tratamento farmacológico , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fosfatase 1 de Especificidade Dupla/biossíntese , Eosinófilos , Humanos , Interleucina-5/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
11.
Am Surg ; 82(1): 85-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26802863

RESUMO

To determine whether a restrictive strategy of red cell transfusion was safe in elderly trauma patients, we compared those treated with a restrictive transfusion strategy versus those who were liberally transfused. We performed a retrospective study of elderly (age ≥ 70 years) trauma patients admitted to our Level I trauma center from 2005 to 2013. Patients with a hemoglobin (Hg) < 10 g/dL after 48 hours were included. We excluded patients with an Injury Severity Score > 25 or active cardiac ischemia. Patients who were transfused for an Hg < 10 g/dL (liberal group) were compared to those who were transfused for an Hg< 7 g/dL (restrictive group). There were 382 patients included, 229 and 153 in the liberal and restrictive transfusion groups, respectively. All patients in the liberal group and 20 per cent of patients in the restrictive group received a transfusion (P < 0.0001). Patients in the liberal group had more overall complications (52 vs 32%, P = 0.0001). On multivariate analysis, receiving a transfusion was an independent risk factor to develop a complication [odds ratio = 2.3 (1.5-3.6), P < 0.0001]. For survivors, patients in the liberal group spent more days in the hospital (nine versus seven days, P = 0.007) and intensive care unit (two versus one day, P = 0.01). There was no difference in mortality (3 vs 4%, P = 0.82). In conclusion, restrictive transfusion appears to be safe in elderly trauma patients and may be associated with decreased complications and shortened length of stay.


Assuntos
Transfusão de Sangue/métodos , Transfusão de Eritrócitos/métodos , Mortalidade Hospitalar , Ferimentos e Lesões/mortalidade , Ferimentos e Lesões/terapia , Centros Médicos Acadêmicos , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Estudos de Coortes , Cuidados Críticos/métodos , Transfusão de Eritrócitos/efeitos adversos , Feminino , Avaliação Geriátrica , Hemoglobinas/metabolismo , Humanos , Escala de Gravidade do Ferimento , Tempo de Internação , Masculino , Prognóstico , Estudos Retrospectivos , Medição de Risco , Taxa de Sobrevida , Reação Transfusional , Centros de Traumatologia , Resultado do Tratamento , Ferimentos e Lesões/diagnóstico
12.
PLoS One ; 10(11): e0143165, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26581097

RESUMO

Invasive pulmonary aspergillosis (IPA) is an opportunistic fungal infection in patients undergoing chemotherapy for hematological malignancy, hematopoietic stem cell transplant, or other forms of immunosuppression. In this group, Aspergillus infections account for the majority of deaths due to mold pathogens. Although early detection is associated with improved outcomes, current diagnostic regimens lack sensitivity and specificity. Patients undergoing chemotherapy, stem cell transplantation and lung transplantation were enrolled in a multi-site prospective observational trial. Proven and probable IPA cases and matched controls were subjected to discovery proteomics analyses using a biofluid analysis platform, fractionating plasma into reproducible protein and peptide pools. From 556 spots identified by 2D gel electrophoresis, 66 differentially expressed post-translationally modified plasma proteins were identified in the leukemic subgroup only. This protein group was rich in complement components, acute-phase reactants and coagulation factors. Low molecular weight peptides corresponding to abundant plasma proteins were identified. A candidate marker panel of host response (9 plasma proteins, 4 peptides), fungal polysaccharides (galactomannan), and cell wall components (ß-D glucan) were selected by statistical filtering for patients with leukemia as a primary underlying diagnosis. Quantitative measurements were developed to qualify the differential expression of the candidate host response proteins using selective reaction monitoring mass spectrometry assays, and then applied to a separate cohort of 57 patients with leukemia. In this verification cohort, a machine learning ensemble-based algorithm, generalized pathseeker (GPS) produced a greater case classification accuracy than galactomannan (GM) or host proteins alone. In conclusion, Integration of host response proteins with GM improves the diagnostic detection of probable IPA in patients undergoing treatment for hematologic malignancy. Upon further validation, early detection of probable IPA in leukemia treatment will provide opportunities for earlier interventions and interventional clinical trials.


Assuntos
Antígenos de Fungos/metabolismo , Proteínas Sanguíneas/metabolismo , Aspergilose Pulmonar Invasiva/diagnóstico , Aspergilose Pulmonar Invasiva/etiologia , Leucemia/complicações , Leucemia/tratamento farmacológico , Algoritmos , Sequência de Aminoácidos , Biomarcadores/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Aprendizado de Máquina , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Modelos Biológicos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Curva ROC , Reprodutibilidade dos Testes
13.
Science ; 349(6253): 1176-7, 2015 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-26359396
14.
J Clin Virol ; 64: 97-106, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25728087

RESUMO

OBJECTIVES: Dengue virus (DENV) infection is a significant risk to over a third of the human population that causes a wide spectrum of illness, ranging from sub-clinical disease to intermediate syndrome of vascular complications called dengue fever complicated (DFC) and severe, dengue hemorrhagic fever (DHF). Methods for discriminating outcomes will impact clinical trials and understanding disease pathophysiology. STUDY DESIGN: We integrated a proteomics discovery pipeline with a heuristics approach to develop a molecular classifier to identify an intermediate phenotype of DENV-3 infectious outcome. RESULTS: 121 differentially expressed proteins were identified in plasma from DHF vs dengue fever (DF), and informative candidates were selected using nonparametric statistics. These were combined with markers that measure complement activation, acute phase response, cellular leak, granulocyte differentiation and viral load. From this, we applied quantitative proteomics to select a 15 member panel of proteins that accurately predicted DF, DHF, and DFC using a random forest classifier. The classifier primarily relied on acute phase (A2M), complement (CFD), platelet counts and cellular leak (TPM4) to produce an 86% accuracy of prediction with an area under the receiver operating curve of >0.9 for DHF and DFC vs DF. CONCLUSIONS: Integrating discovery and heuristic approaches to sample distinct pathophysiological processes is a powerful approach in infectious disease. Early detection of intermediate outcomes of DENV-3 will speed clinical trials evaluating vaccines or drug interventions.


Assuntos
Biomarcadores/análise , Vírus da Dengue/genética , Dengue/diagnóstico , Dengue Grave/diagnóstico , Reação de Fase Aguda , Adulto , Biomarcadores/sangue , Ativação do Complemento , Dengue/genética , Dengue/virologia , Diagnóstico Precoce , Feminino , Humanos , Masculino , Fenótipo , Contagem de Plaquetas , Proteômica , Curva ROC , Dengue Grave/genética , Dengue Grave/virologia , Tropomiosina/análise , Carga Viral , Adulto Jovem , alfa-Macroglobulinas/análise
15.
Clin Transl Sci ; 5(1): 8-20, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22376251

RESUMO

Secondary dengue viral infection can produce capillary leakage associated with increased mortality known as dengue hemorrhagic fever (DHF). Because the mortality of DHF can be reduced by early detection and intensive support, improved methods for its detection are needed. We applied multidimensional protein profiling to predict outcomes in a prospective dengue surveillance study in South America. Plasma samples taken from initial clinical presentation of acute dengue infection were subjected to proteomics analyses using ELISA and a recently developed biofluid analysis platform. Demographics, clinical laboratory measurements, nine cytokines, and 419 plasma proteins collected at the time of initial presentation were compared between the DF and DHF outcomes. Here, the subject's gender, clinical parameters, two cytokines, and 42 proteins discriminated between the outcomes. These factors were reduced by multivariate adaptive regression splines (MARS) that a highly accurate classification model based on eight discriminant features with an area under the receiver operator curve (AUC) of 0.999. Model analysis indicated that the feature-outcome relationship were nonlinear. Although this DHF risk model will need validation in a larger cohort, we conclude that approaches to develop predictive biomarker models for disease outcome will need to incorporate nonparametric modeling approaches.


Assuntos
Dengue/diagnóstico , Dinâmica não Linear , Proteínas/análise , Proteômica , Dengue Grave/diagnóstico , Adolescente , Adulto , Área Sob a Curva , Biomarcadores/sangue , Criança , Pré-Escolar , Cromatografia em Gel , Citocinas/análise , Dengue/sangue , Diagnóstico Diferencial , Análise Discriminante , Diagnóstico Precoce , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Fluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Valor Preditivo dos Testes , Estudos Prospectivos , Proteômica/métodos , Medição de Risco , Fatores de Risco , Dengue Grave/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Venezuela , Adulto Jovem
16.
Comput Inform Nurs ; 29(12): 692-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21825971

RESUMO

The advent of the electronic medical record has brought a new challenge to nursing education. Although most nursing students are proficient in data entry and computer skills, they often do not comprehend how the information they enter becomes a vital component of interdisciplinary team communication. Furthermore, the electronic medical record becomes a repository for information that can be retrieved for the purpose of decision support. Developed by the Cleveland Clinic, the Deans' Roundtable, and University Hospitals of Cleveland, the Student Nurse Portal provides a means of assisting the student to understand how data entered into the computer transforms into information and knowledge, resulting in the wisdom that enables healthcare workers to provide optimal patient care. Current courses present the purpose of the electronic medical record and its roleas a powerful communication tool, but future courses will also help the student develop data entry and retrieval skills. Hosted on the Cleveland Clinic servers and available to students around-the-clock from any computer with Internet access, students have found the Student Nurse Portal to be a valuable tool in preparing for the use of the electronic medical record during their clinical experiences.


Assuntos
Assistência à Saúde/organização & administração , Educação em Enfermagem/organização & administração , Registros Eletrônicos de Saúde , Currículo , Humanos , Pesquisa em Educação de Enfermagem , Informática em Enfermagem
17.
Biochemistry ; 50(25): 5601-14, 2011 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-21615140

RESUMO

Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.


Assuntos
Cisteína/química , Óxido Nítrico/química , Proteômica/métodos , Animais , Compostos de Boro/química , Calorimetria , Cisteína/metabolismo , Feminino , Fluorescência , Hipóxia/metabolismo , Hipóxia/patologia , Isquemia/metabolismo , Isquemia/patologia , Luminescência , Maleimidas/química , Óxido Nítrico/metabolismo , Perfusão , Fosforilação , Distribuição Aleatória , Ratos , Ratos Wistar
18.
J Immunol ; 186(11): 6485-96, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21525390

RESUMO

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CßII (PKCßII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCßII with 4,5-bis(4-fluoroanilino)phtalimide or PKCßII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)ß(2) integrin, which was sensitive to PKCßII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCßII inhibitors. Similarly, inhibition of PKCßII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)ß(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCßII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCßII-mediated L-plastin phosphorylation.


Assuntos
Eosinófilos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteína Quinase C/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Sequência de Aminoácidos , Western Blotting , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Eosinófilos/metabolismo , Humanos , Integrinas/metabolismo , Glicoproteínas de Membrana/química , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Paxilina/metabolismo , Peptídeos/farmacologia , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Ftalimidas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C beta , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Interferência de RNA , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Recombinantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
19.
Mol Imaging ; 10(1): 43-55, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21303614

RESUMO

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.


Assuntos
Portadores de Fármacos/metabolismo , Proteínas Opsonizantes/sangue , Silício/química , Animais , Portadores de Fármacos/química , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Camundongos , Porosidade
20.
Proteomics Clin Appl ; 3(10): 1151-1173, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-21048890

RESUMO

Eosinophils are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood eosinophils from normal human donors primarily employing 2-dimensional gel electrophoresis with protein spot identification by matrix-assisted laser desorption/ionization mass spectrometry. Protein subfractionation methods employed included isoelectric focusing (Zoom(®) Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3,141 proteins which had Mascot expectation scores of 10(-3) or less. Of these 426 were unique and non-redundant of which 231 were novel proteins not previously reported to occur in eosinophils. Ingenuity Pathway Analysis showed that some 70% of the non-redundant proteins could be subdivided into categories that are clearly related to currently known eosinophil biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the eosinophil. This dataset of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

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