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1.
Lasers Surg Med ; 50(9): 908-916, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29799134

RESUMO

OBJECTIVES: To measure and compare microvascular responses within the skin of the upper arm to local stimuli, such as heating or rubbing, through the use of optical coherence tomography angiography (OCTA), and to investigate its impact on blood volume collection. MATERIALS AND METHODS: With the use of heat packs or rubbing, local stimulation was applied to the skin of either the left or right upper arm. Data from the stimulated sites were obtained using OCTA comparing pre- and post-stimulation microvascular parameters, such as vessel density, mean vessel diameter, and mean avascular pore size. Additionally, blood was collected using a newly designed collection device and volume was recorded to evaluate the effect of the skin stimulation. RESULTS: Nineteen subjects were recruited for local stimulation study (including rubbing and heating) and 21 subjects for blood drawn study. Of these subjects, 14 agreed to participate in both studies. OCTA was successful in monitoring and measuring minute changes in the microvasculature of the stimulated skin. Compared to baseline, significant changes after local heating and rubbing were respectively found in vessel density (16% [P = 0.0004] and 33% [P < 0.0001] increase), mean vessel diameter (14% and 11% increase) and mean avascular pore size (5% [P = 0.0068] and 8% [P = 0.0005] decrease) after stimulations. A gradual recovery was recorded for each parameter, with no difference being measured after 30 minutes. Blood collection volumes significantly increased after stimulations of heating (48% increase; P = 0.049) and rubbing (78% increase; P = 0.048). Significant correlations were found between blood volume and microvascular parameters except mean avascular pore size under the heating condition. CONCLUSIONS: OCTA can provide important information regarding microvascular adaptations to local stimuli. With that, both heating and rubbing of the skin have positive effects on blood collection capacity, with rubbing having the most significant effect. Lasers Surg. Med. 50:908-916, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Angiografia , Derme/irrigação sanguínea , Derme/diagnóstico por imagem , Microvasos/diagnóstico por imagem , Estimulação Física , Tomografia de Coerência Óptica , Adulto , Coleta de Amostras Sanguíneas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Extremidade Superior , Adulto Jovem
2.
Blood ; 122(25): 4140-9, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24108461

RESUMO

The Src family kinases (SFKs) c-Src and Yes mediate vascular leakage in response to various stimuli including lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF). Here, we define an opposing function of another SFK, Lyn, which in contrast to other SFKs, strengthens endothelial junctions and thereby restrains the increase in vascular permeability. Mice lacking Lyn displayed increased mortality in LPS-induced endotoxemia and increased vascular permeability in response to LPS or VEGF challenge compared with wild-type littermates. Lyn knockout mice repopulated with wild-type bone marrow-derived cells have higher vascular permeability than wild-type mice, suggesting a role of endothelial Lyn in the maintenance of the vascular barrier. Small interfering RNA-mediated down-regulation of Lyn disrupted endothelial barrier integrity, whereas expression of a constitutively active mutant of Lyn enhanced the barrier. However, down-regulation of Lyn did not affect LPS-induced endothelial permeability. We demonstrate that Lyn association with focal adhesion kinase (FAK) and phosphorylation of FAK at tyrosine residues 576/577 and 925 were required for Lyn-dependent stabilization of endothelial adherens junctions. Thus, in contrast to c-Src and Yes, which increase vascular permeability in response to stimuli, Lyn stabilizes endothelial junctions through phosphorylation of FAK. Therefore, therapeutics activating Lyn kinase may strengthen the endothelial barrier junction and hence have anti-inflammatory potential.


Assuntos
Junções Aderentes/enzimologia , Permeabilidade Capilar/fisiologia , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Quinases da Família src/metabolismo , Junções Aderentes/genética , Animais , Permeabilidade Capilar/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Células Endoteliais/citologia , Endotélio Vascular/citologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Quinases da Família src/genética
3.
EMBO J ; 28(19): 2896-907, 2009 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-19696743

RESUMO

There are at least 11 mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and only 3 major groups of MAPKs, raising the question of whether these phosphatases have non-redundant functions in vivo. Using a modified mouse model of local Shwartzman reaction, we found that deletion of the MKP5 gene, but not the MKP1 gene, led to robust and accelerated vascular inflammatory responses to a single dose of LPS injection. Depletion of neutrophils significantly reduced the vascular injury in Mkp5(-/-) mice, whereas adoptive transfer of Mkp5(-/-) neutrophils replicated the LPS-induced skin lesions in wild-type recipients. Neutrophils isolated from Mkp5(-/-) mice exhibited augmented p38 MAPK activation and increased superoxide generation on activation. The p38 MAPK inhibitor, SB203580, significantly reduced p47(phox) phosphorylation and diminished superoxide production in neutrophils. p38 MAPK phosphorylated mouse p47(phox), and deletion of the p47(phox) gene ablated the LPS-induced vascular injury in Mkp5(-/-) mice. Collectively, these results show an earlier unrecognized and non-redundant function of MKP5 in restraining p38 MAPK-mediated neutrophil oxidant production, thereby preventing LPS-induced vascular injury.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Lipopolissacarídeos/imunologia , Microvasos/lesões , Animais , Fosfatases de Especificidade Dupla/genética , Deleção de Genes , Camundongos , Microvasos/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Proc Natl Acad Sci U S A ; 106(47): 19842-7, 2009 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-19897731

RESUMO

As mediators of innate immunity, neutrophils respond to chemoattractants by adopting a highly polarized morphology. Efficient chemotaxis requires the formation of one prominent pseudopod at the cell front characterized by actin polymerization, while local inhibition suppresses the formation of rear and lateral protrusions. This asymmetric control of signaling pathways is required for directional migration along a chemotactic gradient. Here, we identify the MAGUK protein p55/MPP1 as a mediator of the frontness signal required for neutrophil polarization. We developed a p55 knockout (p55(-/-)) mouse model, and demonstrate that p55(-/-) neutrophils form multiple transient pseudopods upon chemotactic stimulation, and do not migrate efficiently in vitro. Upon agonist stimulation, p55 is rapidly recruited to the leading edge of neutrophils in mice and humans. Total F-actin polymerization, along with Rac1 and RhoA activation, appear to be normal in p55(-/-) neutrophils. Importantly, phosphorylation of Akt is significantly decreased in p55(-/-) neutrophils upon chemotactic stimulation. The activity of immunoprecipitated phosphatidylinositol 3-kinase gamma (PI3Kgamma), responsible for chemoattractant-induced synthesis of PIP(3) and Akt phosphorylation, is unperturbed in p55(-/-) neutrophils. Although the total amount of PIP(3) is normal in p55(-/-) neutrophils, PIP(3) is diffusely localized and forms punctate aggregates in activated p55(-/-) neutrophils, as compared to its accumulation at the leading edge membrane in the wild type neutrophils. Together, these results show that p55 is required for neutrophil polarization by regulating Akt phosphorylation through a mechanism that is independent of PI3Kgamma activity.


Assuntos
Polaridade Celular , Guanilato Quinases/metabolismo , Neutrófilos , Actinas/metabolismo , Animais , Quimiotaxia de Leucócito , Classe Ib de Fosfatidilinositol 3-Quinase , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Ativação Enzimática , Feminino , GTP Fosfo-Hidrolases/metabolismo , Guanilato Quinases/genética , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transplante de Células-Tronco , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteína rhoA de Ligação ao GTP/metabolismo
5.
J Immunol ; 182(12): 7997-8004, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19494325

RESUMO

Bacterial LPS induces rapid thrombocytopenia, hypotension, and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. In this study, we show that LPS stimulates platelet secretion of dense and alpha granules as indicated by ATP release and P-selectin expression, and thus enhances platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including TLR (TLR4), CD14, MD2, and MyD88, and the effect of LPS on platelet activation was abolished by an anti-TLR4-blocking Ab or TLR4 knockout, suggesting that the effect of LPS on platelet aggregation requires the TLR4 pathway. Furthermore, LPS-potentiated thrombin- and collagen-induced platelet aggregation and FeCl(3)-induced thrombus formation were abolished in MyD88 knockout mice. LPS also induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was abolished by inhibitors of NO synthase and the cGMP-dependent protein kinase (PKG). LPS-induced cGMP elevation was inhibited by an anti-TLR4 Ab or by TLR4 deficiency, suggesting that activation of the cGMP/protein kinase G pathway by LPS involves the TLR4 pathway. Taken together, our data indicate that LPS stimulates platelet secretion and potentiates platelet aggregation through a TLR4/MyD88- and cGMP/PKG-dependent pathway.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , GMP Cíclico/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/metabolismo , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Ligação Proteica , Trombina/metabolismo
6.
PLoS One ; 16(9): e0255841, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473717

RESUMO

BACKGROUND: Efforts to minimize COVID-19 exposure during the current SARS-CoV-2 pandemic have led to limitations in access to medical care and testing. The Tasso-SST kit includes all of the components necessary for remote, capillary blood self-collection. In this study, we sought to investigate the accuracy and reliability of the Tasso-SST device as a self-collection device for measurement of SARS-CoV-2 IgG antibodies. METHODS: Capillary blood was obtained via unsupervised and supervised application of the Tasso-SST device, and venous blood was collected by standard venipuncture. Unsupervised self-collected blood samples underwent either extreme summer or winter-simulated shipping conditions prior to testing. Sera obtained by all three methods were tested concurrently using the EuroImmun anti-SARS-CoV-2 S1 IgG assay in a CLIA-certified clinical laboratory. RESULTS: Successful Tasso-SST capillary blood collection by unsupervised and supervised administration was completed by 93.4% and 94.5% of participants, respectively. Sera from 56 participants, 55 with documented (PCR+) COVID-19, and 33 healthy controls were then tested for anti-SARS-CoV-2 IgG antibodies. Compared to venous blood results, Tasso-SST-collected (unstressed) and the summer- and winter-stressed blood samples demonstrated Deming regression slopes of 1.00 (95% CI: 0.99-1.02), 1.00 (95% CI: 0.98-1.01), and 0.99 (95% CI: 0.97-1.01), respectively, with an overall accuracy of 98.9%. CONCLUSIONS: Capillary blood self-collection using the Tasso-SST device had a high success rate. Moreover, excellent concordance was found for anti-SARS-CoV-2 IgG results between Tasso-SST capillary and standard venous blood-derived sera. The Tasso-SST device should enable widespread collection of capillary blood for testing without medical supervision, facilitating epidemiologic studies.


Assuntos
Anticorpos Antivirais/imunologia , Coleta de Amostras Sanguíneas/métodos , Teste para COVID-19/métodos , COVID-19/diagnóstico , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Coleta de Amostras Sanguíneas/instrumentação , COVID-19/epidemiologia , COVID-19/virologia , Teste para COVID-19/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Reprodutibilidade dos Testes , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade , Adulto Jovem
7.
Mol Pharmacol ; 75(1): 227-34, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18931035

RESUMO

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF.


Assuntos
Plaquetas/efeitos dos fármacos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Fator de Ativação de Plaquetas/análogos & derivados , Fator de Ativação de Plaquetas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Animais , Plaquetas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/antagonistas & inibidores
8.
Mol Interv ; 10(2): 86-97, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20368369

RESUMO

A-Kinase Anchoring Proteins (AKAPs) orchestrate and synchronize cellular events by tethering the cAMP-dependent protein kinase (PKA) and other signaling enzymes to organelles and membranes. The control of kinases and phosphatases that are held in proximity to activators, effectors, and substrates favors the rapid dissemination of information from one cellular location to the next. This article charts the inception of the PKA-anchoring hypothesis, the characterization of AKAPs and their nomenclature, and the physiological roles of context-specific AKAP signaling complexes.


Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Enzimas Imobilizadas/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Modelos Biológicos , Modelos Moleculares
9.
J Immunol ; 174(5): 2981-9, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15728511

RESUMO

Lysophosphatidylcholine (LPC) is an oxidized phospholipid present in micromolar concentrations in blood and inflamed tissues. The effects of LPC on neutrophil functions remain incompletely understood, because conflicting reports exist for its stimulatory and inhibitory roles. We report in this study that LPC inhibits superoxide generation in fMLP- and PMA-stimulated neutrophils without affecting fMLP-induced Ca(2+) mobilization and cell viability. This effect was observed with LPC dissolved in ethanol, but not with LPC stock solutions prepared in water or in BSA-containing aqueous solution with sonication. Under the same experimental conditions, platelet-activating factor primed neutrophils for superoxide generation. The inhibitory effect of LPC was observed within 30 s after its application and was maximal at LPC concentrations between 0.1 and 1 muM. Inhibition of superoxide generation was accompanied by a 2.5-fold increase in the intracellular cAMP concentration. In addition, LPC reduced fMLP-stimulated phosphorylation of ERK and Akt and membrane translocation of p67(phox) and p47(phox). The protein kinase A inhibitors H-89 and adenosine 3'5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP) partially restored superoxide production in LPC-treated neutrophils, indicating involvement of protein kinase A in LPC-mediated inhibition. Using an ex vivo mouse lung perfusion model that measures lung weight change and capillary filtration coefficient, we found that LPC prevented lung vascular injury mediated by fMLP-activated neutrophils. Taken together, these results suggest that LPC-induced elevation of intracellular cAMP is partially responsible for its inhibition of neutrophil NADPH oxidase activation. A similar mechanism of inhibition may be used for the control of neutrophil-mediated tissue injury.


Assuntos
AMP Cíclico/fisiologia , Lisofosfatidilcolinas/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oxidantes/biossíntese , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/metabolismo , Permeabilidade Capilar/imunologia , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Técnicas In Vitro , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Camundongos , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Oxidantes/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia
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