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1.
J Vis Exp ; (173)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34279509

RESUMO

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.


Assuntos
Fosfatase Alcalina , Escherichia coli , Animais , Escherichia coli/genética , Imunofluorescência , Lipídeos , Camundongos , Proteínas Recombinantes de Fusão
2.
Cardiovasc Res ; 117(5): 1358-1371, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33038226

RESUMO

AIMS: Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. METHODS AND RESULTS: To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1ß. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. CONCLUSION: Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans.


Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos , Monócitos/efeitos dos fármacos , NADPH Oxidases/metabolismo , Cloreto de Sódio/farmacologia , Transferência Adotiva , Adulto , Idoso , Animais , Antígenos CD/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Ativação Enzimática , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunoglobulinas/metabolismo , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Transgênicos , Pessoa de Meia-Idade , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/transplante , Fenótipo , Receptores de IgG/metabolismo , Cloreto de Sódio na Dieta/farmacologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígeno CD83
3.
Am J Physiol Renal Physiol ; 318(3): F647-F659, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984788

RESUMO

CD148 is a transmembrane protein tyrosine phosphatase (PTP) that is expressed in the renal vasculature, including the glomerulus. Previous studies have shown that CD148 plays a role in the negative regulation of growth factor signals (including epidermal growth factor and vascular endothelial growth factor), suppressing cell proliferation and transformation. However, the role of CD148 in kidney disease remains unknown. Here, we generated an agonistic anti-CD148 antibody and evaluated its effects in murine diabetic nephropathy (DN). Monoclonal antibodies (mAbs) against the mouse CD148 ectodomain sequence were generated by immunizing CD148 knockout (CD148KO) mice. The mAbs that increased CD148 activity were selected by biological (proliferation) and biochemical (PTP activity) assays. The mAb (18E1) that showed strong agonistic activity was injected (10 mg/kg ip) in streptozotocin-induced wild-type and CD148KO diabetic mice for 6 wk, and the renal phenotype was then assessed. The effects of 18E1 mAb in podocyte growth factor signals were also assessed in culture. Compared with control IgG, 18E1 mAb significantly decreased albuminuria and mesangial expansion without altering hyperglycemia and blood pressure in wild-type diabetic mice. Immunohistochemical evaluation showed that 18E1 mAb significantly prevented the reduction of podocyte number and nephrin expression and decreased glomerular fibronectin expression and renal macrophage infiltration. The 18E1 mAb showed no effects in CD148KO diabetic mice. Furthermore, we demonstrated that 18E1 mAb reduces podocyte epidermal growth factor receptor signals in culture and in diabetic mice. These findings suggest that agonistic anti-CD148 mAb attenuates DN in mice, in part by reducing epidermal growth factor receptor signals in podocytes. This antibody may be used for the treatment of early DN.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Nefropatias Diabéticas/terapia , Albuminúria , Animais , Linhagem Celular , Diabetes Mellitus Experimental/complicações , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Imunoglobulina G/uso terapêutico , Camundongos , Camundongos Knockout , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/imunologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Transdução de Sinais
4.
mBio ; 10(5)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575763

RESUMO

Staphylococcus aureus infects every niche of the human host. In response to microbial infection, vertebrates have an arsenal of antimicrobial compounds that inhibit bacterial growth or kill bacterial cells. One class of antimicrobial compounds consists of polyunsaturated fatty acids, which are highly abundant in eukaryotes and encountered by S. aureus at the host-pathogen interface. Arachidonic acid (AA) is one of the most abundant polyunsaturated fatty acids in vertebrates and is released in large amounts during the oxidative burst. Most of the released AA is converted to bioactive signaling molecules, but, independently of its role in inflammatory signaling, AA is toxic to S. aureus Here, we report that AA kills S. aureus through a lipid peroxidation mechanism whereby AA is oxidized to reactive electrophiles that modify S. aureus macromolecules, eliciting toxicity. This process is rescued by cotreatment with antioxidants as well as in a S. aureus strain genetically inactivated for lcpA (USA300 ΔlcpA mutant) that produces lower levels of reactive oxygen species. However, resistance to AA stress in the USA300 ΔlcpA mutant comes at a cost, making the mutant more susceptible to ß-lactam antibiotics and attenuated for pathogenesis in a murine infection model compared to the parental methicillin-resistant S. aureus (MRSA) strain, indicating that resistance to AA toxicity increases susceptibility to other stressors encountered during infection. This report defines the mechanism by which AA is toxic to S. aureus and identifies lipid peroxidation as a pathway that can be modulated for the development of future therapeutics to treat S. aureus infections.IMPORTANCE Despite the ability of the human immune system to generate a plethora of molecules to control Staphylococcus aureus infections, S. aureus is among the pathogens with the greatest impact on human health. One class of host molecules toxic to S. aureus consists of polyunsaturated fatty acids. Here, we investigated the antibacterial properties of arachidonic acid, one of the most abundant polyunsaturated fatty acids in humans, and discovered that the mechanism of toxicity against S. aureus proceeds through lipid peroxidation. A better understanding of the molecular mechanisms by which the immune system kills S. aureus, and by which S. aureus avoids host killing, will enable the optimal design of therapeutics that complement the ability of the vertebrate immune response to eliminate S. aureus infections.


Assuntos
Antibacterianos/farmacologia , Ácido Araquidônico/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Encéfalo/microbiologia , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Feminino , Rim/microbiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Mutação , Neutrófilos/fisiologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Baço/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Ácidos Teicoicos
5.
Cell Rep ; 21(4): 1009-1020, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29069584

RESUMO

Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox, and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin-1ß (IL-1ß) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation, and hypertension involving increased oxidative stress and IsoLG production in DCs.


Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Canais Epiteliais de Sódio/metabolismo , Hipertensão/metabolismo , Amilorida/farmacologia , Animais , Células Cultivadas , Citocinas/genética , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Estresse Oxidativo , Prostaglandinas E/metabolismo , Proteína Quinase C/metabolismo , Sódio/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Superóxidos/metabolismo
6.
Nanoscale Res Lett ; 11(1): 395, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27620193

RESUMO

Efficient mass transport through porous networks is essential for achieving rapid response times in sensing applications utilizing porous materials. In this work, we show that open-ended porous membranes can overcome diffusion challenges experienced by closed-ended porous materials in a microfluidic environment. A theoretical model including both transport and reaction kinetics is employed to study the influence of flow velocity, bulk analyte concentration, analyte diffusivity, and adsorption rate on the performance of open-ended and closed-ended porous sensors integrated with flow cells. The analysis shows that open-ended pores enable analyte flow through the pores and greatly reduce the response time and analyte consumption for detecting large molecules with slow diffusivities compared with closed-ended pores for which analytes largely flow over the pores. Experimental confirmation of the results was carried out with open- and closed-ended porous silicon (PSi) microcavities fabricated in flow-through and flow-over sensor configurations, respectively. The adsorption behavior of small analytes onto the inner surfaces of closed-ended and open-ended PSi membrane microcavities was similar. However, for large analytes, PSi membranes in a flow-through scheme showed significant improvement in response times due to more efficient convective transport of analytes. The experimental results and theoretical analysis provide quantitative estimates of the benefits offered by open-ended porous membranes for different analyte systems.

7.
PLoS One ; 11(5): e0154916, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27149518

RESUMO

CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1st, but not the 2nd and 3rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis in vivo. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.


Assuntos
Trombospondina 1/metabolismo , Animais , Sítios de Ligação , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imunoprecipitação , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Fragmentos de Peptídeos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo
9.
Sci Rep ; 6: 24919, 2016 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27118599

RESUMO

Protein lysine modification by γ-ketoaldehyde isomers derived from arachidonic acid, termed isolevuglandins (IsoLGs), is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by IsoLGs are subject to genetic regulation and the identity of IsoLG-modified proteins remain unclear. Herein we show that Nrf2 and Nox2 are key regulators of IsoLG modification in pulmonary tissue and report on the identity of proteins analyzed by LC-MS following immunoaffinity purification of IsoLG-modified proteins. Gene ontology analysis revealed that proteins in numerous cellular pathways are susceptible to IsoLG modification. Although cells tolerate basal levels of modification, exceeding them induces apoptosis. We found prominent modification in a murine model of radiation-induced pulmonary fibrosis and in idiopathic pulmonary fibrosis, two diseases considered to be promoted by gene-regulated oxidant stress. Based on these results we hypothesize that IsoLG modification is a hitherto unrecognized sequelae that contributes to radiation-induced pulmonary injury and IPF.


Assuntos
Pulmão/química , Pulmão/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/análise , Fibrose Pulmonar/patologia , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Espectrometria de Massas , Camundongos
10.
J Clin Invest ; 126(1): 50-67, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26595812

RESUMO

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(sm/p22phox) mice produced high levels of IL-17A and IFN-γ. Crossing tg(sm/p22phox) mice with lymphocyte-deficient Rag1(-/-) mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg(sm/p22phox) mice. Autologous pulsing with tg(sm/p22phox) aortic homogenates promoted DCs of tg(sm/p22phox) mice to stimulate T cell proliferation and production of IFN-γ, IL-17A, and TNF-α. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.


Assuntos
Hipertensão/etiologia , Inflamação/complicações , Estresse Oxidativo , Rigidez Vascular , Fatores Etários , Animais , Colágeno/metabolismo , Citocinas/biossíntese , Fibrose , Humanos , Masculino , Camundongos , Linfócitos T/fisiologia , Vasculite/complicações
11.
Circ Res ; 117(6): 547-57, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26156232

RESUMO

RATIONALE: Inflammation and adaptive immunity play a crucial role in the development of hypertension. Angiotensin II and probably other hypertensive stimuli activate the central nervous system and promote T-cell activation and end-organ damage in peripheral tissues. OBJECTIVE: To determine if renal sympathetic nerves mediate renal inflammation and T-cell activation in hypertension. METHODS AND RESULTS: Bilateral renal denervation using phenol application to the renal arteries reduced renal norepinephrine levels and blunted angiotensin II-induced hypertension. Bilateral renal denervation also reduced inflammation, as reflected by decreased accumulation of total leukocytes, T cells, and both CD4+ and CD8+ T cells in the kidney. This was associated with a marked reduction in renal fibrosis, albuminuria, and nephrinuria. Unilateral renal denervation, which partly attenuated blood pressure, only reduced inflammation in the denervated kidney, suggesting that this effect is pressure independent. Angiotensin II also increased immunogenic isoketal-protein adducts in renal dendritic cells (DCs) and increased surface expression of costimulation markers and production of interleukin (IL)-1α, IL-1ß, and IL-6 from splenic DCs. Norepinephrine also dose dependently stimulated isoketal formation in cultured DCs. Adoptive transfer of splenic DCs from angiotensin II-treated mice primed T-cell activation and hypertension in recipient mice. Renal denervation prevented these effects of hypertension on DCs. In contrast to these beneficial effects of ablating all renal nerves, renal afferent disruption with capsaicin had no effect on blood pressure or renal inflammation. CONCLUSIONS: Renal sympathetic nerves contribute to DC activation, subsequent T-cell infiltration and end-organ damage in the kidney in the development of hypertension.


Assuntos
Angiotensina II/toxicidade , Hipertensão/imunologia , Imunidade Celular/fisiologia , Rim/imunologia , Rim/inervação , Simpatectomia , Animais , Hipertensão/patologia , Imunidade Celular/efeitos dos fármacos , Rim/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória
12.
J Clin Invest ; 124(10): 4642-56, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25244096

RESUMO

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1ß, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.


Assuntos
Células Dendríticas/imunologia , Hipertensão/patologia , Ativação Linfocitária , Linfócitos T/citologia , Idoso , Aldeídos/química , Angiotensina II/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Proliferação de Células , Estudos de Coortes , Células Dendríticas/citologia , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Estresse Oxidativo , Oxigênio/metabolismo , Superóxidos/metabolismo
13.
Nanoscale Res Lett ; 9(1): 383, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25136285

RESUMO

A porous silicon (PSi) Bloch surface wave (BSW) and Bloch sub-surface wave (BSSW) composite biosensor is designed and used for the size-selective detection of both small and large molecules. The BSW/BSSW structure consists of a periodic stack of high and low refractive index PSi layers and a reduced optical thickness surface layer that gives rise to a BSW with an evanescent tail that extends above the surface to enable the detection of large surface-bound molecules. Small molecules were detected in the sensor by the BSSW, which is a large electric field intensity spatially localized to a desired region of the Bragg mirror and is generated by the implementation of a step or gradient refractive index profile within the Bragg mirror. The step and gradient BSW/BSSW sensors are designed to maximize both resonance reflectance intensity and sensitivity to large molecules. Size-selective detection of large molecules including latex nanospheres and the M13KO7 bacteriophage as well as small chemical linker molecules is reported.

14.
Lab Chip ; 14(2): 315-24, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24257544

RESUMO

Effective point-of-care diagnostics require a biomarker detection strategy that is low-cost and simple-to-use while achieving a clinically relevant limit of detection. Here we report a biosensor that uses secondary flows arising from surface Marangoni stresses in an evaporating drop to concentrate target-mediated particle aggregates in a visually detectable spot. The spot size increases with increasing target concentration within the dynamic range of the assay. The particle deposition patterns are visually detectable and easily measured with simple optical techniques. We use optical coherence tomography to characterize the effect of cross-sectional flow fields on the motion of particles in the presence and absence of target (aggregated and non-aggregated particles, respectively). We show that choice of substrate material and the presence of salts and glycerol in solution promote the Marangoni-induced flows that are necessary to produce signal in the proposed design. These evaporation-driven flows generate signal in the assay on a PDMS substrate but not substrates with greater thermal conductivity like indium tin oxide-coated glass. In this proof-of-concept design we use the M13K07 bacteriophage as a model target and 1 µm-diameter particles surface functionalized with anti-M13 monoclonal antibodies. Using standard microscopy-based techniques to measure the final spot size, the assay has a calculated limit-of-detection of approximately 100 fM. Approximately 80% of the maximum signal is generated within 10 minutes of depositing a 1 µL drop of reacted sample on PDMS enabling a relatively quick time-to-result.


Assuntos
Técnicas Biossensoriais , Biomarcadores/metabolismo , Tomografia de Coerência Óptica
15.
Proc Natl Acad Sci U S A ; 109(6): 1985-90, 2012 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-22308318

RESUMO

CD148 is a receptor-type protein tyrosine phosphatase that is expressed in several cell types, including vascular endothelial cells and duct epithelial cells. Growing evidence demonstrates a prominent role for CD148 in negative regulation of growth factor signals, suppressing cell proliferation and transformation. However, its extracellular ligand(s) remain unknown. To identify the ligand(s) of CD148, we introduced HA-tagged CD148 into cultured endothelial cells and then isolated its interacting extracellular protein(s) by biotin surface labeling and subsequent affinity purifications. The binding proteins were identified by mass spectrometry. Here we report that soluble thrombospondin-1 (TSP1) binds to the extracellular part of CD148 with high affinity and specificity, and its binding increases CD148 catalytic activity, leading to dephosphorylation of the substrate proteins. Consistent with these findings, introduction of CD148 conferred TSP1-mediated inhibition of cell growth to cells which lack CD148 and TSP1 inhibition of growth. Further, we demonstrate that TSP1-mediated inhibition of endothelial cell growth is antagonized by soluble CD148 ectodomain as well as by CD148 gene silencing. These findings provide evidence that CD148 functions as a receptor for TSP1 and mediates its inhibition of cell growth.


Assuntos
Trombospondina 1/metabolismo , Proliferação de Células , Espaço Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Rim/citologia , Ligantes , Microvasos/citologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo
16.
PLoS One ; 6(8): e23813, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21876773

RESUMO

BACKGROUND: Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1) was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42. CONCLUSIONS: Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".


Assuntos
Quimiotaxia/fisiologia , Junções Intercelulares/metabolismo , Receptores de Interleucina-8B/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Quimiotaxia/efeitos dos fármacos , Cromatografia Líquida , Células HEK293 , Células HL-60 , Humanos , Junções Intercelulares/efeitos dos fármacos , Interleucina-8/farmacologia , Espectrometria de Massas , Ligação Proteica/efeitos dos fármacos , Proteômica , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/química
17.
J Cell Sci ; 122(Pt 11): 1882-94, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19435808

RESUMO

Chemotaxis regulates the recruitment of leukocytes, which is integral for a number of biological processes and is mediated through the interaction of chemokines with seven transmembrane G-protein-coupled receptors. Several studies have indicated that chemotactic signaling pathways might be activated via G-protein-independent mechanisms, perhaps through novel receptor-interacting proteins. CXCR2 is a major chemokine receptor expressed on neutrophils. We used a proteomics approach to identify unique ligand-dependent CXCR2-interacting proteins in differentiated neutrophil-like HL-60 cells. Using this approach, vasodilator-stimulated phosphoprotein (VASP) was identified as a CXCR2-interacting protein. The interaction between CXCR2 and VASP is direct and enhanced by CXCL8 stimulation, which triggers VASP phosphorylation via PKA- and PKCdelta-mediated pathways. The interaction between CXCR2 and VASP requires free F-actin barbed ends to recruit VASP to the leading edge. Finally, knockdown of VASP in HL-60 cells results in severely impaired CXCR2-mediated chemotaxis and polarization. These data provide the first demonstration that direct interaction of VASP with CXCR2 is essential for proper CXCR2 function and demonstrate a crucial role for VASP in mediating chemotaxis in leukocytes.


Assuntos
Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Quimiotaxia/fisiologia , Leucócitos/fisiologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Receptores de Interleucina-8B/metabolismo , Actinas/metabolismo , Animais , Moléculas de Adesão Celular/genética , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HL-60 , Humanos , Interleucina-8/metabolismo , Leucócitos/citologia , Proteínas dos Microfilamentos/genética , Fosfoproteínas/genética , Fosforilação , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Receptores de Interleucina-8B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Transdução de Sinais/fisiologia
18.
Methods Enzymol ; 460: 315-30, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19446732

RESUMO

Chemokine-receptor signaling is initiated upon ligand binding to the receptor and continues through the process of endocytic trafficking by the association of a variety of adaptor proteins with the chemokine receptor. In order to define the adaptor proteins that associate with CXCR2 before and after ligand activation, a protocol was developed using differentiated HL-60 cells transfected to express CXCR2 stimulated or not stimulated with ligand for one minute. CXCR2-associating proteins were isolated by immunoprecipitation with CXCR2 antibody and the eluted proteins were electrophoretically run into the separating gel directly without a stacking gel. The stained single band was subjected to in-gel trypsin digestion. The tryptic peptides were subjected to, LC/MS/MS proteomic analysis. Proteins identified in a minimum of three of four separate experiments with multiple peptides were then validated as CXCR2 adaptor proteins by coimmunoprecipitation, GST pull-down studies, and immunocytochemical CXCR2-colocalization experiments using dHL-60-CXCR2 cells. Subsequently, a functional analysis of the interaction between CXCR2 and CXCR2 interacting proteins was performed. This approach can be used to characterize chemokine receptor-associating proteins over time both before and after ligand stimulation, allowing definition of the dynamic spatial and temporal formation of a "chemosynapse."


Assuntos
Proteínas/metabolismo , Proteômica/métodos , Receptores de Interleucina-8B/metabolismo , Células HL-60 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Microscopia Confocal , Ligação Proteica , Receptores de Interleucina-8B/genética
19.
Biosens Bioelectron ; 24(9): 2853-7, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19327975

RESUMO

In this report, the peptide linker connecting scFv V(H) and V(L) domains were genetically modified to contain different amino acids (i.e. cysteine (scFv-cys) or histidines (scFv-his)) to enable the scFv to adsorb or self-assemble onto the gold nanoparticles (NPs). The scFv-cys stabilized gold NPs were used to develop a highly sensitive colorimetric immunosensor. The scFv-cys stabilized gold NPs were characterized by UV-vis spectra, transmission electron microscope (TEM) and FTIR. After adding the antigen rabbit IgG, the solution of scFv-cys stabilized gold NPs shows obvious visible color change from deep red to light purple due to the aggregation of the gold nanoparticles. Based on the colorimetric aggregation of scFv-cys stabilized gold NPs, the immunosensor exhibits high sensitivity with a detection limit of 1.7 nM and good specificity. The good properties of the colorimetric aggregation immunosensor can be attributed to the small size of scFv and the covalent link between the scFv and gold NPs that improve the better orientation and enhance the probe density. With the advantages of speed, simplicity and specificity, the colorimetric immunoassay based on the functionalized scFv stabilized gold NPs represents a promising approach for protein analysis and clinical diagnostics.


Assuntos
Anticorpos/imunologia , Colorimetria/métodos , Ouro/química , Imunoensaio/métodos , Imunoglobulina G/análise , Nanopartículas Metálicas/química , Animais , Anticorpos/química , Anticorpos/genética , Técnicas Biossensoriais/métodos , Humanos , Imunoglobulina G/imunologia , Nanopartículas Metálicas/ultraestrutura , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Espectrofotometria
20.
Anal Chem ; 80(6): 1910-7, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18290668

RESUMO

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors.


Assuntos
Técnicas Biossensoriais , Imunoensaio/métodos , Fragmentos de Imunoglobulinas/imunologia , Engenharia de Proteínas , Adsorção , Sequência de Aminoácidos , Eletroquímica , Fragmentos de Imunoglobulinas/química , Dados de Sequência Molecular
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