RESUMO
Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.
Assuntos
Dipeptídeos/análise , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Microdiálise , Animais , Cromatografia Líquida de Alta Pressão , Dipeptídeos/farmacologia , Humanos , Masculino , Metaloproteinase 1 da Matriz/química , Metaloproteinase 9 da Matriz/química , Inibidores de Metaloproteinases de Matriz , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Protein kinase C-epsilon (PKC-epsilon) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-epsilon mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding epsilonC1 and epsilonC1B domains, or the epsilonC1B point mutant epsilonC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that epsilonC259G, epsilonC1, and epsilonC1B accumulation at phagosomes was significantly less than that of intact PKC-epsilon. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-epsilon translocation. Thus, DAG binding to epsilonC1B is necessary for PKC-epsilon translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-gamma1, and PI-PLC-gamma2 in PKC-epsilon accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-epsilon localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-epsilon accumulation. Although expression of PI-PLC-gamma2 is higher than that of PI-PLC-gamma1, PI-PLC-gamma1 but not PI-PLC-gamma2 consistently concentrated at phagosomes. Macrophages from PI-PLC-gamma2-/- mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-epsilon at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-gamma1 as the enzyme that supports PKC-epsilon localization and phagocytosis. That PI-PLC-gamma1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-gamma1 provides DAG that binds to epsilonC1B, facilitating PKC-epsilon localization to phagosomes for efficient IgG-mediated phagocytosis.
Assuntos
Fagocitose , Fosfolipase C gama/fisiologia , Proteína Quinase C-épsilon/metabolismo , Receptores de IgG/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Diglicerídeos/antagonistas & inibidores , Diglicerídeos/metabolismo , Diglicerídeos/farmacologia , Proteínas de Fluorescência Verde/análise , Imunoglobulina G/metabolismo , Macrófagos/fisiologia , Camundongos , Modelos Biológicos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Fosfolipase D/fisiologia , Proteína Quinase C-épsilon/química , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Proteínas Recombinantes de Fusão/análiseRESUMO
The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.
Assuntos
Meios de Cultura/química , Francisella tularensis/fisiologia , Macrófagos/microbiologia , Adaptação Fisiológica , Animais , Proteínas de Bactérias/análise , Western Blotting , Contagem de Colônia Microbiana , Citocinas/biossíntese , Francisella tularensis/química , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Proteoma/análise , Análise de Sobrevida , Tularemia/microbiologiaRESUMO
Cytokines are important cellular signaling proteins involved in inflammation, wound healing and are thought to direct the foreign body response to implanted materials. In this work, polyurethane tubes (25 mm length, 1.02 mm i.d., and 1.65 mm o.d.) were implanted into subcutaneous tissue of male Sprague-Dawley rats. The tubes served as the biomaterial and a means to collect the interstitial fluid that would be exchanged within the tube lumen and the surrounding tissue. After 3 and 7 days, the tubes were explanted and cytokines in the fluid were quantified with a multiplexed cytokine immunoassay. Six cytokines, interleukin-1beta (IL-1beta), IL-4, IL-6, IL-10, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha), were simultaneously quantified. All cytokine concentrations with the exception of IL-4 and TNF-alpha ranged between low pg/mL to mid ng/mL levels. Neither TNF-alpha nor IL-4 was detected from any sample. These results illustrate the potential of using the tube materials combined with bead-based immunoassays as a direct method for in vivo collection of multiple cytokines in low microliter sample volumes for fixed day biomaterial implant studies.
Assuntos
Citocinas/metabolismo , Líquido Extracelular/metabolismo , Próteses e Implantes , Animais , Quimiocina CCL2/análise , Citocinas/análise , Líquido Extracelular/química , Líquido Extracelular/imunologia , Estudos de Viabilidade , Imunoensaio , Mediadores da Inflamação/análise , Interleucina-10/análise , Interleucina-1beta/análise , Interleucina-4/análise , Interleucina-6/análise , Masculino , Poliuretanos , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/análiseRESUMO
Ligation of Fc receptors for immunoglobulin G (FcgammaRs) inhibits lipopolysaccharide (LPS)-stimulated secretion of interleukin (IL)-12 by macrophages. FcgammaR activation of protein kinase C (PKC) contributes to several functions of this receptor including phagocytosis, activation of the reduced nicotinamide adenine dinucleotide phosphate oxidase, and secretion of certain cytokines. Therefore, we tested the hypothesis that PKC mediates the FcgammaR inhibition of IL-12 secretion by macrophages. In murine macrophages, FcgammaR ligation augmented LPS-stimulated activation of PKC-alpha and PKC-delta but reduced IL-12p40 secretion. Similarly, activation of PKC with phorbol 12-myristate 13-acetate (PMA) depressed LPS-stimulated IL-12p40 secretion, and depletion of PKC augmented LPS-stimulated IL-12p40 secretion. Antisense down-regulation of PKC-delta increased LPS-stimulated IL-12p40 secretion and fully prevented the effects of FcgammaR ligation or PMA on IL-12p40 secretion. In contrast, down-regulation of PKC-epsilon blocked LPS-stimulated secretion of IL-12p40. Down-regulation of PKC-alpha had no effect on LPS-stimulated IL-12p40 secretion. The results suggest a negative role for PKC-delta and a positive role for PKC-epsilon in the regulation of LPS-stimulated IL-12p40 secretion.
Assuntos
Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Proteína Quinase C/fisiologia , Receptores de IgG/imunologia , Animais , Células Cultivadas , Regulação para Baixo , Interleucina-12/antagonistas & inibidores , Isoenzimas/efeitos dos fármacos , Isoenzimas/fisiologia , Ligantes , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/efeitos dos fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Microdialysis sampling probes were used to collect cytokine samples from lipopolysaccharide (LPS)-stimulated macrophages. The probes were immersed into cell culture wells containing either RAW 264.7 or isolated peritoneal macrophages. Dialysates (15 microL) from these wells were subjected to a multiplexed cytokine sandwich immunoassay platform analyzed by flow cytometry that measures up to six separate cytokines, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) in a single 15-muL sample. In vitro microdialysis sampling relative recovery experiments showed that only IFN-gamma, IL-6, MCP-1, and TNF-alpha could be recovered across a commercially-available 100-kDa MWCO microdialysis membrane. Eleven hours after LPS addition (1 microg/mL), RAW 264.7 macrophages secreted greater than 8000 pg/mL of TNF-alpha and greater than 1000 pg/mL MCP-1. With the peritoneal macrophages, greater than 6000 pg/mL of IL-6, MCP-1, and TNF-alpha were obtained. The maximum dialysate concentrations obtained from the RAW macrophages were 1300 pg/mL for TNF-alpha and 55 pg/mL for MCP-1. Maximum cytokine concentrations from peritoneal macrophage dialysates reached approximately 2000 pg/mL, 1100 pg/mL and 500 pg/mL for TNF-alpha, MCP-1 and IL-6, respectively. Microdialysis sampling allowed for 20-min samples to be collected during the cytokine release from the activated macrophages. These results demonstrate that microdialysis sampling can be used for collection of selected cytokines with improved temporal resolution.
Assuntos
Citocinas/análise , Ativação de Macrófagos , Macrófagos Peritoneais/metabolismo , Animais , Linhagem Celular , Quimiocina CCL2/análise , Quimiocina CCL2/metabolismo , Meios de Cultura/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Imunoensaio/métodos , Interleucina-6/análise , Interleucina-6/metabolismo , Lipopolissacarídeos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Microdiálise , Peso Molecular , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cytokines are proteins that mediate communication between cells of the immune system as well as certain other non-immune host cells. These proteins are produced by many cell types and they mediate immune and inflammatory responses. However, the direct site analysis of these critical proteins is hampered by the lack of site-specific tools available for such direct measurements. In this study, both in vitro and in vivo microdialysis sampling of different cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], interleukin-6 [IL-6], IL-12p70, and macrophage chemoattractant protein-1 [MCP-1]) was performed. A mouse model of bacterial lipopolysaccharide (LPS) administration and response pattern was used for in vivo studies. Three cytokines, TNF-alpha, IL-6, and MCP-1 were quantified in the serum from mice given LPS. In vivo studies demonstrated the ability to monitor increasing levels of these cytokines (TNF-alpha, IL-6, and MCP-1) via microdialysis probes placed in the peritoneal cavity of mice given LPS. All three cytokines were quantified simultaneously in 15 muL of dialysate using a multiplexed bead-based immunoassay for flow cytometry. The detected dialysate cytokine concentrations varied between 200 pg/mL and 1500 pg/mL for TNF-alpha, between 600 pg/mL and 3000 pg/mL for MCP-1, and between 2700 pg/mL and more than 5000 pg/mL for IL-6. The detected serum cytokine concentrations ranged from 5700 pg/mL to 35,000 pg/mL for TNF-alpha, from 40,000 pg/mL to 65,000 pg/mL for MCP-1, and greater than than 100,000 pg/mL for IL-6. This work demonstrates that microdialysis sampling can be used in vivo to collect temporal profiles of cytokine production.
Assuntos
Citocinas/análise , Lipopolissacarídeos/farmacologia , Microdiálise/métodos , Animais , Líquido Ascítico/química , Quimiocina CCL2/análise , Citocinas/biossíntese , Citocinas/sangue , Citometria de Fluxo/métodos , Interferon gama/análise , Interleucina-12/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas Microbiológicas/instrumentação , Microdiálise/instrumentação , Subunidades Proteicas/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
Lipopolysaccharide (LPS) increases serum TNF-alpha levels due to TNF-alpha secretion by macrophages. The serum TNF-alpha response to LPS was augmented 10x when FcgammaR ligation was induced by the intravenous injection of Gig-coated erythrocytes (IgG) prior to the administration of LPS. The macrophage population responsible for the augmented TNF-alpha secretion was determined by isolating Kupffer cells, splenic macrophages and peritoneal macrophages from mice that had been given ElgG prior to LPS and determining TNF-alpha secretion ex vivo. The intravenous injection of ElgG augmented LPS-stimulate TNF-alpha secretion by Kupffer cells and splenic macrophages. In contrast, LPS-stimulated TNF-alpha secretion by peritoneal macrophages was not altered by either the intravenous or intraperitoneal injection of ElgG. In vitro phagocytosis of ElgG by isolated peritoneal macrophages also did not augment LPS-stimulated TNF-alpha secretion. These results show that FcgammaR ligation augments LPS-stimulated TNF-alpha secretion by Kupffer cells and splenic macrophages but not by peritoneal macrophages. Thus, the ability of FcgammaR ligation to influence TNF-alpha secretion may be specific to the tissue source of the macrophages.
Assuntos
Fígado/metabolismo , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Baço/efeitos dos fármacosRESUMO
Fc gamma receptor (Fc gammaR) signaling mediates several important macrophage functions including cytokine secretion and respiratory burst. The present study describes the development of a model using the macrophage cell line, RAW 264.7 for studying Fc gammaR-stimulated tumor necrosis factor-alpha (TNF-alpha) secretion and hydrogen peroxide (H2O2) production. In unprimed cells these functions were low but pretreatment with interferon-gamma augmented Fc gammaR-stimulated TNF-alpha secretion and H2O2 production to levels that were about half that caused by lipopolysaccharide (LPS) and zymosan, respectively. Studies on the signaling pathways found that TNF-alpha secretion stimulated by either Fc gammaR or LPS was decreased by inhibitors of PKC, MAPK p42/p44, and MAPK p38. TNF-alpha secretion was also reduced by the combination of PLC and PLD inhibitors but not by the individual inhibitors alone. H2O2 production stimulated by either Fc gammaR or zymosan was blocked by inhibitors of PKC, PLC, PLD, and MAPK p42/44 but not by MAPK p38. Thus, interferon-gamma treated RAW 264.7 cells are a model of inflammatory macrophages and are well suited for further study of these signaling pathways.
Assuntos
Macrófagos/metabolismo , Receptores de IgG/fisiologia , Explosão Respiratória/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Estrenos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Pirrolidinonas/farmacologia , Explosão Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
In RAW 264.7 cells, PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-εâ»/â» cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-εâ»/â» macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-εâ»/â» macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-εâ»/â» BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.
Assuntos
Membrana Celular/metabolismo , Imunoglobulina G/farmacologia , Macrófagos/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Proteína Quinase C-épsilon/fisiologia , Pseudópodes/fisiologia , Animais , Células da Medula Óssea/metabolismo , Citometria de Fluxo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Técnicas de Patch-Clamp , Plasmídeos , Transporte Proteico , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Atherosclerosis is responsible for the death of thousands of Americans each year. The carotid constriction model of plaque development has recently been presented as a model for unstable plaque formation in mice. In this study we 1) validate ultrasound biomicroscopy (UBM) for the determination of carotid plaque size, percent stenosis, and plaque development in live animals, 2) determine the sensitivity of UBM in detecting changes in blood flow induced by carotid constriction and 3) test whether plaque formation can be predicted from blood flow parameters measured by UBM. Carotid plaques were induced by surgical constriction in Apo Eâ»/â» mice. Arteries were imaged bi-weekly by UBM, at which time PW-Doppler measurements of proximal blood flow, as well as plaque length and percent stenosis were determined. Histology was performed 9 weeks post surgery. When compared to whole mount post-mortem measurements, UBM accurately reported carotid plaque length. Percent stenosis, based on transverse B-mode UBM measurements, correlated well with that calculated from histological sections. PW-Doppler revealed that constriction reduced maximum systolic velocity (v(max)) and duration of the systolic velocity peak (t(s)/t(t)). Pre-plaque (2 week post-surgery) PW-Doppler parameters (v(max) and t(s)/t(t)) were correlated with plaque length at 9 weeks, and were predictive of plaque formation. Correlation of initiating PW-Doppler parameters (v(max) and t(s)/t(t)) with resulting plaque length established the degree of flow disturbance required for subsequent plaque development and demonstrated its power for predicting plaque development.
Assuntos
Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/patologia , Determinação de Ponto Final , Microscopia Acústica/métodos , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/patologia , Animais , Velocidade do Fluxo Sanguíneo/fisiologia , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/fisiopatologia , Progressão da Doença , Implantes Experimentais , Camundongos , Camundongos Endogâmicos C57BL , Sensibilidade e Especificidade , Ultrassonografia Doppler em CoresRESUMO
Protein kinase C (PKC) is a family of kinases that are implicated in a plethora of diseases, including cancer and cardiovascular disease. PKC isoforms can have different, and sometimes opposing, effects in these disease states. Toll-like receptors (TLRs) are a family of pattern recognition receptors that bind pathogens and stimulate the secretion of cytokines. It has long been known that PKC inhibitors reduce LPS-stimulated cytokine secretion by macrophages, linking PKC activation to TLR signaling. Recent studies have shown that PKC-α, -δ, -ε, and -ζ are directly involved in multiple steps in TLR pathways. They associate with the TLR or proximal components of the receptor complex. These isoforms are also involved in the downstream activation of MAPK, RhoA, TAK1, and NF-κB. Thus, PKC activation is intimately involved in TLR signaling and the innate immune response.
RESUMO
Modulation of the foreign body reaction is considered to be an important step toward creation of implanted sensors with reliable long-term performance. In this work, microdialysis probes were implanted into the subcutaneous space of Sprague-Dawley rats. The probe performance was evaluated by comparing collected endogenous glucose concentrations with internal standard calibration (2-deoxyglucose, antipyrine, and vitamin B12). Probes were tested until failure, which for this work was defined as loss of fluid flow. In order to determine the effect of fibrous capsule formation on probe function, monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) was delivered locally via the probe to increase capsule thickness and dexamethasone 21-phosphate was delivered to reduce capsule thickness. Probes delivering MCP-1 had a capsule that was twice the thickness (500-600 µm) of control probes (200-225 µm) and typically failed 2 days earlier than control probes. Probes delivering dexamethasone 21-phosphate had more fragile capsules and the probes typically failed 2 days later than controls. Unexpectedly, extraction efficiency and collected glucose concentrations exhibited minor differences between groups. This is an interesting result in that the foreign body capsule formation was related to the duration of probe function but did not consistently relate to probe calibration.
Assuntos
Automonitorização da Glicemia/métodos , Sistemas de Liberação de Medicamentos/métodos , Reação a Corpo Estranho/terapia , Microdiálise/instrumentação , Animais , Antipirina/análise , Calibragem , Quimiocina CCL2/uso terapêutico , Desoxiglucose/análise , Dexametasona/administração & dosagem , Dexametasona/análogos & derivados , Reação a Corpo Estranho/prevenção & controle , Masculino , Modelos Estatísticos , Ratos , Ratos Sprague-Dawley , Vitamina B 12/análise , Xenobióticos/farmacologiaRESUMO
Stroke is a leading cause of death in the United States. As â¼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases (MMP, higher in vulnerable), MMP modulators (inhibitors: lower, activators: higher in vulnerable), activating Fc receptors (FcγR, higher in vulnerable) and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions. In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR â macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated.
Assuntos
Biomarcadores/metabolismo , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Macrófagos/metabolismo , Receptores de IgG/genética , Idoso , Complexo Antígeno-Anticorpo , Doenças das Artérias Carótidas/metabolismo , Feminino , Humanos , Ligadura , Macrófagos/citologia , Masculino , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/genética , Receptores de IgG/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The level at which implanted sensors and sampling devices maintain their calibration is an important research area. In this work, microdialysis probes with identical geometry and different membranes, polycarbonate/polyether (PC) or polyethersulfone (PES), were used with internal standards (Vitamin B(12) (MW 1355), antipyrine (MW 188) and 2-deoxyglucose (2-DG, MW 164)) and endogenous glucose to investigate changes in their long-term calibration after implantation into the subcutaneous space of Sprague-Dawley rats. Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for antipyrine and 2-DG was not altered throughout the implant lifetime for either PC- or PES membranes. Yet, Vitamin B(12) extraction efficiency and collected glucose concentrations decreased during the implant lifetime. Antipyrine was administered i.v. and its concentrations obtained in both PC- and PES-membrane probes were significantly reduced between the implant day and seven (PC) or 10 (PES) days post-implantation suggesting that solute supply is critical for in vivo extraction efficiency. For the low molecular weight solutes such as antipyrine and glucose, localized delivery is not affected by the foreign body reaction, but recovery is significantly reduced. For Vitamin B(12), a larger solute, the fibrotic capsule formed around the probe significantly restricts diffusion from the implanted microdialysis probes.
Assuntos
Técnicas Biossensoriais , Calibragem/normas , Microdiálise , Animais , Antipirina/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/normas , Desoxiglucose/metabolismo , Glucose/análise , Implantes Experimentais , Injeções Subcutâneas , Microdiálise/instrumentação , Microdiálise/métodos , Microdiálise/normas , Polímeros/química , Polímeros/metabolismo , Ratos , Ratos Sprague-Dawley , Vitamina B 12/metabolismoRESUMO
Microdialysis sampling is a method that has promise for collection of important signaling proteins such as cytokines that are involved in every aspect of the immune response. The objective of this study was to determine the role of membrane and tissue alterations on the reduction of interleukin-6 (IL-6) relative recovery of microdialysis probes implanted for 3 and 7 days versus probes implanted on day 0 (acute implant or control probe). Lipopolysaccharide (LPS), a bacterial endotoxin, was used to elicit IL-6 production in the animals. Within the same animal, the recovery of IL-6 through control probes implanted the day of sample collection was compared to the 3- or 7-day implanted probes. Two hours post-LPS administration, the IL-6 concentrations obtained from either the 3-day or 7-day implanted probe were reduced by more than 8-fold when compared to the control probe. The IL-6 concentrations obtained for the 3-day versus control probes 2-h post-LPS injection were 730 +/- 310 and 6440 +/- 1550 pg/mL (mean +/- SD, n = 3), respectively. For the 7-day implant, the IL-6 concentration in the dialysis probe obtained at 2-h post-LPS injection was 990 +/- 590 versus 5520 +/- 1430 pg/mL (mean +/- SD, n = 3) for the control. In vitro recovery experiments and scanning electron microscopy images combined with the in vivo data suggest that the decreased IL-6 content in the dialysate was caused principally by tissue alterations or tissue encapsulation rather than membrane blockage with biological components (membrane biofouling).
Assuntos
Interleucina-6/isolamento & purificação , Microdiálise/métodos , Pele/imunologia , Animais , Membrana Celular/ultraestrutura , Lipopolissacarídeos/imunologia , Microscopia Eletrônica de Varredura , Ratos , Pele/ultraestruturaRESUMO
The virulence of Francisella tularensis LVS is determined in part by its ability to invade and replicate within macrophages and stimulate the production of inflammatory cytokines. The present study determined the effects of growing F. tularensis in macrophages on its ability to stimulate cytokine secretion by macrophages. F. tularensis grown in Mueller-Hinton broth (FtB) stimulated the secretion of large amounts of TNF-alpha, IL-12p40, IL-6 and MCP-1/CCL2 when incubated with macrophages overnight. In contrast, F. tularensis released from infected macrophages (FtMac) stimulated very little secretion of these cytokines by primary cultures of murine peritoneal macrophages, human monocytes or macrophage cell lines. Stimulation of nitric oxide production by FtMac was also less than that elicited by FtB. FtMac killed with gentamicin or paraformaldehyde also stimulated low levels of cytokine secretion. FtMac recovered the ability to stimulate cytokine secretion after overnight culture in broth. Infection of macrophages with FtMac inhibited the cytokine response to subsequent stimulation with LPS from Escherichia coli but did not affect Fcgamma receptor-mediated phagocytosis. FtMac were ingested by macrophages at about half the rate of FtB, however, this did not account for the lower cytokine secretion. FtMac and FtB replicated at similar rates within macrophages. Finally, Mice infected with FtMac had a higher mortality rate than those infected with FtB. These results reveal that growth in macrophages causes a reversible phenotypic change in F. tularensis that is associated with decreased stimulation of cytokine secretion, inhibition of LPS-stimulated secretion of inflammatory cytokines by macrophages and increased lethality in mice.