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1.
Can J Microbiol ; 63(1): 35-45, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27900876

RESUMO

The goal of this study was to use a microencapsulation technology to prepare air-dried concentrated cultures of Lactobacillus rhamnosus R0011. The cultures were microencapsulated in alginate beads, which were added to a growth medium to allow cell multiplication inside the matrix; the beads were recovered, dipped in protective solutions, and air-dried. The effects of fermentation technology and of the composition of the protective solutions on subsequent survival during air-drying were examined. The cells prepared under a constant pH of 6.2 had only 2.5% survival to air-drying at 25 °C when the protective solution was composed of sucrose and phosphate. Allowing the pH to drop to 4.2 during the biomass production step and using a protective medium composed of glycerol, maltodextrin, yeast extract, and ascorbate increased survival to 20%. If the ingredients of the protective medium at the beginning of drying were concentrated at a water activity of 0.96 rather than 0.98, survival during air-drying increased further to 56%. This rate was similar to that of a traditional freeze-drying process. These data suggest that applying a combination of acid and osmotic stresses to L. rhamnosus R0011 cells improves their subsequent stability during the air-drying process. Dried microencapsulated cultures having 2.6 × 1011 CFU·g-1 were obtained.


Assuntos
Alginatos/química , Composição de Medicamentos/métodos , Lactobacillus rhamnosus/química , Lactobacillus rhamnosus/crescimento & desenvolvimento , Probióticos/química , Meios de Cultura/metabolismo , Composição de Medicamentos/instrumentação , Fermentação , Liofilização , Lactobacillus rhamnosus/metabolismo , Viabilidade Microbiana
2.
Food Microbiol ; 46: 176-183, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25475282

RESUMO

The goals of this study were to evaluate the precision and accuracy of flow cytometry (FC) methodologies in the evaluation of populations of probiotic bacteria (Lactobacillus rhamnosus R0011) in two commercial dried forms, and ascertain the challenges in enumerating them in a chocolate matrix. FC analyses of total (FC(T)) and viable (FC(V)) counts in liquid or dried cultures were almost two times more precise (reproducible) than traditional direct microscopic counts (DCM) or colony forming units (CFU). With FC, it was possible to ascertain low levels of dead cells (FC(D)) in fresh cultures, which is not possible with traditional CFU and DMC methodologies. There was no interference of chocolate solids on FC counts of probiotics when inoculation was above 10(7) bacteria per g. Addition of probiotics in chocolate at 40 °C resulted in a 37% loss in viable cells. Blending of the probiotic powder into chocolate was not uniform which raised a concern that the precision of viable counts could suffer. FCT data can serve to identify the correct inoculation level of a sample, and viable counts (FCV or CFU) can subsequently be better interpreted.


Assuntos
Cacau/microbiologia , Citometria de Fluxo/métodos , Lactobacillus rhamnosus/citologia , Lactobacillus rhamnosus/crescimento & desenvolvimento , Viabilidade Microbiana
3.
Can J Microbiol ; 60(5): 287-95, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24773334

RESUMO

The aim of this study is to evaluate the effects of defatted colostrum (Col), defatted decaseinated colostrum whey, cheese whey, and spray-dried porcine plasma (SDPP) as supplements of a growth medium (de Man - Rogosa - Sharpe (MRS) broth) on the multiplication of lactic acid bacteria, probiotic bacteria, and potentially pathogenic Escherichia coli. Using automated spectrophotometry (in vitro system), we evaluated the effect of the 4 supplements on maximum growth rate (µ(max)), lag time (LagT), and biomass (OD(max)) of 12 lactic acid bacteria and probiotic bacteria and of an E. coli culture. Enrichment of MRS broth with a Col concentration of 10 g/L increased the µ(max) of 5 of the 12 strains by up to 55%. Negative effects of Col or SDPP on growth rates were also observed with 3 probiotic strains; in one instance µ(max) was reduced by 40%. The most effective inhibitor of E. coli growth was SDPP, and this effect was not linked to its lysozyme content. The positive effect of enrichment with the dairy-based ingredient might be linked to enrichment in sugars and increased buffering power of the medium. These in vitro data suggest that both Col and SDPP could be considered as supplements to animal feeds to improve intestinal health because of their potential to promote growth of probiotic bacteria and to inhibit growth of pathogenic bacteria such as E. coli.


Assuntos
Ração Animal , Bactérias/crescimento & desenvolvimento , Meios de Cultura/química , Escherichia coli/crescimento & desenvolvimento , Probióticos , Animais , Bovinos , Queijo , Colostro , Suplementos Nutricionais , Feminino , Concentração de Íons de Hidrogênio , Muramidase/farmacologia , Plasma , Suínos , Tetraciclina/farmacologia
4.
Int J Food Sci ; 2014: 749630, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-26904647

RESUMO

The goal of the study was to compare the dissolution of chocolate to other lipid-based matrices suitable for the microencapsulation of bioactive ingredients in simulated gastrointestinal solutions. Particles having approximately 750 µm or 2.5 mm were prepared from the following lipid-based matrices: cocoa butter, fractionated palm kernel oil (FPKO), chocolate, beeswax, carnauba wax, and paraffin. They were added to solutions designed to simulate gastric secretions (GS) or duodenum secretions (DS) at 37°C. Paraffin, carnauba wax, and bees wax did not dissolve in either the GS or DS media. Cocoa butter, FPKO, and chocolate dissolved in the DS medium. Cocoa butter, and to a lesser extent chocolate, also dissolved in the GS medium. With chocolate, dissolution was twice as fast as that with small particles (750 µm) as compared to the larger (2.5 mm) ones. With 750 µm particle sizes, 90% dissolution of chocolate beads was attained after only 60 minutes in the DS medium, while it took 120 minutes for 70% of FPKO beads to dissolve in the same conditions. The data are discussed from the perspective of controlled release in the gastrointestinal tract of encapsulated ingredients (minerals, oils, probiotic bacteria, enzymes, vitamins, and peptides) used in the development of functional foods.

5.
Appl Microbiol Biotechnol ; 95(3): 745-56, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22350318

RESUMO

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a (w)) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a (w) = 0.96 or 0.99 were then recovered by centrifugation, added to a sucrose-phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a (w) = 0.96. Growing the cells in complex media adjusted at a (w) = 0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose-phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.


Assuntos
Bifidobacterium/fisiologia , Dessecação , Lactobacillus/fisiologia , Viabilidade Microbiana , Água/química , Ar , Bifidobacterium/metabolismo , Meios de Cultura/química , Glicerol/metabolismo , Lactobacillus/metabolismo , Pressão Osmótica , Cloreto de Sódio/metabolismo , Sacarose/metabolismo
6.
Arthritis Rheum ; 64(3): 826-34, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21953548

RESUMO

OBJECTIVE: Previous studies have demonstrated that, once released into the extracellular environment, the systemic sclerosis (SSc)-associated autoantigen DNA topoisomerase I (topo I) binds specifically to the surface of fibroblasts via an unknown receptor. We extended these results by identifying topo I-mediated cellular effects and characterizing the specific target of topo I on fibroblast surfaces. METHODS: Purified topo I was used to investigate intracellular signaling pathway activation and tested for cell migration. To demonstrate the expression of specific chemokine receptors on fibroblasts, we performed immunoblotting and flow cytometry. To evaluate the direct interaction between chemokine receptor and topo I, a protein-protein based enzyme-linked immunosorbent assay (ELISA) was used. Finally, topo I coupled to the fluorochrome phycoerythrin (PE) was used to investigate competition of topo I specific binding on fibroblast surfaces with chemokine ligand. RESULTS: Topo I stimulated the phosphorylation of phospholipase Cγ1, c-Raf, ERK-1/2, and p38 MAPK, intracellular signaling pathways that stimulated fibroblast migration via a G(αi) protein-coupled receptor. CCR7 was found to interact directly with topo I. Furthermore, its ligand, CCL21, competed in vitro for this interaction and in vivo with the binding of PE-coupled topo I to fibroblast surfaces. CONCLUSION: These new roles of topo I in fibroblast physiology and the identification of its target on the cell surface demonstrate that topo I is a bifunctional autoantigen and open up new perspectives of study in the field of SSc-associated anti-topo I autoantibodies.


Assuntos
DNA Topoisomerases Tipo I/farmacologia , Fibroblastos/efeitos dos fármacos , Receptores CCR7/efeitos dos fármacos , Escleroderma Sistêmico , Autoanticorpos/imunologia , Autoantígenos/imunologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL21/imunologia , Quimiocina CCL21/metabolismo , DNA Topoisomerases Tipo I/imunologia , DNA Topoisomerases Tipo I/metabolismo , Derme/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores CCR7/imunologia , Receptores CCR7/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Arthritis Rheum ; 64(5): 1632-41, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22095242

RESUMO

OBJECTIVE: Previous studies have demonstrated that the systemic sclerosis (SSc)-associated autoantigen DNA topoisomerase I (topo I) binds specifically to the surface of fibroblasts when released in the extracellular environment and recruits anti-topo I autoantibodies, which subsequently leads to the adhesion and activation of monocytes. This study aimed to characterize the molecular interactions of topo I with fibroblast surfaces in order to elucidate the pathogenic role of topo I/anti-topo I immune complexes (ICs) in SSc. METHODS: Topo I directly coupled to fluorochromes was used to follow its binding to fibroblast surfaces by flow cytometry and fluorescence microscopy. Purified IgG from normal subjects or SSc patients was added with topo I to the cells; unfractionated heparin (UFH) and low molecular weight heparin (LMWH) were used to determine their effects on the binding of topo I and topo I/anti-topo I IC to fibroblast surfaces. RESULTS: Heparan sulfate (HS) proteoglycans on fibroblast surfaces were found to act as coreceptors for topo I binding. The addition of anti-topo I autoantibodies from SSc sera led to the amplification of topo I binding to HS chains. UFH and LMWH were shown to inhibit topo I and topo I/anti-topo I IC binding to HS chains. CONCLUSION: This study is the first to show that topo I binds specifically to HS proteoglycans on fibroblast surfaces and that anti-topo I autoantibodies from SSc patients amplify topo I binding to HS chains. The accumulation of topo I on cell surfaces by anti-topo I autoantibodies could contribute to the initiation of an inflammatory cascade stimulating the fibrosis. UFH and LMWH inhibited the binding of topo I/anti-topo I IC to fibroblasts, suggesting a potential therapeutic role in SSc-associated fibrosis.


Assuntos
Complexo Antígeno-Anticorpo/efeitos dos fármacos , DNA Topoisomerases Tipo I/imunologia , Fibrinolíticos/farmacologia , Fibroblastos/efeitos dos fármacos , Heparina/farmacologia , Heparitina Sulfato/imunologia , Complexo Antígeno-Anticorpo/imunologia , Antígenos de Superfície/imunologia , Autoanticorpos/imunologia , Autoanticorpos/farmacologia , Células Cultivadas , Derme/patologia , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Ligação Proteica , Escleroderma Sistêmico/imunologia
8.
J Cell Physiol ; 226(7): 1907-14, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21506121

RESUMO

Our hypothesis is that the development of lesional areas of skin in patients with systemic sclerosis (SSc) originates from the selection of profibrotic cell subpopulations within their non-lesional skin areas, due to their greater resistance to apoptosis. Sensitivity to apoptosis of early-stage or late-stage SSc fibroblasts as well as of healthy cells was compared using extrinsic or intrinsic apoptotic pathway-inducers. Subpopulations of non-lesional SSc cells and healthy cells obtained after repeated Fas-induced apoptosis were compared with respect to their fibrotic parameters such as collagen and MMP secretion. Only late-stage lesional SSc cells were more resistant to Fas-induced apoptosis than their non-lesional counterparts isolated from the same patient. This result correlated with an increase in the levels of the anti-apoptotic proteins cFLIPs and cIAP in lesional cells compared to non-lesional cells. Healthy and non-lesional cell populations could be selected to generate a subpopulation that was more resistant to apoptosis. However, only the late-stage non-lesional SSc fibroblast populations showed a significant decrease in MMP secretion, one of parameters of the fibrosis. Our results show that resistance to apoptosis is an important characteristic of the late-stage lesional SSc fibroblast phenotype. We thus hypothesized that a selection of specific fibroblast subpopulations from late-stage non-lesional SSc skin areas could be at the origin of lesional populations. These cells should become independent of any exogenous stimuli and can induce or maintain SSc skin lesions.


Assuntos
Apoptose , Proteína Ligante Fas/metabolismo , Fibroblastos/enzimologia , Metaloproteinases da Matriz Secretadas/metabolismo , Esclerodermia Difusa/enzimologia , Transdução de Sinais , Pele/enzimologia , Adulto , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Regulação para Baixo , Fibroblastos/patologia , Fibrose , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Pessoa de Meia-Idade , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Esclerodermia Difusa/diagnóstico , Esclerodermia Difusa/patologia , Pele/patologia , Receptor fas/metabolismo
9.
Food Microbiol ; 27(8): 1104-11, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20832691

RESUMO

An assessment of various methods to determine viable counts (CFU) in freeze-dried and dried microencapsulated (ME) probiotic cultures was carried out. Microencapsulation was done by spray-coating of dried Lactobacillus rhamnosus R0011 or Bifidobacterium longum ATCC 15708 cultures with fat. Rehydration of the ME powders was incomplete when they were added to water and gently agitated. As a result analytical methods based on vortexing of rehydrated ME cultures and which did not incorporate a high-shear homogenization (HSH) step underestimated the viable counts. The CFU of ME cultures were identical when methods using either blender or generator probes high-shear homogenization (HSH) were carried out. Furthermore HSH reduced the variability of the CFU results of both free-cell and ME cultures by a factor of three. The addition of an emulsifier (Tween 80) in the rehydrating medium to dissolve fat did not improve CFU counts when generator probes were used for HSH. The presence of fat in the ME product, or when added to the rehydration medium, improved CFU counts of B. longum but not of L. rhamnosus.


Assuntos
Bifidobacterium/crescimento & desenvolvimento , Contagem de Colônia Microbiana/métodos , Composição de Medicamentos , Lactobacillus rhamnosus/crescimento & desenvolvimento , Viabilidade Microbiana , Probióticos/química , Bifidobacterium/química , Liofilização , Lactobacillus rhamnosus/química , Tamanho da Partícula
10.
Arthritis Rheum ; 60(9): 2805-16, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19714638

RESUMO

OBJECTIVE: We have previously found that the CENP-B nuclear autoantigen, which is specifically targeted by autoantibodies in the limited cutaneous form of systemic sclerosis, behaved as a potent migratory factor for human pulmonary artery smooth muscle cells (PASMCs). Other recent studies have shown that several disease-associated autoantigens induced cell migration by interacting with various chemokine receptors. Prompted by this hypothesis, we undertook this study to determine whether CENP-B interacts with chemokine receptors on the surface of human PASMCs, to explore the relevant signaling pathways, and to characterize the effects of anti-CENP-B binding on SMC stimulation. METHODS: To demonstrate the expression of specific chemokine receptors by human PASMCs at both the messenger RNA and protein levels, reverse transcription-polymerase chain reaction, immunoblotting, and flow cytometry analyses were performed. Desensitization studies and specific inhibitors were used to further identify the CENP-B target on the surface of human PASMCs. RESULTS: Our data strongly suggested that CENP-B used chemokine receptor 3 (CCR3) to mediate human PASMCs signaling. Moreover, several lines of evidence indicated that CENP-B binding subsequently stimulated the cross-talk between CCR3 and epidermal growth factor receptor (EGFR) via a matrix metalloprotease-dependent mechanism that involved the processing of heparin-binding EGF-like growth factor. Transactivation of the EGFR through CCR3 was found to be a critical pathway that elicits MAP kinase activation and secretion of cytokines such as interleukin-8. Finally, anti-CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular smooth muscle cells. CONCLUSION: The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti-CENP-B autoantibodies.


Assuntos
Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Receptores ErbB/metabolismo , Músculo Liso Vascular/metabolismo , Receptores CCR3/metabolismo , Ativação Transcricional/fisiologia , Autoanticorpos/farmacologia , Células Cultivadas , Humanos , Interleucina-8/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Artéria Pulmonar/citologia , RNA Mensageiro/metabolismo , Receptor Cross-Talk/fisiologia , Escleroderma Sistêmico/metabolismo , Transdução de Sinais/fisiologia
11.
J Pathol ; 217(4): 534-42, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19086038

RESUMO

We set out to examine the pathophysiological mechanisms of fibrosis in diffuse systemic sclerosis (SSc) using a tissue engineering approach. Skin fibroblasts were isolated from lesional skin of SSc patients with a disease duration of less than 1 year (early-stage SSc) or more than 10 years (late-stage SSc). Fibroblasts were also isolated from non-lesional skin and compared with normal fibroblasts isolated from healthy adults. Cells were cultured using a tissue engineering method to reconstruct a human dermis, and histologically observed. Dermal thickness was measured, as it reflects the global and intrinsic capacity of cells to reconstitute matrix. Collagen I, MMP-1, and MMP activity were evaluated. Cells were treated with TGFbeta1 or CTGF during dermis formation to study their fibrogenic role. Clinical severity of skin involvement was measured by a modified Rodnan score. Thickness of the dermis generated with non-lesional early-stage SSc fibroblasts was similar to normal cells. In contrast, reconstructed dermis from lesional early-stage SSc fibroblasts and non-lesional late-stage SSc cells was thinner, while lesional late-stage SSc fibroblasts made a thicker dermis. Dermis was always thicker when produced with TGFbeta1-treated cells, except when lesional late-stage SSc fibroblasts from patients with high Rodnan skin scores were used. CTGF did not affect dermal thickness. Measurements of collagen I and collagenases in the culture medium of the various reconstructed dermis could explain some of the changes observed. We conclude that the fibrotic phenotype of SSc fibroblasts varies with disease duration and with severity of skin involvement, and this is clearly visualized during in vitro dermis reconstruction.


Assuntos
Escleroderma Sistêmico/patologia , Pele/patologia , Adulto , Idoso , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/análise , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Progressão da Doença , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Masculino , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinases da Matriz/análise , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Fenótipo , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/farmacologia
12.
Arthritis Rheum ; 58(12): 3902-12, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19035499

RESUMO

OBJECTIVE: To identify in patients with Raynaud's phenomenon (RP) independent markers that predict progression to definite systemic sclerosis (SSc) and to determine in patients with progression to SSc the type and sequence of microvascular damage and its relationship to SSc-specific autoantibodies. METHODS: Consecutive patients referred for evaluation of RP who had no definite connective tissue disease were evaluated for microvascular damage by nailfold capillary microscopy (NCM) and for anticentromere (anti-CENP-B), anti-Th/To, anti-topoisomerase I, and anti-RNA polymerase III (anti-RNAP III) autoantibodies by specific assays. Patients were studied prospectively. RESULTS: Of the 586 patients who were followed up for 3,197 person-years, 74 (12.6%) developed definite SSc. A characteristic sequence of microvascular damage was identified, starting with enlarged capillaries, followed by capillary loss, and then by capillary telangiectases. Definite SSc was diagnosed in close temporal relationship to capillary loss. Enlarged capillaries, capillary loss, and SSc-specific autoantibodies independently predicted definite SSc. Anti-CENP-B and anti-Th/To antibodies predicted enlarged capillaries; these autoantibodies and anti-RNAP III predicted capillary loss. Each autoantibody was associated with a distinct time course of microvascular damage. At followup, 79.5% of patients with 1 of these autoantibodies and abnormal findings on NCM at baseline had developed definite SSc. Patients with both baseline predictors were 60 times more likely to develop definite SSc. The data validated the proposed criteria for early SSc. CONCLUSION: In RP evolving to definite SSc, microvascular damage is dynamic and sequential, while SSc-specific autoantibodies are associated with the course and type of capillary abnormalities. Abnormal findings on NCM at baseline together with an SSc-specific autoantibody indicate a very high probability of developing definite SSc, whereas their absence rules out this outcome.


Assuntos
Autoanticorpos/sangue , Microvasos/imunologia , Doença de Raynaud/imunologia , Doença de Raynaud/patologia , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Adulto , Especificidade de Anticorpos , Árvores de Decisões , Progressão da Doença , Diagnóstico Precoce , Feminino , Seguimentos , Humanos , Incidência , Masculino , Microvasos/patologia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Doença de Raynaud/classificação , Doença de Raynaud/epidemiologia , Escleroderma Sistêmico/classificação , Escleroderma Sistêmico/epidemiologia , Estudos Soroepidemiológicos , Adulto Jovem
13.
Arthritis Rheum ; 56(11): 3814-26, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17968937

RESUMO

OBJECTIVE: A growing number of intracellular autoantigenic polypeptides have been found to play a second biologic role when they are present in the extracellular medium. We undertook this study to determine whether the CENP-B nuclear autoantigen could be added to this set of bifunctional molecules. METHODS: Purified CENP-B or CENP-B released from apoptotic cells was tested for surface binding to a number of human cell types by cell-based enzyme-linked immunosorbent assay, flow cytometry, and indirect immunofluorescence. The biologic effects of CENP-B on the migration, interleukin secretion, and signaling pathways of its specific target cells were evaluated. RESULTS: CENP-B was found to bind specifically to the surface of human pulmonary artery smooth muscle cells (SMCs) and not to fibroblasts or endothelial cells (ECs). Furthermore, CENP-B bound preferentially to SMCs of the contractile type rather than to SMCs of the synthetic type. Binding of CENP-B to SMCs stimulated their migration during in vitro wound healing assays, as well as their secretion of interleukins 6 and 8. The mechanism by which CENP-B mediated these effects involved the focal adhesion kinase, Src, ERK-1/2, and p38 MAPK pathways. Finally, CENP-B released from apoptotic ECs was found to bind to SMCs, thus indicating a plausible in vivo source of extracellular CENP-B. CONCLUSION: These novel biologic roles of the nuclear autoantigen CENP-B open up a new perspective for studying the pathogenic role of anti-CENP-B autoantibodies.


Assuntos
Autoantígenos/imunologia , Proteína B de Centrômero/imunologia , Músculo Liso Vascular/imunologia , Miócitos de Músculo Liso/imunologia , Artéria Pulmonar/citologia , Adulto , Apoptose , Autoantígenos/metabolismo , Divisão Celular , Movimento Celular , Células Cultivadas , Proteína B de Centrômero/metabolismo , Citocinas/metabolismo , Epitopos , Fibroblastos/citologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Imunofenotipagem , Pulmão/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fosforilação , Ligação Proteica/imunologia , Quinases da Família src/metabolismo
14.
Arthritis Rheum ; 54(3): 963-73, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16508979

RESUMO

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis due to excessive and dysregulated collagen production by fibroblasts. Previously, we reported that anti-DNA topoisomerase I (anti-topo I) antibodies bound specifically to fibroblast surfaces; however, we had not identified their antigenic target. We undertook this study to characterize the target of anti-topo I antibodies on fibroblasts and the effects of their binding. METHODS: Purified topo I or topo I released from apoptotic cells was tested for surface binding to a number of human cell types by cell-based enzyme-linked immunosorbent assay, flow cytometry, and indirect immunofluorescence. Antibodies purified from SSc patient and normal control sera were used to detect topo I binding. The consequences of topo I and anti-topo I binding to fibroblasts were assessed by coculture with THP-1 monocytes. RESULTS: The autoantigen topo I itself was found to bind specifically to fibroblasts in a dose-dependent and saturable manner, where it was recognized by anti-topo I from SSc patients. The binding of anti-topo I subsequently stimulated adhesion and activation of cocultured monocytes. Topo I released from apoptotic endothelial cells was also found to bind specifically to fibroblasts. CONCLUSION: The findings of this study thus confirm and extend the findings of our previous study by showing that topo I binding to fibroblast surfaces is both necessary and sufficient for anti-topo I binding. Second, topo I-anti-topo I complex binding can then trigger the adhesion and activation of monocytes, thus providing a plausible model for the amplification of the fibrogenic cascade in anti-topo I-positive SSc patients.


Assuntos
Autoanticorpos/imunologia , Adesão Celular/imunologia , DNA Topoisomerases Tipo I/imunologia , Fibroblastos/imunologia , Monócitos/imunologia , Escleroderma Sistêmico/imunologia , Apoptose , Células Cultivadas , Eletroforese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Microscopia Confocal
16.
Medicine (Baltimore) ; 84(4): 231-49, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16010208

RESUMO

Our objective was to improve the currently imperfect classifications of idiopathic inflammatory myopathies (IIM). In clinical practice, overlap features are common in IIM. This provided a rationale for positioning overlap clinical features at the core of a new classification system. We conducted a longitudinal study of 100 consecutive adult French Canadian patients with IIM. Clinical and laboratory data were obtained by retrospective chart review. Sera were analyzed for autoantibodies (aAbs) by protein A-assisted immunoprecipitation and double immunodiffusion. Overlap aAbs encompassed aAbs to synthetases, systemic sclerosis-associated aAbs, anti-signal recognition particle (SRP) and anti-nucleoporins. Patients were classified both at IIM diagnosis, based on data at presentation, and at the end of follow-up, based on cumulative findings. Three classifications were used: 1) the Bohan and Peter original classification, 2) a new version of that classification as modified by us, and 3) a novel clinicoserologic classification. As investigators were blinded to aAb results, the modified classification is strictly a clinical classification. Its core concept is the attribution of diagnostic significance to the presence of overlap features, that is, their presence resulted in a diagnosis of overlap myositis (OM). This approach allowed direct comparison with the original Bohan and Peter classification. By integrating aAb results to the modified classification, we also defined the clinicoserologic classification, which allowed to examine the added value of aAbs to diagnostic, therapeutic and prognostic stratification. Whereas polymyositis (PM) was the most common IIM according to the original classification, accounting for 45% of the cohort at diagnosis, its frequency fell to 14% with the modified classification. Conversely, while the frequency of myositis associated with connective tissue disease was 24% according to the original classification, the frequency of OM was 60% when using the modified classification. At last follow-up, the frequency of PM fell further to only 9%, while the frequency of OM rose to 67%. Systemic sclerosis was the most common connective tissue disease associated with IIM, accounting for 42.6% of OM patients and 29% of the cohort. The frequencies of overlap aAbs in the cohort and in OM patients were 48% and 70.5% (n =48/68), respectively. The presence of overlap aAbs at IIM diagnosis identified additional OM patients unrecognized by the modified classification. The sensitivity of the modified classification for OM at diagnosis was 87%, suggesting that clinicians may rely on the modified classification for identification of most OM patients, while awaiting results of aAb assays. The new classifications predicted the response to prednisone and IIM course. Using stringent definitions, IIM was classified as responsive or refractory after an adequate initial corticosteroid therapy, and the disease course as monophasic or chronic after a single adequate trial of prednisone. PM was always chronic and was associated with the highest rate (50%) of refractoriness to initial corticosteroid treatment. Dermatomyositis was almost always chronic (92% rate); however, its responsiveness to initial corticosteroid treatment was high (87%). OM was almost always responsive to corticosteroids (89%-100% rates). When OM patients were divided according to aAb subsets, anti-synthetase, SRP, or nucleoporin aAbs were markers for chronic myositis, whereas aAbs to U1RNP, Pm-Scl, or Ku were markers for monophasic myositis. We conclude that the original Bohan and Peter classification should be abandoned as it leads to misclassification of patients. Much of IIM is composed of OM. The proposed modified and clinicoserologic classifications have diagnostic, prognostic, and therapeutic value.


Assuntos
Autoanticorpos/análise , Miosite/classificação , Adolescente , Adulto , Idoso , Anti-Inflamatórios/uso terapêutico , Estudos de Coortes , Dermatomiosite/classificação , Dermatomiosite/imunologia , Feminino , Seguimentos , Humanos , Ligases/imunologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Miosite/imunologia , Complexo de Proteínas Formadoras de Poros Nucleares/imunologia , Polimiosite/classificação , Polimiosite/imunologia , Prednisona/uso terapêutico , Quebeque , Estudos Retrospectivos , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/imunologia , Partícula de Reconhecimento de Sinal/imunologia , Método Simples-Cego , Síndrome , Resultado do Tratamento
17.
J Immunol ; 174(9): 5740-9, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15843576

RESUMO

Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-x(L) overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-x(L) protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of alpha-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.


Assuntos
Apoptose/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/fisiopatologia , Transdução de Sinais/imunologia , Adulto , Sequência de Aminoácidos , Diferenciação Celular/imunologia , Linhagem Celular , Sulfatos de Condroitina/farmacologia , Meios de Cultivo Condicionados , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Fibroblastos/imunologia , Proteoglicanas de Heparan Sulfato/fisiologia , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Estrutura Terciária de Proteína , Escleroderma Sistêmico/imunologia
19.
Arthritis Rheum ; 50(10): 3265-74, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15476238

RESUMO

OBJECTIVE: Fibroblasts play a crucial role in the development of systemic sclerosis (SSc), and antifibroblast antibodies (AFAs) capable of inducing a proinflammatory phenotype in fibroblasts have been detected in the sera of SSc patients. This study examined the prevalence of AFAs in SSc and other diseases and the possible correlation between AFAs and known antinuclear antibody specificities in SSc patients. METHODS: Sera from 99 patients with SSc, 123 patients with other autoimmune and nonautoimmune diseases, and 30 age- and sex-matched healthy controls were examined. AFA prevalence was assessed by flow cytometry and further characterized by indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Anti-topoisomerase I (anti-topo I) from SSc sera were purified by affinity chromatography on topo I. RESULTS: AFAs were more common in SSc patients (26.3%) than in any other disease groups studied. The presence of AFA was significantly associated with pulmonary involvement and death. AFA-positive sera from SSc patients bound to all human and rodent fibroblasts tested, but not to human primary endothelial cells or smooth muscle cells. All SSc AFAs strongly reacted with topo I by ELISA and immunoblotting. The binding intensity of SSc AFAs correlated strongly with reactivity against topo I on immunoblots of fibroblast extracts and with the immunofluorescence pattern typical of anti-topo I on permeabilized cells. Total IgG and affinity-purified anti-topo I from AFA-positive SSc sera were found to react with the surface of unpermeabilized fibroblasts by flow cytometry as well as by immunofluorescence and confocal microscopy. CONCLUSION: This is the first report establishing that AFAs in SSc are strongly correlated with anti-topo I and, furthermore, that anti-topo I antibodies themselves display AFA activity by reacting with determinants at the fibroblast surface.


Assuntos
Antígenos de Superfície/imunologia , Autoanticorpos/imunologia , DNA Topoisomerases Tipo I/imunologia , Fibroblastos/imunologia , Escleroderma Sistêmico/imunologia , Células Endoteliais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Pulmão/fisiopatologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Miócitos de Músculo Liso/imunologia , Escleroderma Sistêmico/fisiopatologia
20.
Arthritis Rheum ; 50(10): 3221-31, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15476243

RESUMO

OBJECTIVE: To determine whether anti-endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted. METHODS: AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity-purified on this antigen and incubated with ECs to determine their physiologic effects. Anti-Hsp60 antibody titers were determined by enzyme-linked immunosorbent assay. The relationship of anti-Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed. RESULTS: Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60-kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti-Hsp60 antibodies from commercial sources or affinity-purified from SLE sera that bound ECs. Incubation of ECs with these anti-Hsp60 antibodies induced apoptosis in a time- and dose-dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti-Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti-Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients. CONCLUSION: The presence of Hsp60 at the surface of ECs serves as a target for the anti-Hsp60 antibodies in SLE sera. These anti-Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.


Assuntos
Apoptose/imunologia , Autoanticorpos/imunologia , Chaperonina 60/imunologia , Células Endoteliais/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/análise , Inibidor de Coagulação do Lúpus/análise , Masculino , Pessoa de Meia-Idade , Trombose/imunologia
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