RESUMO
BACKGROUND: IL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the "nucleopod," which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators. OBJECTIVE: Determine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin. METHODS: Blood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed. RESULTS: After 10 minutes in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30-60 minutes, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. CONCLUSIONS AND CLINICAL RELEVANCE: Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target.
Assuntos
Proteínas ADAM/metabolismo , Moléculas de Adesão Celular/metabolismo , Quimiotaxia de Leucócito/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Interleucina-5/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/antagonistas & inibidores , Adesão Celular , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Imunofluorescência , Humanos , Interleucina-5/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Tiofenos/farmacologiaRESUMO
BACKGROUND: Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. OBJECTIVE: We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. RESULTS: Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. CONCLUSIONS AND CLINICAL RELEVANCE: We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.
Assuntos
Degranulação Celular/imunologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Interleucina-3/metabolismo , Antígeno de Macrófago 1/metabolismo , Receptores de IgG/metabolismo , Anticorpos Monoclonais/farmacologia , Biomarcadores , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Eosinófilos/efeitos dos fármacos , Humanos , Interleucina-3/farmacologia , Receptores de IgG/antagonistas & inibidoresRESUMO
Activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) is characterized by increased expression and activator of signal transducer and activator of transcription 3 (STAT3). ABC DLBCL cells require STAT3 for growth in culture. In ABC DLBCL cells, eosinophils and perhaps all cells, four variant STAT3 mRNAs (Sα, ΔSα, Sß and ΔSß) are present as a result of two alternative splicing events, one that results in the inclusion of a 55-residue C-terminal transactivation domain (α) or a truncated C-terminal domain with 7 unique residues (ß) and a second that includes (S) or excludes (ΔS) the codon for Ser-701 in the linker between the SH2 and C-terminal domains. A substantial literature indicates that both α and ß variants are required for optimal STAT3 function, but nothing is known about functions of ΔS variants. We used a knockdown/re-expression strategy to explore whether survival of ABC DLBCL cells requires that the four variants be in an appropriate ratio. No single variant rescued survival as well as STAT3Sα-C, Sα with activating mutations (A661C and N663C) in the SH2 domain. Better rescue was achieved when all four variants were re-expressed or Sα and ΔSα or Sß and ΔSß were re-expressed in pairs. Rescue correlated with expression of STAT3-sensitive genes NFKBIA and NFKBIZ. We consider a variety of explanations why a mix of S and ΔS variants of STAT3 should enable survival of ABC DLBCL cells.
RESUMO
BACKGROUND: IL-5 activates α(M) ß(2) integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated ß(2) -integrins. OBJECTIVE: To identify roles for IL-5 in regulating human eosinophil integrins in vivo. METHODS: Blood and BAL eosinophils were analysed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. RESULTS: Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated ß(2) -integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil ß(2) , α(M) and α(L) integrin subunits increased modestly post challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of ß(2) , α(M) , α(L) and α(D) and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity α(M) ß(2) , were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of ß(1) -integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of ß(1) -integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared with eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. CONCLUSION AND CLINICAL RELEVANCE: IL-5 supports a heterogeneous population of circulating eosinophils with partially activated ß(2) -integrins and is responsible for up-regulation of ß(2) -integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on properties of eosinophils in BAL. Dampening of ß(2) -integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antígenos CD18/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Interleucina-5/imunologia , Adulto , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Asma/imunologia , Asma/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos CD18/imunologia , Eosinófilos/efeitos dos fármacos , Epitopos/imunologia , Feminino , Humanos , Interleucina-5/antagonistas & inibidores , Contagem de Leucócitos , Masculino , Glicoproteínas de Membrana/metabolismo , Adulto JovemRESUMO
Thrombospondins are large secreted, multimodular, calcium-binding glycoproteins that have complex roles in mediating cellular processes. Determination of high-resolution structures of thrombospondins has revealed unique and interesting protein motifs. Here, we review this progress and discuss implications for function. By combining structures of modules from thrombospondins and related extracellular proteins it is now possible to prepare an overall model of the structure of thrombospondin-1 and thrombospondin-2 and discern features of other thrombospondins. (Part of a multi-author Review).
Assuntos
Trombospondinas/química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Trombospondinas/genética , Trombospondinas/metabolismoRESUMO
Fibronectin is a component of subendothelial matrices and abundant in plasma. A role of fibronectin in thrombogenesis has been suspected for three decades. Soluble fibronectin is assembled by adherent fibroblasts and platelets and thus converted to an insoluble form that mediates cell adhesion. Recently, in vivo studies using intravital videomicroscopy revealed that plasma fibronectin is important for stabilization of platelet aggregates after vascular injury. This review goes over roles of fibronectin in platelet functions with a focus on fibronectin assembly within developing platelet thrombi.
Assuntos
Plaquetas/patologia , Fibronectinas/fisiologia , Trombose/etiologia , Sítios de Ligação , Coagulação Sanguínea , Fibronectinas/biossíntese , Fibronectinas/química , Humanos , Agregação Plaquetária , Trombose/patologiaRESUMO
BACKGROUND: Various types of laminin (LN) are ubiquitous components of basement membrane and exposed to blood upon localized damage of vascular endothelial cells. Fibronectin is a plasma protein that is insolubilized into fibrils in a regulated fashion by, for example, lysophosphatidic acid (LPA)-stimulated fibroblasts or platelets spread on supportive adhesive ligands. OBJECTIVE: To study assembly of plasma fibronectin by LPA-activated platelets adherent to LN-111 via alpha6beta1 integrin. RESULTS: Platelets adherent to LN-111-bound plasma fibronectin or its N-terminal 70 kD fragment in fibrillar arrays at the periphery of spread platelets under static but not shear conditions. Formation of fibronectin arrays under static conditions was inhibited by co-incubation with the N-terminal 70 kD fragment or with a 49-amino acid peptide that binds to the N-terminal region of fibronectin. Approximately 7000 fibronectin dimers bound per adherent platelet with a K(d) of 50 nm. Bound 70 kD fragment was readily solubilized with deoxycholate (DOC), whereas bound fibronectin became progressively insoluble. Bound 70 kD fragment became resistant to DOC extraction after treatment with a cell-impermeable, reducible crosslinker. Crosslinked 70 kD fragment was found in a high molecular weight complex. As with fibroblasts, signaling molecules modulating actin cytoskeletal organization controlled expression of binding sites for the N-terminal 70 kD region of fibronectin on adherent platelets. CONCLUSIONS: These results indicate that platelets adherent to LN-111 via alpha(6)beta(1) support subsequent assembly of fibronectin, but possibly only under conditions of intermittent or stagnant blood flow.
Assuntos
Plaquetas/metabolismo , Adesão Celular , Fibronectinas/metabolismo , Laminina/metabolismo , Adsorção , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Fluoresceína-5-Isotiocianato , Humanos , Cinética , Lisofosfolipídeos/farmacologia , Ligação ProteicaRESUMO
BACKGROUND: Thrombospondin-1 (TSP-1) has been implicated in many different processes based in part on inhibitory activities of anti-TSP-1 monoclonal antibodies (mAbs). OBJECTIVE: To map epitopes of 13 anti-TSP-1 mAbs to individual modules or groups of modules spanning TSP-1 and the closely related TSP-2 homolog. RESULTS: The mapping has led to assignment or reassignment of the epitopes of four mAbs, refinement of the epitopes of six mAbs, and confirmation of the epitopes of the remaining three mAbs. ESTs10, P12, and MA-II map to the N-terminal domain; 5G11, TSP127.6, and ESTs12 to the third properdin module; C6.7, HB8432, and P10 to epidermal growth factor (EGF)-like modules 1 and/or 2; and A6.1, mAb133, MA-I, and D4.6 to the calcium-binding wire module. A6.1, which recognizes a region of the wire that is identical in mouse and human TSP-1, reacts with TSP-1 from both species, and also reacts weakly with human TSP-2. Two other mouse antihuman TSP-1 mAbs, A4.1 and D4.6, also react with mouse TSP-1. CONCLUSIONS: Consideration of previous literature and mapping of epitopes of inhibitory mAbs suggest that biological activities are present throughout TSP-1, including the EGF-like modules that have not been implicated in the past. Because the epitopes for 10 of the antibodies likely are within 18 nm of one another in calcium-replete TSP-1, some of the inhibitory effects may result from steric hindrance. Such seems to be the case for mAb133, which binds the calcium-binding wire but is still able to interfere with the activation of latent TGF-beta by the properdin modules.
Assuntos
Anticorpos Monoclonais/farmacologia , Trombospondina 1/antagonistas & inibidores , Trombospondina 1/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Mapeamento de Epitopos , Humanos , Camundongos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trombospondina 1/química , Trombospondina 1/genética , Trombospondinas/antagonistas & inibidores , Trombospondinas/química , Trombospondinas/genética , Trombospondinas/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are lipids that bind G-protein coupled receptors and differentially promote transmigration of endothelial cells. OBJECTIVE: To determine if endothelial cell transmigration stimulated by LPA, not S1P, is dependent on the extracellular matrix. METHODS: Bovine pulmonary artery (BPAE) endothelial cell transmigration and locomotion were measured using a modified-Boyden chamber and video microscopy, respectively. Results were related to strength of adhesion and characteristics of cell adhesive contacts. RESULTS AND CONCLUSIONS: BPAEs responded to LPA by transmigration through gelatin- or collagen-coated filters, but not through fibronectin-, vitronectin-, or fibrinogen-coated filters. Fewer cells adhered to collagen or gelatin than to fibronectin in a static cell adhesion assay or after application of a g-force to detach cells. Video microscopy revealed that S1P stimulates large lamellipodia on two-dimensional fibronectin substrate. LPA stimulated lamellipodia on fibronectin, but the trailing edge remained attached, resulting in sting ray-shaped cells in video microscopy. LPA-treated cells on gelatin released the trailing edge. To understand how the extracellular matrix may regulate endothelial cell shape during movement, we surveyed changes in focal adhesion proteins. More Hic-5, a paxillin homolog, was detected in the detergent insoluble fraction of BPAEs attached to gelatin than fibronectin. No such difference was found in paxillin. In BPAEs, Hic-5 was localized to smaller punctate structures on fibronectin and longer, thinner focal adhesions on gelatin. These results indicated that localization of Hic-5 and strength of adhesion correlate with endothelial cell transmigration stimulated by LPA, but not with transmigration stimulated by S1P.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Lisofosfolipídeos/farmacologia , Esfingosina/análogos & derivados , Animais , Bovinos , Adesão Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desintegrinas/farmacologia , Endotélio Vascular/citologia , Fibronectinas/metabolismo , Gelatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Microscopia de Vídeo , Oligopeptídeos/farmacologia , Paxilina , Fosfoproteínas/metabolismo , Esfingosina/farmacologiaRESUMO
The thrombospondin (TSP) family of extracellular glycoproteins consists of five members in vertebrates, TSP1 to -4 and TSP5/cartilage oligomeric matrix protein, and a single member in Drosophila. TSPs are modular multimeric proteins. The C-terminal end of a monomer consists of 3-6 EGF-like modules; seven tandem 23-, 36-, or 38-residue aspartate-rich, Ca(2+)-binding repeats; and an approximately 230-residue C-terminal sequence. The Ca(2+)-binding repeats and C-terminal sequence are spaced almost exactly the same in different TSPs and share many blocks of identical residues. We studied the C-terminal portion of human TSP2 from the third EGF-like module through the end of the protein (E3CaG2). E3CaG2, CaG2 lacking the EGF module, and Ca2 composed of only the Ca(2+)-binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca(2+) in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca(2+). Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the 18 cysteines in the Ca(2+)-binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern.
Assuntos
Dissulfetos/metabolismo , Trombospondinas/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Trombospondinas/químicaRESUMO
Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.
Assuntos
Adesinas Bacterianas/metabolismo , Fibronectinas/metabolismo , Streptococcus pyogenes/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Fibronectinas/química , Heparina/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are agonists of the endothelial differentiation gene (Edg) family of G-protein-coupled receptors. LPA and S1P are generated by platelet activation during blood coagulation. Both lipids induce assembly of exogenous fibronectin (FN) by fibroblasts. This study examined whether LPA and S1P stimulate binding and assembly of fluoresceinated FN (FITC-FN) by adherent platelets. LPA enhanced deposition of FITC-FN into linear arrays overlying platelet surfaces and on edges of platelets adherent to FN or vitronectin (VN). Deposition was greater when platelets were adherent to FN than to VN and was elicited by platelet agonists with the following order of potency: thrombin > LPA = ADP (adenosine diphosphate) > S1P. The linear pattern of FITC-FN deposition was different from the more diffuse pattern of Alexa-fibrinogen (Alexa-FGN) binding to adherent platelets. FITC-FN was deposited by adherent platelets that had dense arrays of cytoskeletal actin when stained with rhodamine-phalloidin. The 70-kd N-terminal fragment of FN or L8 monoclonal antibody to a self-association domain of FN abolished deposition of FITC-FN but had no effect on binding of Alexa-FGN. Conversely, integrilin did not attenuate deposition of FITC-FN but abolished binding of Alexa-FGN. RGDS (Arg-Gly-Asp-Ser) or antibodies to alpha5beta1 or alphaIIbbeta3 integrins caused a partial decrease in LPA-induced deposition of FITC-FN. Correlative electron microscopy with anti-FITC coupled to gold beads revealed linear arrays on platelet surfaces associated with less than 20-nm-diameter filaments. These observations demonstrate that LPA, thrombin, ADP, and S1P induce adherent platelets to bind and assemble FN and suggest that platelets may contribute to early deposition of FN matrix after vascular injury.
Assuntos
Plaquetas/efeitos dos fármacos , Fibronectinas/metabolismo , Lisofosfolipídeos/agonistas , Lisofosfolipídeos/farmacologia , Difosfato de Adenosina/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Eptifibatida , Matriz Extracelular/metabolismo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Hemostáticos/farmacologia , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Peptídeos/farmacologia , Adesividade Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Trombina/farmacologia , Transglutaminases/farmacologiaRESUMO
We did a retrospective analysis on the safety and efficacy of sequential infusion fludarabine and cytosine arabinoside (ara-C) in treating refractory, recurrent or poor prognosis acute leukemia in adult patients. Forty-five adult patients with acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL) received a total of 68 courses of sequential continuous infusion of fludarabine for 2 days (total dose 71.5 mg/m(2) ) followed by 3 days of ara-C (total dose 7590 mg/m(2) ). Thirty-nine patients had refractory or recurrent disease, and six had other adverse prognostic features. Thirty-six patients had AML, seven had ALL, and two had CML in blastic phase. Complete remission was seen in 20 patients (44%), and partial remission in 5 patients (11%), giving a total response rate of 56%, similar for both AML and ALL. Duration of response to prior therapy did not affect the response rate. All 3 patients with Philadelphia chromosome positive ALL obtained complete remission. Median remission duration was 4.7 months (range 0.6-36.6), and median overall survival was 5.0 months (0.7-40+). Median overall survival was 10.1 months in responders. Pulmonary toxicity was seen in 8 patients, of whom 2 died from adult respiratory distress syndrome. No cardiac toxicity was observed, but 3 patients had transient cerebellar toxicity. Profound myelosuppression was seen in all patients. We conclude that the sequential infusion of fludarabine and ara-C is an effective non-cardiotoxic regimen for adults with refractory, recurrent or poor prognosis acute leukemia, may be particularly useful for resistant Philadelphia chromosome positive ALL, and may warrant further investigation in this subset. Pulmonary rather than neurological toxicity may be a unique side effect of the regimen.
Assuntos
Citarabina/administração & dosagem , Leucemia/complicações , Leucemia/tratamento farmacológico , Vidarabina/administração & dosagem , Doença Aguda , Adolescente , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Estudos de Coortes , Citarabina/toxicidade , Análise Citogenética , Intervalo Livre de Doença , Esquema de Medicação , Feminino , Doenças Hematológicas/etiologia , Humanos , Infecções/etiologia , Bombas de Infusão , Leucemia/genética , Pneumopatias/etiologia , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/etiologia , Prognóstico , Recidiva , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento , Vidarabina/análogos & derivados , Vidarabina/toxicidadeRESUMO
F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC(50) of approximately 10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.
Assuntos
Adesinas Bacterianas/química , Fibronectinas/metabolismo , Streptococcus pyogenes/patogenicidade , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Divisão Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Adesões Focais/ultraestrutura , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos/farmacologia , Fibras de Estresse/ultraestrutura , Células Tumorais CultivadasRESUMO
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 microM whereas the concentration in plasma is considerably less. The SPP concentration in serum is 0.5 microM, approximately two-fold higher than the plasma concentration. The lipids are present during tissue injury and promote cellular processes involved in wound repair. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.
Assuntos
Matriz Extracelular/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Colágeno/metabolismo , Fibronectinas/metabolismo , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismo , Esfingosina/análogos & derivadosRESUMO
GD25 cells lacking beta 1 integrins or expressing beta 1A with mutations of conserved cytoplasmic tyrosines (Y783, Y795) to phenylalanine have poor directed migration to platelet-derived growth factor or lysophosphatidic acid when compared with GD25 cells expressing wild-type beta 1A. We studied the effects of v-src on these cells. Transformation with v-src caused tyrosine and serine phosphorylation of wild-type beta1 A but not of Y783/795F doubly mutated beta 1A. v-src-transformed cells had rounded and/or fusiform morphology and poor assembly of fibronectin matrix. Adhesion to fibronectin or laminin and coupling of focal contacts to actin-containing cytoskeleton were preserved in transformed Y783/795F cells but lost on transformation when beta 1A was wild type. Transformed Y783/795F cells also retained ability, albeit limited, to migrate across filters, whereas transformed cells with wild-type beta 1A were unable to transverse filters. Studies of single tyrosine mutants showed that the more important tyrosine for retaining ability to adhere, assemble focal contacts, and migrate is Y783. These results suggest that overactive phosphorylation of cytoplasmic residues of beta 1A, particularly Y783, accounts in part for the phenotype of v-src-transformed cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Integrina beta1/metabolismo , Proteína Oncogênica pp60(v-src)/fisiologia , Tirosina/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Adesões Focais/metabolismo , Integrina beta1/fisiologia , Camundongos , FosforilaçãoRESUMO
Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 repeats (TSRs), modules of approximately 60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an alpha-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is well described in epidermal growth factor-like modules. Both C- and O-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.
Assuntos
Fucose/metabolismo , Manose/metabolismo , Trombospondinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Glicosilação , Humanos , Dados de Sequência Molecular , Trombospondinas/químicaRESUMO
Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.
Assuntos
Epitopos/química , Epitopos/metabolismo , Inibidor da Proteína C/química , Inibidor da Proteína C/metabolismo , Proteína C/química , Proteína C/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Epitopos/imunologia , Humanos , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteína C/imunologia , Inibidor da Proteína C/biossíntese , Inibidor da Proteína C/imunologia , Conformação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
There has been considerable interest in the effect that gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) can have in asthma, given that inhalation of LPS has been shown to cause bronchial hyperresponsiveness. Further, there is evidence that the endotoxin-binding protein CD14 may be a marker for asthma. Inhaled LPS has been shown to cause an influx of eosinophils into the nasal airway and to increase the survival of CD16-negatively selected eosinophils in vitro. In this study, we compared survival of eosinophils isolated via CD16-negative selection with eosinophils that were isolated using both CD16- and CD14-negative selection criteria. Survival of CD16-negatively selected eosinophils was enhanced by LPS in a dose-dependent manner and was inhibited by the endotoxin antagonists polymyxin B or lipid X. In contrast, depletion of CD14(+) cells within the eosinophil preparations (CD14/CD16-negatively selected eosinophils) decreased the effect of LPS on survival. Preincubation of CD16-negatively selected eosinophils with antibody 60bd, which blocks LPS binding to CD14, prevented the survival-enhancing effect of LPS. However, CD14 was not detected on eosinophils by flow cytometry, even after incubation with LPS for up to 24 h. These results suggest that the survival-enhancing effect of LPS on eosinophils requires the presence of CD14(+) cells in the population. It is our hypothesis that enhanced eosinophil survival with LPS involves the contribution of another cell type.