RESUMEN
A simple lab-on-a-chip method for blood plasma separation was developed by combining stereolithographic 3D printing with inkjet printing, creating a completely sealed microfluidic device. In some approaches, one dilutes the blood sample before separation, reducing the concentration of a target analyte and increasing a contamination risk. In this work, a single drop (8 µl) of heparinized whole blood could be efficiently filtered using a capillary effect without any external driving forces and without dilution. The blood storage in heparin tubes during 24 h at 4 °C initiated the formation of small crystals that formed auto-filtration structures in the sample upon entering the 3D-printed device, with pores smaller than the red blood cells, separating plasma from the cellular content. The total filtration process took less than 10 s. The presented printed plasma filtration microfluidics fabricated with a rapid prototyping approach is a miniaturized, fast and easy-to-operate device that can be integrated into healthcare/portable systems for point-of-care diagnostics.
RESUMEN
Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor (NNRTI), is part of first-line therapy for the treatment of human immunodeficiency virus type 1 infection (HIV-1/AIDS). This drug shows relatively low oral absorption and bioavailability, as well as high intra- and inter-subject variability. Several studies have shown that treatment failure and adverse effects are associated with low and high EFV plasma concentrations, respectively. Some studies suggest different EFV formulations to minimize inter-patient variability and improve its solubility and dissolution; however, all of these formulations are complex, using for instance, cyclodextrins, dendrimers and polymeric nanoparticles, rendering them inviable industrially. The aim of this work was to prepare simple and low-cost suspensions of EFV for improvement of solubility and dissolution rate by using colloid mill, spray or freeze-drying, and characterization of the powders obtained. The results demonstrated an increase in the dissolution rate of EFV, using 0.2% of sodium lauryl sulfate (SLS) and 0.2% of hydroxypropylcellulose (HPC) or hydroxypropylmetilcellulose (HPMC) in both freeze and spray dried powders. The pharmacokinetic studies demonstrated improved pharmacokinetic parameters for the formulation containing SLS and HPC. The powders obtained, which present enhanced dissolution properties, can be incorporated in a solid dosage form for treatment of AIDS in paediatric patients with promising results.
Asunto(s)
Benzoxazinas/química , Benzoxazinas/farmacocinética , Coloides/química , Alquinos , Animales , Celulosa/análogos & derivados , Celulosa/química , Química Farmacéutica/métodos , Ciclopropanos , Composición de Medicamentos , Nariz Electrónica , Liofilización/métodos , Masculino , Nanopartículas/química , Tamaño de la Partícula , Polvos/química , Polvos/farmacocinética , Ratas , Ratas Wistar , Dodecil Sulfato de Sodio/química , Solubilidad , Suspensiones/química , Suspensiones/farmacocinéticaRESUMEN
Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.
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Factor Estimulante de Colonias de Macrófagos/genética , Animales , Femenino , Operón Lac , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Embarazo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase phi (PTP phi). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTP phi and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTP phi induces cell rounding and ruffle formation, while C325S PTP phi has no effect. In contrast, in CSF-1-starved cells, C325S PTP phi behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTP phi increases adhesion in cycling cells, while WT PTP phi enhances motility. In WT PTP phi-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTP phi-expressing cells. Moreover, a complex containing PTP phi, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTP phi selectively binds paxillin and Pyk2 in vitro. Although PTP phi and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTP phi in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.
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Proteínas del Citoesqueleto/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Tirosina/metabolismo , Western Blotting , Encéfalo/metabolismo , Dominio Catalítico , Adhesión Celular , División Celular , Línea Celular , Movimiento Celular , Supervivencia Celular , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Riñón/metabolismo , Microscopía Fluorescente , Microscopía de Contraste de Fase , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Paxillin , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Factores de Tiempo , Cicatrización de HeridasRESUMEN
OBJECTIVES: The aims of this study were to measure levels of colony stimulating factor (CSF-1) in patients with trophoblastic disease, to determine whether such measurement may be useful to supplement measurement of the prognostically reliable human chorionic gonadotrophin (hCG), and to assess whether measurement of CSF-1 may be helpful in predicting requirement for chemotherapy in patients with hydatidiform mole. METHODS: Serial weekly serum samples were selected for CSF-1 assay from representative diagnostic groups of patients with trophoblastic disease: hydatidiform-mole with spontaneous resolution, low-risk post-hydatidiform-mole trophoblastic tumor, partial hydatidiform mole, high-risk metastatic gestational trophoblastic tumor, primary ovarian choriocarcinoma, and placental site trophoblastic tumor. hCG was measured by an in-house radioimmunoassay that measures all parts of the hCG molecule. CSF-1 was measured by radioimmunoassay with (125)I-labeled recombinant CSF-1. The upper level of normal CSF-1 was taken as 8 ng/ml. RESULTS: In this study of 45 patients with trophoblastic disease, some very high levels of CSF-1 were encountered. In a few patients there was dramatic correlation with hCG. Generally, however, CSF-1 and hCG did not correlate. CSF-1 was frequently not elevated when hCG was still significantly elevated and conversely CSF-1 was elevated when hCG was negative. CONCLUSION: The measurement of CSF-1 does not appear to be useful in managing trophoblastic disease as it does not correlate with the level of hCG. Occasionally, high levels of CSF-1 were found in patients with trophoblastic disease.
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Factor Estimulante de Colonias de Macrófagos/sangre , Neoplasias Trofoblásticas/sangre , Neoplasias Uterinas/sangre , Adolescente , Adulto , Anciano , Gonadotropina Coriónica/sangre , Femenino , Humanos , Mola Hidatiforme/sangre , Mola Hidatiforme/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Embarazo , Radioinmunoensayo , Factores de RiesgoRESUMEN
We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10(-4) M IDP and 10(-4) M AZ, as well as 10(-5) M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl- cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10(-4) M IDP as well as 10(-5) M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.
Asunto(s)
Benzotiadiazinas , Resorción Ósea/prevención & control , Indapamida/farmacología , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Simportadores , Animales , Secuencia de Bases , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Proteínas Portadoras/metabolismo , División Celular/efectos de los fármacos , Técnicas de Cocultivo , Cartilla de ADN , Diuréticos , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Técnicas de Cultivo de Órganos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simportadores del Cloruro de SodioRESUMEN
BACKGROUND: Effacement of podocyte foot processes occurs early in many glomerular diseases associated with proteinuria and is accompanied by a reorganization of the actin cytoskeleton. The molecular mechanisms regulating these structural changes are poorly understood. METHODS: To address these questions, we analyzed the effect of the polycation, protamine sulfate (PS), and puromycin aminonucleoside (PA) on the morphology, cytoskeleton, and tyrosine phosphorylation of differentiated process-bearing cultured podocytes. RESULTS: PS and PA induced similar profound morphological alterations, including retraction and detachment of podocyte processes from the extracellular matrix (ECM). The effects of PS occurred within six hours, whereas PA showed its most severe effects after 72 hours. Structural changes included reorganization of the actin cytoskeleton and focal contacts and were accompanied by an increase in tyrosine phosphorylation. The same effects were induced by application of vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), suggesting that PTPs regulate podocyte process structure. Since disruption of the actin cytoskeleton with cytochalasin B protected the cells from PS-induced effacement and detachment, cytoplasmic PTPs were implicated in these events. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the expression of four cytoplasmic PTPs in podocytes: SHP-2, PTP-PEST, PTP-1B, and PTP-36. CONCLUSIONS: These studies indicate an important role for cytoplasmic PTPs as regulators of podocyte process dynamics. Future studies will aim at restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria by modulating the activity of cytoplasmic PTPs.
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Glomérulos Renales/citología , Proteínas Tirosina Fosfatasas/fisiología , Actinas/metabolismo , Animales , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/fisiología , Ratones , Fosforilación , Protaminas/farmacología , Puromicina Aminonucleósido/farmacología , Tirosina/metabolismo , Vanadatos/farmacologíaRESUMEN
Dorsal closure (DC) in the Drosophila embryo requires the coordinated interaction of two different functional domains of the epidermal cell layer-the leading edge (LE) and the lateral epidermis. In response to activation of a conserved c-Jun amino-terminal kinase (JNK) signaling module, the dorsal-most layer of cells, which constitute the LE of the stretching epithelial sheet, secrete Dpp, a member of the TGFbeta superfamily. Dpp and other LE cell-derived signaling molecules stimulate the bilateral dorsal elongation of cells of the dorsolateral epidermis over the underlaying amnioserosa and the eventual fusion of their LEs along the dorsal midline. We have found that flies bearing a Shark tyrosine kinase gene mutation, shark(1), exhibit a DC-defective phenotype. Dpp fails to be expressed in shark(1) mutant LE cells. Consistent with these observations, epidermal-specific reconstitution of shark function or overexpression of an activated form of c-Jun in the shark(1) mutant background, rescues the DC defect. Thus, Shark regulates the JNK signaling pathway leading to Dpp expression in LE cells. Furthermore, constitutive activation of the Dpp pathway throughout the epidermis fails to rescue the shark(1) DC defect, suggesting that Shark may function in additional pathways in the LE and/or lateral epithelium.
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Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Morfogénesis/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Animales , Embrión no Mamífero/fisiología , Epidermis/embriología , Metanosulfonato de Etilo , Femenino , Proteínas de Insectos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis , Mutación Puntual , Transducción de Señal , Cromosoma XRESUMEN
SHP-1 plays key roles in the modulation of hematopoietic cell signaling. To ascertain the impact of SHP-1 on colony-stimulating factor-1 (CSF-1)-mediated survival and proliferative signaling, we compared the CSF-1 responses of primary bone marrow macrophages (BMM) from wild-type and SHP-1-deficient motheaten (me/me) mice. CSF-1-induced protein tyrosine phosphorylation levels were similar in wild-type and me/me BMM, except for the constitutive hyperphosphorylation of a 62-kDa phosphoprotein (pp62) in me/me macrophages. pp62 was identified as the RASGAP-associated p62(DOK) and was shown to be the major CSF-1R-associated tyrosine-phosphorylated protein in CSF-1-treated BMM. p62(DOK) was found to be constitutively associated with SHP-1 in BMM and in 293T cells, co-expressing p62(dok) and either wild-type or catalytically inert SHP-1 (SHP-1 C453S). In both cell types, the interaction of SHP-1 with p62(DOK) occurred independently of p62(DOK) tyrosine phosphorylation, but only the tyrosine-phosphorylated p62(DOK) was bound by SHP-1 C453S in a far Western analysis. These findings suggest a constitutive association of SHP-1 and p62(DOK) that is either conformation-dependent or indirect as well as a direct, inducible association of the SHP-1 catalytic domain with tyrosine-phosphorylated p62(DOK). p62(DOK) hyperphosphorylation is not associated with altered CSF-1-induced RAS signaling or proliferation. However, whereas wild-type macrophages undergo cell death following CSF-1 removal, me/me macrophages exhibit prolonged survival in the absence of growth factor. Thus, p62(DOK) is a major SHP-1 substrate whose tyrosine phosphorylation correlates with growth factor-independent survival in macrophages.
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Proteínas de Unión al ADN , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Unión al ARN , Animales , Células de la Médula Ósea/citología , Línea Celular , Células Cultivadas , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Mutantes , Fosforilación , Fosfotirosina/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transfección , Dominios Homologos srcRESUMEN
Colony-stimulating factor-1 (CSF-1) activation of the CSF-1 receptor (CSF-1R) causes Cbl protooncoprotein tyrosine phosphorylation, Cbl-CSF-1R association and their simultaneous multiubiquitination at the plasma membrane. The CSF-1R is then rapidly internalized and degraded, whereas Cbl is deubiquitinated in the cytoplasm without being degraded. We have used primary macrophages from gene-targeted mice to study the role of Cbl. Cbl-/- macrophages form denser colonies and, at limiting CSF-1 concentrations, proliferate faster than Cbl+/+ macrophages. Their CSF-1Rs fail to exhibit multiubiquitination and a second wave of tyrosine phosphorylation previously suggested to be involved in preparation of the CSF-1-CSF-1R complex for endocytosis. Consistent with this result, Cbl-/- macrophage cell surface CSF-1-CSF-1R complexes are internalized more slowly, yet are still lysosomally degraded, and the CSF-1 utilization by Cbl-/- macrophages is reduced approximately 2-fold. Thus, attenuation of proliferation by Cbl is associated with its positive regulation of the coordinated multiubiquitination and endocytosis of the activated CSF-1R, and a reduction in the time that the CSF-1R signals from the cell surface. The results provide a paradigm for studies of the mechanisms underlying Cbl attenuation of proliferative responses induced by ligation of receptor tyrosine kinases.
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Endocitosis , Macrófagos/citología , Complejo de la Endopetidasa Proteasomal , Proteínas Proto-Oncogénicas/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismo , Animales , División Celular , Tamaño de la Célula , Supervivencia Celular , Transformación Celular Neoplásica , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Eliminación de Gen , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/fisiología , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo , Proteínas ras/metabolismoRESUMEN
Addition of colony stimulating factor-1 (CSF-1) to macrophages stimulates the rapid, transient tyrosine phosphorylation, membrane association and multiubiquitination of Cbl (Wang et al. [1996] J. Biol. Chem. 271:17-20). Kinetic analysis reveals that the tyrosine phosphorylation of Cbl is coincident with its plasma membrane translocation and association with the activated tyrosine phosphorylated CSF-1 R, p85, Grb2, and tyrosine phosphorylated p58Shc and that these events precede the simultaneous multiubiquitination of Cbl and the CSF-1 R. Tyrosine phosphorylation and multiubiquitination of the cell surface CSF-1 R are stoichiometric and the multiubiquitinated CSF-1 R is degraded. Similarly, the membrane associated Cbl is almost stoichiometrically ubiquitinated, but the ubiquitinated Cbl is not degraded, being recovered, deubiquitinated, in the cytosol 3-10 min after stimulation at 37 degrees C. In the membrane fraction of cells stimulated at 4 degrees C, the association of p58Shc and Grb2 with Cbl is stable, whereas its association with Sos and p85 is transient and their dissociation occurs at the time CSF-1 R and Cbl multiubiquitination commence. The membrane translocation and the pattern of association of Sos with the CSF-1R, p85, Grb2, and p58Shc resemble those of Cbl but Sos is not tyrosine phosphorylated, nor multiubiquitinated and the coprecipitation of these proteins, other than Grb2, with Sos is much less. Complexes formed by Sos and Cbl are largely independent and membrane complexes of Cbl with other tyrosine phosphorylated proteins, p85 and Grb2 also contain CSF-1 R. These data raise the possibility that the predicted negative regulatory role of Cbl in macrophages is its enhancement of ligand-induced CSF-1 R internalization/degradation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Tirosina/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Proteína Adaptadora GRB2 , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Receptor de Factor Estimulante de Colonias de Macrófagos/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The SH2 domain-containing cytoplasmic protein tyrosine phosphatase, SHP-1, negatively regulates hematopoietic cell signaling. SHP-1 is associated with a tyrosine phosphorylated, plasma membrane-spanning glycoprotein, pp130, in colony stimulating factor-1 stimulated or unstimulated macrophages. This association is phosphotyrosine dependent and is mediated by the amino-terminal SH2 domain of SHP-1. pp130 behaves as a substrate of SHP-1 in vitro and is hyperphosphorylated on tyrosine in SHP-1 deficient macrophages from viable-motheaten mice. Co-immunoprecipitation data indicate that pp130 is the product of the mouse p91/PIR-B gene that encodes a member of the killer cell inhibitory receptor (KIR)/leukocyte immunoglobulin-like receptor (LIR) family. By analogy to the KIRs, p91/PIR-B may represent a novel class of macrophage receptors which act to suppress macrophage activation. These observations identify SHP-1 interactions with and regulation of p91/PIR-B as a potential mechanism for inhibiting the signaling cascades linking extracellular stimuli to macrophage activation and/or development.
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Antígenos CD , Macrófagos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores Inmunológicos/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Péptidos y Proteínas de Señalización Intracelular , Receptor Leucocitario Tipo Inmunoglobulina B1 , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/fisiología , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Receptores Inmunológicos/genética , Receptores KIR , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transducción de SeñalRESUMEN
An approximately 37-kDa cytoplasmic protein is rapidly tyrosine-phosphorylated in the response of mouse BAC1.2F5 macrophages to colony stimulating factor-1 (CSF-1). pp37 was purified from the cytosolic fraction by anti-Tyr(P) affinity chromatography, size exclusion chromatography, and C4 reverse phase high pressure liquid chromatography. The sequences of four peptides derived from the purified protein matched portions of an expressed sequence tag (EST) sequence, and the EST clone was used to obtain cDNA clones encoding the pp37 protein, which shares sequence similarity with the PST PIP (proline, serine, threonine phosphatase interacting protein)/CDC15 family of protein-tyrosine phosphatase substrates. pp37 is predicted to contain a Fes/CIP4 homology (FCH) domain and an actin-binding domain-like sequence. It is expressed selectively in macrophages, macrophage cell lines, and at low levels in macrophage-containing tissues. pp37 is predominantly found in the cytosol, where it is associated with actin. However, approximately 4% resides in the membrane fraction, and the trace amount in the cytoskeletal fraction is increased by CSF-1 stimulation. Termed macrophage actin-associated tyrosine-phosphorylated protein (MAYP), p37 is the major F-actin-associated protein that is tyrosine-phosphorylated in macrophages and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto , Factor Estimulante de Colonias de Macrófagos/farmacología , Proteínas de Microfilamentos/química , Proteínas Tirosina Fosfatasas/metabolismo , Tirosina/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Clonación Molecular , Citoesqueleto/metabolismo , Ratones , Proteínas de Microfilamentos/aislamiento & purificación , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Peso Molecular , FosforilaciónRESUMEN
The synthesis and degradation of type I and type III interstitial collagens releases several antigenic metabolites, whose measurement allows the metabolism of connective tissue to be evaluated under a variety of different conditions. In this study we investigated the influence of benign and malignant ovarian neoplasms on the metabolism of these collagens. The study population comprised patients with benign (n = 53), borderline (n = 6) or malignant (n = 36) ovarian neoplasms. We quantified the serum, cyst fluid and peritoneal/ascitic fluid concentrations of the amino-terminal propeptide of type I (PINP) and III (PIIINP) procollagens, indicators of the synthesis of type I and III collagen, respectively and the cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), an indicator of type I collagen degradation. Macrophage colony-stimulating factor 1 (CSF-1) concentration was also assayed as its serum level is increased in ovarian cancer and CSF-1 may be involved in the regulation of collagen metabolism. The concentration of each antigen was significantly higher in patients with malignant tumour than with benign neoplasm in each comparison, except for ICTP in peritoneal fluid and for CSF-1 in cyst fluid. The high ascitic fluid concentration of PINP, PIIINP or CSF-1 correlated with malignancy, and the low cyst fluid concentration of any of the four markers was indicative of benign tumour. Levels of CSF-1 did not correlate with the levels of any of the markers of collagen turnover. The concentration of PINP in ascites was about 50 times higher and in cyst fluid about eight times higher than that in the serum from patients with malignant tumour, whereas the respective ratios for ICTP were only 2.5 and 1.3. In such patients, the ratio of ascitic fluid to serum concentration was also about 80-fold higher for PIIINP and about 20-fold higher for PINP than for ICTP. The different distributions of PIIINP, PINP and ICTP suggests dominance of synthetic processes or retarded elimination of PIIINP and PINP in ovarian cancer. In advanced malignancies, the accumulation of PINP and PIIINP in abdominal space, possibly due to increased synthesis and/or failed resorption, may promote ascites formation. This study shows that both accelerated synthesis and breakdown of fibrillar collagens are characteristic of ovarian malignancy, and suggests that measurements of cyst fluid or ascitic fluid concentrations of collagen metabolites or CSF-1 could be used in the differential diagnosis of benign and malignant ovarian neoplasms.
Asunto(s)
Colágeno/metabolismo , Neoplasias Ováricas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Femenino , Humanos , Factor Estimulante de Colonias de Macrófagos/análisis , Persona de Mediana Edad , Fragmentos de Péptidos/análisis , Procolágeno/análisisRESUMEN
Stimulation of macrophages with colony-stimulating factor-1 (CSF-1) results in the protein tyrosine phosphorylation of the CSF-1 receptor (CSF-1R) and many other, primarily cytosolic, proteins. Stimulation by CSF-1 at 4 degreesC was used to facilitate the purification and identification of the proteins of the cytosolic anti-phosphotyrosine (PY)-reactive fraction (alphaPY-RF) involved in downstream signaling pathways. Confocal microscopy revealed that the PY proteins are in close proximity to the CSF-1R at the plasma membrane. The alphaPY-RF contained pre-existing complexes of PY proteins and non-PY proteins which generally increased in size and PY protein content following CSF-1 stimulation. PY proteins identified by microsequencing and Western blotting include Cbl, STAT3, STAT5a, STAT5b, SHP-1, Shc, and two novel proteins pp57 and pp37. Other proteins included cytoskeletal/contractile proteins (paxillin, vimentin, elongation factor-1alpha, F-actin, tropomyosin, and myosin regulatory light chain), Ras family signaling proteins (p85 (phosphoinositide 3-kinase), Vav, Ras-GTPase-activating protein SH3 domain-binding protein, and Grb2), DnaJ-like protein, and glyceraldehyde-3-phosphate dehydrogenase. CSF-1 induced the de novo recruitment of Cbl, STAT3, STAT5a, STAT5b, p85, SHP-1, Shc, vimentin, and Grb2 to complexes and caused pre-existing complexes involving Vav, elongation factor-1alpha, and F-actin to increase in size. These studies indicate that CSF-1-induced protein tyrosine phosphorylation is associated with the reorganization of complexes of cytoskeletal, signaling, and other proteins that mediate CSF-1-regulated motility and growth.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Citosol/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Transducción de Señal , Actinas/metabolismo , Secuencia de Aminoácidos , Biopolímeros , Western Blotting , Línea Celular , Cromatografía en Gel , Citocalasina D/farmacología , Proteínas del Citoesqueleto/aislamiento & purificación , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Macrófagos/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
Colony-stimulating factor-1 (CSF-1), the primary regulator of mononuclear phagocyte (Mphi) production, exists as either a circulating or cell surface, membrane-spanning molecule. To establish transplacental transfer of maternal CSF-1, gestational day-17 mothers were injected intravenously with 125I-mouse CSF-1 or human rCSF-1, and the 125I-cpm or human CSF-1 concentrations were measured in fetal tissue, placenta, and fetal/maternal sera. Biologically active CSF-1 crossed the placenta and peaked in fetal tissue, placenta, and serum 10 minutes after injection. The role of CSF-1 in perinatal Mphi development was examined by studying the CSF-1-deficient osteopetrotic (csfmop/csfmop) mouse. Fetal/neonatal mice, derived from matings of either +/csfmop females with csfmop/csfmop males or the reciprocal pairings, were genotyped and tissue Mphi identified and quantified. In the presence of circulating maternal CSF-1 (+/csfmop mother), Mphi development in csfmop/csfmop liver was essentially complete at birth relative to +/csfmop littermates, but significantly reduced in spleen, kidney, and lung. In the absence of circulating maternal CSF-1 (csfmop/csfmop mother), Mphi numbers at birth were reduced in csfmop/csfmop liver relative to the offspring of +/csfmop mothers, but were similar in spleen, kidney, and lung. We conclude that CSF-1 is required for the perinatal development of most Mphi in these tissues. Compensation for total absence of local CSF-1 production by circulating, maternal CSF-1 is tissue-specific and most prominent in liver, the first fetal organ perfused by placental blood. However, because some Mphi developed in the complete absence of CSF-1, other factors must also be involved in the regulation of macrophage development.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/farmacología , Osteopetrosis/patología , Fagocitos/efectos de los fármacos , Animales , Femenino , Sangre Fetal/química , Enfermedades Fetales/sangre , Enfermedades Fetales/patología , Heterocigoto , Homocigoto , Humanos , Riñón/patología , Hígado/patología , Pulmón/patología , Factor Estimulante de Colonias de Macrófagos/análisis , Factor Estimulante de Colonias de Macrófagos/deficiencia , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/farmacocinética , Masculino , Intercambio Materno-Fetal , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Especificidad de Órganos , Osteoclastos/patología , Osteopetrosis/sangre , Osteopetrosis/embriología , Placenta/metabolismo , Embarazo/sangre , Proteínas Recombinantes/análisis , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Bazo/patologíaRESUMEN
Macrophages are found throughout the male reproductive tract and its accessory glands. Mice homozygous for a null mutation (csfm(op)) in the gene for the mononuclear phagocytic growth factor colony-stimulating factor-1 (CSF-1) have a significantly lower density of macrophages, defined by the mononuclear phagocytic antigen F4/80, in the testis, cauda and caput epididymis, prostate, seminal vesicles, and vas deferens. These data indicate that CSF-1 is the major growth factor regulating the occurrence of macrophages in male reproductive tissues. The residual macrophages were correctly located in the tissue except in the caput epididymis, where they failed to take up positions adjacent to the tubular epithelium. Restoration of circulating CSF-1 concentrations in csfm(op)/csfm(op) males totally restored F4/80+ cell density in the testis and caput and cauda epididymis and partially restored their density in the vas deferens and seminal vesicles but failed to affect density in the prostate. This failure to correct all populations with circulating CSF-1 suggests the requirement for local synthesis of CSF-1 at appropriate developmental stages and/or its expression in a cell surface-associated form. The absence of macrophages in the testis and epididymis of csfm(op)/csfm(op) mice correlates with dysfunction in these tissues, suggesting that macrophages play important nonimmunological roles in these tissues.
Asunto(s)
Genitales Masculinos/patología , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/patología , Osteopetrosis/genética , Osteopetrosis/patología , Animales , Epidídimo/patología , Epidídimo/fisiopatología , Genitales Masculinos/fisiopatología , Factor Estimulante de Colonias de Macrófagos/deficiencia , Macrófagos/fisiología , Masculino , Ratones , Ratones Noqueados , Osteopetrosis/fisiopatología , Próstata/patología , Próstata/fisiopatología , Vesículas Seminales/patología , Vesículas Seminales/fisiopatología , Conducto Deferente/patología , Conducto Deferente/fisiopatologíaRESUMEN
CSF-1 is a hemopoietic growth factor that regulates the survival, proliferation, and differentiation of mononuclear phagocytes, cells that are critical in the inflammatory response. In the case of Gram-negative infection, LPS plays an important role by inducing several cell types to produce the proinflammatory cytokines, IL-1, IL-6, and TNF-alpha. In this study, we examined the effects of i.p. administration of LPS on CSF-1 expression in the mouse. Two- to sevenfold increases in the CSF-1 concentrations determined by RIA were evident within hours of LPS administration in serum, liver, kidney, lung, spleen, brain, intestine, and heart. While alterations in the CSF-1 receptor-mediated clearance of CSF-1 appeared not to account for the increased growth factor concentrations in LPS-treated animals, there was an early LPS-induced reduction of splenic [125I]CSF-1 uptake consistent with tissue-specific down-modulation of CSF-1 receptors. The results of Northern analysis revealed increased expression of a CSF-1 mRNA species in liver, lung, kidney, spleen, intestine, and heart following LPS treatment, demonstrating that increased synthesis was responsible for the increased tissue CSF-1 concentrations. The increased expression and synthesis of CSF-1 in response to LPS may be essential for mobilizing and activating mononuclear phagocytes in the inflammatory response.
Asunto(s)
Lipopolisacáridos/administración & dosificación , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Femenino , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Especificidad de ÓrganosRESUMEN
The SJL/J mouse strain has a high spontaneous incidence of a B-cell neoplasm, reticulum cell neoplasm type B (RCN B). In addition, following irradiation, 10% to 30% of these mice develop acute myelomonocytic leukemia (radiation-induced acute myeloid leukemia [RI-AML]), an incidence that can be increased to 50% by treatment of the mice with corticosteroids after irradiation. The role played by the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), in the development of RI-AML in SJL/J mice was investigated. Mice dying of RI-AML, but not those dying of RCN B or without disease, possessed elevated concentrations of circulating CSF-1. In addition, in mice developing RI-AML with a more prolonged latency, circulating CSF-1 concentrations were increased before overt expression of RI-AML. First-passage tumors from 14 different RI-AMLs all contained high concentrations of CSF-1, and six of six different first- or second-passage tumors expressed the CSF-1 receptor (CSF-1 R). Furthermore, in vitro colony formation by first- or second-passage tumor cells from 20 of 20 different RI-AMLs was blocked by neutralizing anti-CSF-1 antibody, and four of four of these tumors were inhibited by anti-CSF-1R antibody. The results of these antibody neutralization studies, coupled with the observation of elevated circulating CSF-1 in mice developing RI-AML, show an autocrine role for CSF-1 in RI-AML development in SJL/J mice. Southern blot analysis of tumor DNA from six of six of these tumors failed to reveal any rearrangements in the genes for CSF-1 or the CSF-1R. Studies in humans have shown that patients with AML possess elevated levels of circulating CSF-1 and that AML cells can express CSF-1 and the CSF-1R. Thus, RI-AML in the SJL/J mouse appears to be a useful model for human AML.