RESUMEN
OBJECTIVE: To determine the efficacy and safety of pamapimod in adult patients with active rheumatoid arthritis (RA) who had an inadequate clinical response to methotrexate (MTX). METHODS: Patients receiving stable doses of MTX were randomised to one of six dose groups and received 12 weeks of double-blind pamapimod (up to 300 mg once daily) or matching placebo. The primary efficacy measure was the proportion of patients with > or =20% improvement in RA based on the American College of Rheumatology criteria (ACR20) at 12 weeks. Secondary measures were ACR50, Disease Activity Score (DAS)/European League Against Rheumatism (EULAR) responses and the individual ACR core set of parameters. Safety measures included adverse events (AEs), laboratory testing and immunology assessments. RESULTS: On a background of MTX, the percentage of patients with an ACR20 response at week 12 in the pamapimod groups (31% to 43%) was not significantly different from placebo (34%). Secondary efficacy end points showed a similar pattern. AEs were typically mild and included infections, gastrointestinal disturbances, dizziness and rashes; AEs resulting in discontinuation of study drug were primarily attributed to infections. CONCLUSION: In patients with active RA receiving stable doses of MTX, pamapimod showed non-significant improvement in efficacy outcomes compared to placebo.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Metotrexato/uso terapéutico , Piridonas/uso terapéutico , Pirimidinas/uso terapéutico , Adolescente , Adulto , Anciano , Antirreumáticos/efectos adversos , Artritis Reumatoide/sangre , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Método Doble Ciego , Quimioterapia Combinada , Humanos , Metotrexato/efectos adversos , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/efectos adversos , Pirimidinas/efectos adversos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto Joven , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase, is the active agent of the immunosuppressive drug, mycophenolate mofetil (MMF). Previous studies have shown that MPA inhibits DNA synthesis in T and B lymphocytes by blocking de novo guanosine synthesis, and that MPA induces monocyte differentiation. MMF is being used for prevention of organ graft rejection and has also shown efficacy in rheumatoid arthritis trials. This study was designed to determine if apoptosis also plays a role in the immunosuppressive and anti-inflammatory effects of MMF. METHODS: Cultured human T lymphocytic (MOLT-4) and monocytic (THP-1 and U937) cell lines were treated with MPA. Apoptosis, cell viability, DNA content, lipid content, cell volume, and lysosomes were measured by a variety of microscopic, flow cytometric, and biochemical techniques. RESULTS: MPA inhibits proliferation, arrests cell cycle in S phase, and increases apoptosis in all three cell lines. Exogenous guanosine added within 24 hr of MPA treatment, but not later, partially reversed MPA-induced apoptosis in MOLT-4 cells. MPA increased lipid droplets in all three cell lines and increased both cell volumes and numbers of lysosomes in the monocytic cell lines. In both monocytic cell lines, MPA also reduced the number of nuclei containing nucleoli and greatly increased neutral lipids, primarily triacylglycerols, suggesting that these cells were differentiating. CONCLUSIONS: Increased apoptosis and terminal differentiation of both lymphocytes and monocytes may promote the antiproliferative, immunosuppressive, and anti-inflammatory effects of MMF seen clinically in transplantation and rheumatoid arthritis.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos/citología , Monocitos/citología , Ácido Micofenólico/farmacología , Línea Celular , Tamaño de la Célula , Relación Dosis-Respuesta a Droga , Guanosina/farmacología , Humanos , Lípidos/análisis , Lisosomas/efectos de los fármacos , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citología , Células U937/química , Células U937/citologíaRESUMEN
The granulomatous pathology in human intestinal schistosomiasis is induced primarily by the egg antigens of schistosome, a parasitic trematode. Glycolipids and glycoproteins were extracted from the eggs of the two major species which infect human, Schistosoma mansoni and Schistosoma japonicum, for structural characterization based on highly sensitive mass spectrometric analysis coupled with chemical derivatization. Here, we demonstrate that a series of uniquely multifucosylated glycosphingolipids constitute the major egg glycolipids of S. mansoni but not of S. japonicum. The S. mansoni glycosphingolipids were found to be extended by varying numbers of an unusual repeating unit, -->4(Fuc1-->2Fuc1-->3)GlcNAc1-->, and terminating as +/-Fuc1-->2Fuc1-->3GalNAc1--> at the nonreducing terminus. The similarity of these fucosylated structures, particularly the nonreducing terminal sequence, to the previously identified multifucosylated O-linked oligosaccharides of the cercarial glycocalyx, suggests that they may constitute the cross-reacting epitopes between the egg antigens and cercariae of S. mansoni. On the other hand, the difucosylated GalNAc terminal epitope was not found on the glycosphingolipids of S. japonicum. Thus, it qualifies for a possible role as a species-specific recognition glycan.
Asunto(s)
Glicoesfingolípidos/química , Oligosacáridos/química , Óvulo/química , Schistosoma japonicum , Schistosoma mansoni , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ácidos Grasos/análisis , Femenino , Fucosa , Glicoesfingolípidos/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The structural diversity of the glycans from Schistosoma mansoni and Schistosoma japonicum egg glycoproteins was investigated using high sensitivity fast atom bombardment mass spectrometric screening of glycan pools released enzymically or chemically from egg extracts. The egg glycoproteins from the two species carry a comparable range of high mannose and complex type N-glycans with both lacNAc and lacdiNAc constituting the backbones of the antennae in the latter class. Truncated N-glycans similar to those found on nematodes, insects, and plants were also identified. Sequential digestion with peptide N-glycosidase F and peptide N-glycosidase A afforded effective release and separation of N-glycans with nonfucosylated or alpha6-monofucosylated trimannosyl N,N'-diacetyl-chitobiose cores from those carrying core alpha3-, alpha6-difucosylation, all of which were found to be present in both species. Remarkably, a portion of the N-glycans from S. mansoni eggs was shown to be based on a xylosylated, alpha6-fucosylated trimannosyl core, whereas a portion of those from S. japonicum contains a xylosylated alpha3-, alpha6-difucosylated core which has not been previously described in any organism. O-Glycans were chemically released from the de-N-glycosylated glycopeptides and found to carry terminal sequences similar to those in the N-glycans. This study provides further evidence that multi-fucosylated terminal HexNAc units, previously identified on the cercarial glycocalyx O-glycans and egg glycosphingolipids, and now on the egg N- and O-glycans, are unique features of S. mansoni glycans. These multifucosylated terminal structures, which were not detected on the egg glycans of S. japonicum, are likely to constitute the cross-reacting epitopes between the eggs and cercariae of S. mansoni. Interestingly other HexNAc termini, including an unusual stretch of HexNAc3, were found to be common to both species. The mapping studies reported in this article provide an important foundation for further structural work in this challenging and important area of glycobiology.
Asunto(s)
Glicoproteínas/química , Oligosacáridos/química , Óvulo/química , Schistosoma japonicum , Schistosoma mansoni , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicoproteínas/aislamiento & purificación , Insectos , Manosa/análisis , Datos de Secuencia Molecular , Nematodos , Oligosacáridos/aislamiento & purificación , Plantas , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Especificidad de la Especie , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
It was predicted from the amino acid sequence of the bone anabolic peptides, parathyroid hormone (PTH) (1-34) and PTH related protein (PTHrP) (1-34), that the C-terminal amino acids form an amphipathic alpha-helix. Therefore, we substituted a model amphipathic alpha-helical peptide (MAP) sequence in the C-terminal region of hPTHrP(1-34), obtaining RS-66271 ([MAP1-10]22-31 hPTHrP(1-34)-NH2). The anabolic activities of RS-66271 and hPTHrP(1-34) were evaluated in 3-month-old, ovariectomized (OVX) osteopenic rats. Subcutaneous injection of hPTHrP(1-34) at 80 micrograms/kg/day partially reversed estrogen depletion trabecular bone loss but was ineffective in the cortex. In contrast, RS-66271 dose-relatedly reversed loss at both sites and, at 80 micrograms/kg/day, returned both trabecular and cortical bone calcium to the level of sham-operated controls. Histomorphometric analysis showed significantly elevated bone formation rates over vehicle-treated OVX in both trabecular and cortical tibial bone following treatment with RS-66271. Electron microscopy showed an increase in the relative surface area of vertebral trabeculae covered by osteoblasts in animals treated with RS-66271. These studies demonstrate that the C-terminal amino acids of hPTHrP(1-34) can be replaced by a model amphipathic helix and that the new chemical entity has greater anabolic activity than the parent peptide. The results suggest that RS-66271 may be a candidate molecule for the treatment of human osteoporosis.
Asunto(s)
Enfermedades Óseas Metabólicas/tratamiento farmacológico , Fémur/efectos de los fármacos , Ovario/fisiología , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/química , Proteínas/química , Columna Vertebral/efectos de los fármacos , Teriparatido/análogos & derivados , Tibia/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Femenino , Fémur/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Ovariectomía , Ratas , Columna Vertebral/metabolismo , Teriparatido/farmacología , Tibia/metabolismoRESUMEN
Schistosomes metabolize large quantities of glucose obtained from the host serum by facilitated diffusion through the tegument. Here we have used rabbit antibodies affinity purified against the carboxyl terminus of a facilitated glucose transporter, SGTP4, to localize the antigen in both schistosomula and adults. By ultrastructural immunocytochemical analysis, SGTP4 was localized to both lipid bilayers that cover the tegumental surface of adults and schistosomula. In the inner bilayer of adults, SGTP4 was apparently oriented with the carboxyl terminus on the internal side of the bilayer. SGTP4 was also present in the discoid and multilamellar bodies in adults and the membranous bodies in schistosomula. Finally, the affinity purified antibodies against SGTP4 bound nonspecifically to the head glands and postacetabular glands in schistosomula. The localization of the antigen in the two surface lipid bilayers suggests that SGTP4 may be responsible for transporting glucose from mammalian host serum into the tegument.
Asunto(s)
Proteínas de Transporte de Monosacáridos/análisis , Schistosoma mansoni/química , Secuencia de Aminoácidos , Animales , Femenino , Secciones por Congelación , Membrana Dobles de Lípidos/química , Masculino , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/química , Orgánulos/química , Schistosoma mansoni/ultraestructuraAsunto(s)
Proteínas del Helminto/análisis , Proteínas de la Membrana/análisis , Schistosoma mansoni/química , Animales , Western Blotting , ADN de Helmintos/biosíntesis , Matriz Extracelular/química , Matriz Extracelular/ultraestructura , Secciones por Congelación , Proteínas del Helminto/química , Proteínas del Helminto/genética , Inmunohistoquímica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Inmunoelectrónica , Reacción en Cadena de la Polimerasa , Schistosoma mansoni/ultraestructuraRESUMEN
Leflunomide is a novel immunosuppressive compound that is effective in the treatment of animal models of autoimmune disease and human rheumatoid arthritis. The mechanism of action is unknown. Here we show that leflunomide blocked 1) increases in nucleolar size and number, 2) upregulation of the nuclear protein antigens (PCNA and Ki-67), 3) increases in uridine incorporation and total RNA and DNA content, 4) cell cycle progression and 5) proliferation in mitogen-stimulated rat spleen mononuclear cells and human peripheral blood mononuclear cells (HPBMC). Exogenous uridine reversed the leflunomide-dependent inhibition of the normal increase in total RNA and DNA content in mitogen-stimulated HPBMC and rat spleen cells. Uridine reversed the leflunomide-dependent inhibition of cell cycle progression in stimulated rat cell cultures. Either uridine or cytidine, which can be converted to uridine by cytidine deaminase, reversed the antiproliferative effect of leflunomide in HPBMC. Dihydroorotate accumulated in leflunomide-treated human T-lymphoblastoid cells, suggesting that the compound inhibited the fourth enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase. The results support the hypothesis that the in vitro effects of leflunomide on T-lymphocytes are due to inhibition of de novo pyrimidine synthesis.
Asunto(s)
Inmunosupresores/farmacología , Isoxazoles/farmacología , Activación de Linfocitos/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Pirimidinas/biosíntesis , Animales , Ciclo Celular/efectos de los fármacos , Nucléolo Celular/ultraestructura , Células Cultivadas , ADN/metabolismo , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Antígeno Ki-67 , Leflunamida , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN/metabolismo , Ratas , Linfocitos T/efectos de los fármacos , Linfocitos T/ultraestructura , Uridina/metabolismoRESUMEN
RS-23581, a synthetic analog of human PTH-related protein-(1-34), and the amino-terminal 34 amino acids of bovine PTH [bPTH-(1-34)] increase bone mineral density. We wished to determine how quickly the ultrastructure of the osteogenic cells, i.e. osteoblasts and lining cells, of the cancellous bone of the second lumbar vertebra of ovariectomized rats was altered in response to the initiation and cessation of treatment. Ovariectomized rats were injected daily with 80 micrograms/kg RS-23581, bPTH-(1-34), or vehicle for 19 days. Animals were killed throughout the treatment period and during the ensuing 10 days. By 5 days after the initiation of treatment with either peptide, the cells on the trabecular surface were predominantly (> 90%) osteoblasts, with only a small increase in the total cell number. Throughout the dosing period, the relative area of the cytoplasm of osteogenic cells from rats treated with RS-23581 or bPTH-(1-34) was greater than that of cells from the ovariectomized control group, suggesting a relationship between bone formation and cytoplasmic mass. By 7 days after the cessation of treatment, the trabecular surface was covered predominantly by lining cells without a change in cell number. Thus, these peptides apparently promote the osteoblast phenotype; the osteoblasts revert to lining cells after the peptides are withdrawn.
Asunto(s)
Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/ultraestructura , Osteogénesis , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Animales , Bovinos , División Celular/efectos de los fármacos , Femenino , Microscopía Electrónica , Osteoblastos/efectos de los fármacos , Osteoblastos/ultraestructura , Ovariectomía , Ratas , Ratas Sprague-Dawley , TeriparatidoRESUMEN
The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R-->Gal beta-1-->3GalNAc, and type 2, R-->Gal beta 1-->3(R-->GlcNAc beta-1-->6)GalNAc, core structures via repeat units of (3GalNAc beta 1-->4(Fuc alpha 1-->2Fuc alpha 1-->2Fuc alpha 1-->3)GlcNAc beta-1-->3Gal alpha-->)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1-->4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.
Asunto(s)
Glicoproteínas/química , Polisacáridos/química , Schistosoma mansoni/química , Animales , Secuencia de Carbohidratos , Glicoproteínas/fisiología , Glicósido Hidrolasas/farmacología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Polisacáridos/fisiología , alfa-L-Fucosidasa/farmacologíaRESUMEN
Adult parasites of Schistosoma mansoni reside within vertebrate mesenteric veins where they consume immense quantities of host glucose after transporting the sugar through their surface syncytium or tegument. Previously we obtained cDNA clones encoding two functional facilitated diffusion glucose transporter proteins expressed by S. mansoni adult worms (Skelly et al. 1994). Antibodies specific for one transporter (SGTP1) have been generated against an extrafacial and an internal domain of the protein and used to localize the protein by light and electron microscopy. By light microscopy both antibodies stain a linear structure approximately 1-5 microns from the surface of the tegument of adult male and female schistosomes. Electron microscopic examination of frozen thin sections show binding of the antibodies to membranes in the base of the tegument and not to the membranes covering the outer surface or their invaginations. Analysis of the gold distribution suggests that the extrafacial domain is disposed toward the interstitial space beneath the tegument and the internal domain faces the syncytial plasm. The localization suggests that SGTP1 may function to transport free glucose from within the tegument and into the interstitial fluids that bathe the internal organs of these parasites.
Asunto(s)
Membrana Celular/química , Proteínas de Transporte de Monosacáridos/análisis , Schistosoma mansoni/química , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos , Secuencia de Bases , Línea Celular , Membrana Celular/ultraestructura , Difusión , Femenino , Cuerpos de Inclusión/inmunología , Masculino , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas de Transporte de Monosacáridos/inmunología , Péptidos/síntesis química , Péptidos/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Schistosoma mansoni/inmunología , Schistosoma mansoni/ultraestructura , SpodopteraAsunto(s)
Receptores de IgE/análisis , Esquistosomiasis mansoni/patología , Bazo/patología , Animales , Basófilos/ultraestructura , Gránulos Citoplasmáticos/ultraestructura , Femenino , Mastocitos/ultraestructura , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Bazo/inmunología , Bazo/ultraestructuraRESUMEN
The glycocalyx (GCX) that covers schistosomal cercariae is a complex molecule that has immunomodulating properties. Here, we purified milligram amounts of GCX using Anguilla lectin which binds to the GCX covering the cercarial body and tail. Typically, 10 million cercariae were extracted with phenol, dialyzed, and chromatographed on a Sepharose 2B-CL column. An average of 39 mg of total carbohydrate eluted near the void volume from which 31 mg of glycogen-like material was further separated by lectin affinity chromatography. Its identity was established by compositional analysis, sensitivity to amylase digestion, and its nuclear magnetic resonance spectrum. The lectin-bound GCX was eluted with 0.1 M fucose with a final yield of 5.3 mg carbohydrate. Fucose composed 40% of the total GCX carbohydrate with lesser but approximately equal amounts of galactose, glucosamine, and galactosamine present. NMR data indicated that the amino sugars were N-acetylated. Glucose was also present but in varying amounts in different preparations of GCX. Oligosaccharides were released from GCX by hydrazinolysis and separated by electrophoresis after reductive amination to 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). Bands comigrating with standards containing 11, 12, 16, and 17 sugar residues were detected. Thus, the GCX is a complex structure composed of oligosaccharides, probably linked to a peptide.
Asunto(s)
Glucógeno/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Polisacáridos/aislamiento & purificación , Schistosoma mansoni/química , Amino Azúcares/análisis , Animales , Cromatografía de Afinidad , Cromatografía en Capa Delgada , Fucosa/análisis , Glucógeno/química , Glicoproteínas/química , Proteínas del Helminto/análisis , Lectinas/metabolismo , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Monosacáridos/análisis , Oligosacáridos/análisis , Polisacáridos/química , Schistosoma mansoni/metabolismoRESUMEN
OBJECTIVE: To test the hypothesis that the effects of estrogen reduction on uterine leiomyoma regression are mediated through changes in cell density or the extracellular matrix. METHODS: Uterine myomas were obtained from 20 women who had received randomly either the GnRH agonist leuprolide acetate depot for 3 months or placebo. The biochemical and morphologic characteristics studied included: total protein, DNA, and amino acid concentrations; histologic appearance; collagen content; and nuclear density. RESULTS: The absolute and relative concentrations of hydroxylysine, hydroxyproline, glycine, and proline were significantly greater (P < .05) in uterine myomas from patients pretreated with a GnRH agonist compared with placebo-treated controls. The GnRH agonist was also associated with trends toward increased mean total protein, DNA, and nuclear density, but the differences did not reach statistical significance. CONCLUSIONS: The concentrations of the amino acids contained in collagen were significantly greater in uterine myomas from patients treated with the GnRH agonist compared to myomas from placebo-treated controls. In addition, our observations suggest that the reduction in uterine myoma volume associated with GnRH agonist therapy is associated with alterations in the extracellular matrix.
Asunto(s)
Colágeno/análisis , Leiomioma/tratamiento farmacológico , Leuprolida/uso terapéutico , Neoplasias Uterinas/tratamiento farmacológico , Aminoácidos/análisis , Colágeno/biosíntesis , ADN de Neoplasias/análisis , Método Doble Ciego , Femenino , Humanos , Leiomioma/química , Leiomioma/metabolismo , Leiomioma/patología , Neoplasias Uterinas/química , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
Human low-density lipoproteins (LDL) bind specifically and saturably to the surface of the trematode parasite, Schistosoma mansoni, in vitro. Here we have tested whether human monocytes process the bound LDL. Monocytes obtained by leukapheresis generate H2O2, kill schistosomula, and were seen here endocytosing fluorescently labeled human LDL that was bound to the surface of the parasites. Compounds known to inhibit uptake of LDL via the scavenger receptor, namely, acetylated LDL, polyinosinic acid, dextran sulfate, fucoidan, and polyvinyl sulfate, inhibited both endocytosis of LDL and cell-mediated killing. Non-functional analogs of these inhibitors, namely, polycytidylic acid and dextran, did not inhibit either endocytosis or killing. Monocytes obtained from whole blood after venipuncture neither killed the parasite nor endocytosed LDL from the worm surface. Thus, human monocyte killing of schistosomula may involve removal of LDL from the parasite surface via scavenger receptors.
Asunto(s)
Proteínas de la Membrana , Monocitos/fisiología , Receptores Inmunológicos/fisiología , Receptores de Lipoproteína , Schistosoma mansoni/fisiología , Animales , Carbocianinas , Endocitosis , Colorantes Fluorescentes , Humanos , Lipoproteínas LDL/metabolismo , Monocitos/metabolismo , Monocitos/parasitología , Receptores Depuradores , Receptores Depuradores de Clase B , Schistosoma mansoni/metabolismo , Schistosoma mansoni/parasitologíaRESUMEN
Low density lipoproteins (LDL) bound to the surface of Schistosoma mansoni may protect the parasite from assault by the immune system and provide essential lipids for the parasite in human schistosomiasis. Here we have characterized the LDL binding sites on the surface of schistosomula by comparing the binding of fluorescently labeled LDL to the parasite with LDL binding proteins as seen by ligand blotting before and after enzymatic treatment of viable parasites. Ligand blotting revealed two LDL binding bands, 17.8 +/- 0.8 and 15.7 +/- 0.6 kDa, in intact schistosomula. Trypsinization eliminated all of the specific and approximately two-thirds of the total LDL binding capacity of schistosomula in a time and concentration-dependent manner. LDL did not bind to any bands on blots of trypsinized, viable worms. Specific LDL binding was also eliminated by phosphatidylinositol-specific phospholipase C (PIPLC). PIPLC treatment removed both LDL binding bands from the worms and caused the appearance of an LDL binding band, 16.6 +/- 0.3 kDa, in the culture medium. LDL binding to the parasite recovered within 24 to 48 h after trypsinization but the recovery was inhibited by either monensin or puromycin. Both LDL binding bands reappeared in ligand blots of cultured worms within 24 h; the reappearance was blocked by puromycin but not by monensin. These studies suggest that the specific binding of human LDL to schistosomula is mediated by GPI-linked low molecular weight proteins that are continually synthesized and transported to the parasite surface.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Helminto/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana/metabolismo , Schistosoma mansoni/metabolismo , Marcadores de Afinidad , Animales , Carbocianinas , Proteínas Portadoras/efectos de los fármacos , Colorantes Fluorescentes , Glucolípidos/metabolismo , Glicosilfosfatidilinositoles , Lipoproteínas LDL/antagonistas & inhibidores , Proteínas de la Membrana/efectos de los fármacos , Péptido Hidrolasas/farmacología , Fosfatidilinositol Diacilglicerol-Liasa , Fosfatidilinositoles/metabolismo , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/farmacología , Schistosoma mansoni/efectos de los fármacos , Tripsina/farmacologíaRESUMEN
OBJECTIVE: To determine the consequences of mast cell (MC)-chondrocyte interactions. METHODS: Cocultured cells were analyzed histochemically, morphologically, biochemically, and functionally. RESULTS: Cocultured MC adhered to the chondrocytes and remained viable. Chondrocytes cocultured with nonactivated MC produced more proteoglycans than did chondrocytes cultured alone, and these proteoglycans possessed an intact hyaluronic acid-binding region. In contrast, most of the proteoglycans produced by chondrocytes cocultured with activated MC were degraded. CONCLUSION: These studies indicate that a complex interaction occurs in which the nonactivated MC stimulates biosynthesis and the activated MC degrades cartilage proteoglycans.
Asunto(s)
Cartílago Articular/citología , Mastocitos/citología , Proteoglicanos/metabolismo , Animales , Cartílago Articular/metabolismo , Supervivencia Celular , Células Cultivadas/ultraestructura , Condrosarcoma/patología , Ratas , Ratas Endogámicas , Radioisótopos de Azufre , Factores de Tiempo , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here.
Asunto(s)
Antígenos de Superficie/fisiología , Moléculas de Adhesión Celular/fisiología , Linfocitos/fisiología , Glicoproteínas de Membrana/fisiología , Anticuerpos Monoclonales , Antígenos CD/fisiología , Antígenos de Superficie/inmunología , Antígenos CD58 , Adhesión Celular , Moléculas de Adhesión Celular/inmunología , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Movimiento Celular , Aparato de Golgi/fisiología , Aparato de Golgi/ultraestructura , Humanos , Molécula 1 de Adhesión Intercelular , Células Asesinas Naturales/inmunología , Membrana Dobles de Lípidos , Linfocitos/inmunología , Linfocitos/ultraestructura , Glicoproteínas de Membrana/inmunología , Microscopía ElectrónicaRESUMEN
The efficacy of human peripheral blood monocytes (PBM) in killing of schistosomula is controversial. The purpose of this study was to determine the schistosomulacidal activity of human monocytes isolated by two different techniques. Peripheral blood monocytes were obtained either by venipuncture (PBMv) or plasmapheresis (PBMp), purified on Ficoll-Paque, and cultured briefly. The cells then were incubated with schistosomula (cell parasite ratio of 10(4):1) for 16 to 18 hours with or without interferon-gamma IFN-gamma (600 U/ml) or sera from patients with schistosomiasis as a source of antischistosomal antibodies (HASA). Freshly isolated PBMv treated with IFN-gamma or HASA did not kill schistosomula. Freshly isolated PBMp alone killed 22 +/- 13% (mean +/- standard deviation [SD]; n = 9) of worms over background and after incubation with IFN-gamma and HASA, 30 +/- 17%. PBMp cultured in vitro for 7 days killed 50 +/- 15% (mean +/- SD; n = 12) of the schistosomula. Pretreatment of the cells with IFN-gamma and incubation with HASA did not significantly enhance the parasite killing beyond this level. Electron microscopy showed that freshly isolated PBMp attached to the worms and fused occasionally with the outer tegumental membrane. Granules constituted 1.4% of the cytoplasmic volume. Degranulation onto the parasite surface was not observed. Peripheral blood monocytes obtained by plasmapheresis accumulated glycogen during in vitro culture with the parasite and released threefold more H2O2 than PBMv after exposure to phorbol myristate acetate. Thus plasmapheresis increases the schistosomulacidal activity of PBM, enhances the generation of H2O2 and promotes the accumulation of glycogen.
Asunto(s)
Monocitos/fisiología , Plasmaféresis , Schistosoma mansoni , Animales , Venodisección , Separación Celular , Supervivencia Celular , Células Cultivadas , Humanos , Peróxido de Hidrógeno/metabolismo , Microscopía Electrónica , Monocitos/metabolismo , Monocitos/ultraestructura , Schistosoma mansoni/crecimiento & desarrolloRESUMEN
Schistosomula of Schistosoma mansoni develop the ability to ingest and digest red blood cells after the fourth day post-transformation. Here, we have used fluorescently-labeled dextrans and two plasma proteins, albumin and IgG, to test whether day-old schistosomula can ingest and process macromolecules prior to the time that they eat red cells. Worms ingested dextrans of molecular weights 4,000, 70,000 and 2 x 10(6) in a time- and concentration-dependent manner. The dextran remained in the cecal lumen for up to 2 days after feeding. Parasites ingested both fluorescein-conjugated bovine serum albumin and rabbit IgG, but neither of these proteins remained confined to the cecum over time. Instead, fluorescence redistributed to the acetabular glands within a few hours. Thin-layer chromatography indicated that schistosomula degraded fluorescein-conjugated albumin to fluorescein-conjugated peptides approximately 10-15 amino acids long. The volume of the cecum was estimated to be 2431 microns 3 and the surface area 299 microns 2. These results demonstrate that larval schistosomes can ingest both proteins and complex carbohydrates shortly after transformation, before they can ingest red cells. Further, the gut apparently releases proteases that cleave plasma proteins, but not saccharidases that cleave dextran.