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1.
Scand J Immunol ; 79(2): 90-7, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24313893

RESUMEN

Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the anti-apoptotic protein Bcl-xL , known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour-progressive activity in a murine lymphoma model. Bcl-xL overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl-xL protection of tumours from NK cell-mediated attack did not involve protection from NK cell-mediated cytotoxicity. Instead, Bcl-xL -blocked apoptosis resulting from hypoxia and/or nutrient loss associated with the inhibition of angiogenesis caused by NK cell-secreted IFN-γ. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.


Asunto(s)
Interferón gamma/fisiología , Células Asesinas Naturales/inmunología , Linfoma/inmunología , Neovascularización Patológica/prevención & control , Escape del Tumor , Proteína bcl-X/fisiología , Animales , Línea Celular Tumoral , Ciclohexanos/farmacología , Citotoxicidad Inmunológica , Humanos , Ratones , Ratones Endogámicos C57BL , O-(Cloroacetilcarbamoil) Fumagilol , Sesquiterpenos/farmacología
2.
Osteoarthritis Cartilage ; 18(8): 1096-103, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20510384

RESUMEN

OBJECTIVE: Mineralization has been observed in osteoarthritic cartilage but the mechanisms are incompletely understood. Vitamin K is an essential cofactor in post-translational modification of proteins where specific Glu residues become modified to Ca(++) binding gamma-carboxyglutamic acid residues (Gla). One such protein, matrix Gla protein (MGP), is a known mineralization inhibitor. This study determined if synthesis of MGP and formation of a fetuin-MGP protein complex was altered in chondrocytes and vesicles from osteoarthritis (OA) cartilage. METHODS: Chondrocytes and vesicles were isolated from normal and OA human articular cartilage and lysates prepared. Specific antibodies were used in immunoblotting to detect the mature fully gamma-carboxylated form of MGP (cMGP) and non-gamma-carboxylated MGP (ucMGP) as well as fetuin and MGP-fetuin complexes. gamma-carboxylase activity was measured by (14)CO(2) incorporation into the carboxylase peptide substrate FLEEL. Immunocytochemistry was used to examine fetuin in cartilage sections and uptake of biotin-labeled fetuin by isolated chondrocytes. RESULTS: Chondrocytes and vesicles from osteoarthritic tissue produced significantly less cMGP compared to those from normal cartilage. This correlated with significantly less vitamin K-dependent gamma-carboxylase enzyme activity in OA chondrocytes. Fetuin was found to be present in articular cartilage and cultured chondrocytes were capable of fetuin uptake. A fetuin-MGP complex was identified in normal chondrocytes and in vesicles shed from these cells but not in OA cells or vesicles. CONCLUSIONS: The absence of cMGP and of the cMGP-fetuin complex in OA cells and OA vesicles may be an important mechanism for increased mineralization of osteoarthritic cartilage.


Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Osteoartritis/metabolismo , Vitamina K/biosíntesis , alfa-Fetoproteínas/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Proteína Gla de la Matriz
3.
Eur J Clin Nutr ; 57(9): 1052-9, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12947422

RESUMEN

OBJECTIVE: To study the possibility of increasing the very long-chain n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in humans by means of consumption of a common food product, Scandinavian caviar paste, suitable for strategic enrichment with a high concentration of these fatty acids, and to measure the potential inducement of lipid peroxidation. DESIGN: A randomized double blind repeated measures experiment. SUBJECTS AND INTERVENTIONS: In total, 16 healthy, nonsmoking subjects (eight men and eight women, age 42+/-12 y) were included in the study. Eight consumed 25 g ordinary caviar paste daily for 3 weeks, and eight the same amount of caviar paste enriched with a very stable fish oil (7%, wt/wt). Blood lipids, plasma phospholipid fatty acids and lipid peroxidation were measured. RESULTS: alpha-Linoleic acid was significantly decreased after intake of both ordinary (-8%, P<0.05) and fish oil caviar (-10%, P<0.05), as was the sum of all n-6 fatty acids (-6%, P<0.05 and -8%, P<0.001, respectively). The fatty acids EPA and DHA, as well as the sum of all n-3 fatty acids, increased significantly in both caviar groups but more in the group given fish oil caviar paste (EPA: +51%, P<0.05 and +100%, P<0.001, respectively; DHA: +24%, P<0.01 and +29%, P<0.001, respectively; sum of n-3:+27%, P<0.05 and +40%, P<0.001, respectively). Lipid peroxidation, measured as the thiobarbituric acid-malondialdehyde adduct, was increased by 26% (P<0.05) after intake of ordinary caviar paste, but was unchanged after intake of fish oil-enriched caviar paste. CONCLUSION: Scandinavian caviar paste is a spread naturally enriched with n-3 polyunsaturated fatty acids that can be included in the diet to achieve an increase in these fatty acids. However, changing to caviar paste enriched with stable fish oil will lead to a considerably greater increase in EPA and DHA. SPONSORSHIP: Swedish Medical Research Council; Cardinova AB, Uppsala, Sweden.


Asunto(s)
Huevos , Ácidos Grasos/sangre , Aceites de Pescado/farmacología , Alimentos Fortificados , Peroxidación de Lípido/efectos de los fármacos , Fosfolípidos/sangre , Adulto , Anciano , Animales , Método Doble Ciego , Femenino , Aceites de Pescado/administración & dosificación , Productos Pesqueros , Humanos , Peroxidación de Lípido/fisiología , Masculino , Persona de Mediana Edad , Valores de Referencia , Países Escandinavos y Nórdicos , Estadísticas no Paramétricas
4.
J Thromb Haemost ; 1(1): 178-85, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12871556

RESUMEN

The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Here we present additional evidence that the Ca2+-induced conformer of the vitamin K-dependent Gla region in MGP is involved in BMP-2 binding. Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly gamma-carboxylated and did not bind BMP-2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP-2/MGP complex exists in vivo, consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-gamma-carboxylation of MGP, allowing unopposed BMP-2 activity.


Asunto(s)
Enfermedades de la Aorta/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Calcinosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de la Matriz Extracelular , Factor de Crecimiento Transformador beta , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , Calcio/química , Proteínas de Unión al Calcio/farmacología , Bovinos , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Hormona del Crecimiento/química , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Datos de Secuencia Molecular , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Mucosa Nasal/ultraestructura , Tabique Nasal/anomalías , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes , Vitamina K/farmacología , Proteína Gla de la Matriz
5.
J Leukoc Biol ; 70(6): 903-10, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11739553

RESUMEN

The administration of cAMP-elevating agents affects a number of autoimmune and inflammatory conditions. Because dendritic cells (DCs) play a pivotal role in autoimmunity and inflammation, the isolated effects of cAMP-elevating agents on the function of DCs was examined. In a dose-dependent manner, 8-Bromo cAMP, prostaglandin E(2), and 3-isobutyl-1-methylxanthine inhibited tumor necrosis factor alpha release and suppressed antigen presentation by DCs. The same effect was observed with rolipram, a specific inhibitor of phosphodiesterase type 4, but not with inhibitors of other phosphodiesterases. The decreased antigen presentation by DCs was associated with an enhanced production of interleukin (IL)-10 and with lower major histocompatibility complex type II (MHC II) expression. Furthermore, the inhibition of antigen presentation and MHC II expression was significantly reversed by treatment of DCs with neutralizing antibody against IL-10, suggesting the involvement of an IL-10-dependent mechanism. Taken together, these results might explain why certain cAMP-elevating agents such as rolipram are effective in blocking autoimmunity and inflammation.


Asunto(s)
Presentación de Antígeno/efectos de los fármacos , AMP Cíclico/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Células Cultivadas , Interleucina-10/inmunología , Ratones , Ratones Endogámicos BALB C , Inhibidores de Fosfodiesterasa/farmacología , Rolipram/farmacología , Factor de Necrosis Tumoral alfa/inmunología
6.
Clin Exp Allergy ; 31(10): 1583-93, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11678859

RESUMEN

BACKGROUND: The yeast Malassezia furfur (M. furfur), present in the normal microflora of human skin, can act as an allergen that incites specific IgE reactivity and T cell proliferation in atopic dermatitis (AD) patients. The role of antigen presenting dendritic cells (DCs) in the onset and maintenance of AD is not well established. OBJECTIVE: The objective of the present study was to assess whether the interaction of M. furfur with human DCs will result in DC maturation, cytokine production and lymphocyte proliferation. METHODS: Monocyte-derived dendritic cells (MDDCs) were generated from human peripheral blood. Immature MDDCs were cultured with or without M. furfur or plastic beads, and with or without CD40L stimulation. Interaction of yeast cells by MDDCs was studied by time-lapse photography and cytokines were detected in culture supernatants with ELISA. The ability of MDDCs pre-incubated with M. furfur to induce proliferation in autologous lymphocytes was measured by [(3)H]-thymidine incorporation. RESULTS: Time-lapse photography showed that the majority of immature MDDCs internalized whole M. furfur yeast cells within 1 h. The presence of M. furfur induced maturation (CD83 expression) of MDDCs, and up-regulation of the costimulatory molecules CD80 and CD86. Production of TNF-alpha, IL-1 beta and IL-18 by MDDCs increased significantly (P < 0.05 for TNF-alpha and IL-1 beta, and P < 0.01 for IL-18) after the addition of M. furfur, while IL-10 and IL-12p70 levels remained unaltered. The CD40L-stimulated IL12p70 production by MDDCs was decreased in the presence of M. furfur (P < 0.05). Finally, immature MDDCs pre-incubated with M. furfur induced a proliferative response in autologous CD14-depleted peripheral blood mononuclear cells, in a dose-dependent manner. CONCLUSION: The data indicate that immature MDDCs can internalize the opportunistic yeast M. furfur. This process was associated with MDDC maturation, production of pro-inflammatory and immunoregulatory cytokines, which might favour induction of a Th2-type immune response, and a capacity to stimulate lymphocyte proliferation. This chain of events most likely contributes to the inflammatory reaction in AD.


Asunto(s)
Alérgenos/efectos adversos , Células Dendríticas/citología , Malassezia , Levaduras , Antígenos CD/fisiología , Ligando de CD40/biosíntesis , División Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Humanos , Activación de Linfocitos/inmunología , Linfocitos/inmunología , Proyectos Piloto , Valores de Referencia , Factores de Tiempo
7.
FASEB J ; 15(13): 2542-4, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11641264

RESUMEN

Warfarin targets vitamin K 2,3-epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for g-carboxylation of vitamin K-dependent proteins. To identify VKOR, we used 4'-azido-warfarin-3H-alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B5. Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K-dependent g-carboxylation system. The results were repeated in COS-1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin-resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km-values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.


Asunto(s)
Anticoagulantes/farmacología , Warfarina/farmacología , Animales , Células COS , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos/genética , Expresión Génica , Hígado/metabolismo , Pulmón/metabolismo , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/metabolismo , Miocardio/metabolismo , Etiquetas de Fotoafinidad , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transfección , Vitamina K Epóxido Reductasas
8.
J Immunol ; 167(4): 2068-73, 2001 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-11489989

RESUMEN

NK cells provide a line of defense against tumors and virus-infected cells that have lost the expression of one or more MHC class I isoforms. Here, we investigate whether inhibitors of apoptosis can block the rejection of tumors mediated by NK cells, by introducing the long form of Fas-associated death domain-like IL-1beta-converting enzyme-associated inhibitory protein (FLIP(L)) and poxvirus cytokine response modifier A (CrmA) into the MHC class I-deficient T lymphoma cell line RMA-S. RMA-S cells do not normally express Fas in vitro, and it was previously postulated that the rejection of these tumors by NK cells is strictly perforin dependent. We show that perforin-deficient NK cells directly mediate Fas up-regulation on RMA-S cells and thereafter kill the cells in a Fas-dependent manner, and that RMA-S FLIP(L) and RMA-S CrmA are protected from such killing. When injected in immunocompetent recipients, RMA-S cells up-regulate Fas, rendering in vivo-passed mock-transduced cells sensitive to Fas-mediated apoptosis. Moreover, RMA-S FLIP(L) and RMA-S CrmA cells establish aggressive tumors, in contrast to RMA-S mock cells that are rejected. These results demonstrate that FLIP(L) and CrmA function as tumor progression factors by protecting MHC class I-deficient tumors from rejection mediated by NK cells. Moreover, our data indicate that death receptor-mediated apoptosis has a more prominent role in the clearance of NK-sensitive tumors than previously suggested.


Asunto(s)
Apoptosis/inmunología , Proteínas Portadoras/fisiología , Rechazo de Injerto/inmunología , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/inmunología , Serpinas/fisiología , Animales , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/genética , Inhibidores de Cisteína Proteinasa/farmacología , Citotoxicidad Inmunológica/genética , Progresión de la Enfermedad , Vectores Genéticos/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Humanos , Linfoma de Células T/genética , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , Perforina , Proteínas Citotóxicas Formadoras de Poros , Poxviridae/genética , Serpinas/genética , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/trasplante , Regulación hacia Arriba/inmunología , Proteínas Virales/genética , Receptor fas/biosíntesis
9.
Artículo en Inglés | MEDLINE | ID: mdl-11427037

RESUMEN

The effect of a combination of aspirin and fish oil on eicosanoids was studied. Four subjects were given 37.5 mg aspirin orally, and 6 weeks later they received a natural, stable fish oil daily for 1 week before taking the same single dose of aspirin. Blood samples for determination of whole blood production of eicosanoids were taken before and after each experimental period. Serum thromboxane A(2)was decreased by 40% (P<0.05) after aspirin alone, but by 62% (P<0.01) after fish oil + aspirin. Serum prostacyclin (measured as 6-keto PGF(1a)) was decreased by aspirin in both cases. The sum of 6-keto PGF(1a)and its equipotent fish oil-derived analogue Delta(17)-6-keto PGF(1a)was reduced after aspirin intake (55%, NS), but after fish oil + aspirin the reduction was smaller (33%, NS). Leukotriene B(4)was increased by 19% (P<0.05) after aspirin, and decreased by 69% (P<0.05) after fish oil + aspirin. A combination of stable fish oil and aspirin thus improves the eicosanoid pattern more than aspirin alone.


Asunto(s)
Aspirina/farmacología , Eicosanoides/sangre , Ácido Eicosapentaenoico/análogos & derivados , Aceites de Pescado/farmacología , Leucotrieno B4/análogos & derivados , 6-Cetoprostaglandina F1 alfa/sangre , Adulto , Aspirina/administración & dosificación , Interacciones Farmacológicas , Eicosanoides/biosíntesis , Ácido Eicosapentaenoico/sangre , Aceites de Pescado/administración & dosificación , Humanos , Leucotrieno B4/sangre , Persona de Mediana Edad , Tromboxano B2/sangre
10.
Med Res Rev ; 21(4): 274-301, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11410932

RESUMEN

The causes of arterial calcification are beginning to be elucidated. Macrophages, mast cells, and smooth muscle cells are the primary cells implicated in this process. The roles of a variety of bone-related proteins including bone morphogenetic protein-2 (BMP-2), matrix Gla protein (MGP), osteoprotegerin (OPG), osteopontin, and osteonectin in regulating arterial calcification are reviewed. Animals lacking MGP, OPG, smad6, carbonic anhydrase isoenzyme II, fibrillin-1, and klotho gene product develop varying extents of arterial calcification. Hyperlipidemia, vitamin D, nicotine, and warfarin, alone or in various combinations, produce arterial calcification in animal models. MGP has recently been discovered to be an inhibitor of bone morphogenetic protein-2, the principal osteogenic growth factor. Many of the forces that induce arterial calcification may act by disrupting the essential post-translational modification of MGP, allowing BMP-2 to induce mineralization. MGP requires gamma-carboxylation before it is functional, and this process uses vitamin K as an essential cofactor. Vitamin K deficiency, drugs that act as vitamin K antagonists, and oxidant stress are forces that could prevent the formation of GLA residues on MGP. The potential role of arterial apoptosis in calcification is discussed. Potential therapeutic options to limit the rate of arterial calcification are summarized.


Asunto(s)
Arterias/patología , Calcinosis , Modelos Animales , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/fisiopatología , Animales
11.
Acta Anaesthesiol Scand ; 45(5): 583-9, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-11309008

RESUMEN

BACKGROUND: Slow-release formulations of local anaesthetics may produce nerve blocks of long duration. The present study aimed at investigating the in vitro and in vivo properties of a polar lipid formulation for slow release of lignocaine and the effects on nerve block duration by inclusion of dexamethasone into the system. METHODS: In vitro release of lignocaine from the lipid formulation was studied in a US Pharmacopoeia rotating apparatus. Sciatic nerve blocks were induced in rats by 0.1 ml of test formulations containing lignocaine HCl 20 mg. ml-1 in aqueous solution, lignocaine base 20, 100 or 200 mg. ml-1 in lipid formulation or the last formulation with dexamethasone 0.05, 0.5 or 5 mg. ml-1. The durations of sensory and motor block and the arterial blood concentrations of lignocaine were investigated. RESULTS: In vitro there was a sustained release of lignocaine from the lipid formulation, with 50% release at around 48 h. In vivo lignocaine base 20 mg. ml-1 in lipid formulation produced sciatic nerve blocks of significantly shorter duration than lignocaine HCl 20 mg. ml-1 in aqueous solution, while lignocaine base 100 and 200 mg. ml-1 in lipid formulation produced blocks lasting two and three times longer, respectively, than the lignocaine HCl solution. Addition of dexamethasone did not affect the duration of nerve block. Following administration of lignocaine base 200 mg. ml-1 in lipid formulation, as compared to lignocaine HCl 20 mg. ml-1 in aqueous solution, the maximal blood concentration of lignocaine was only three times higher in spite of the ten-fold difference in dose, and the mean terminal half-life was three times longer, reflecting the slow release from the formulation. CONCLUSIONS: Our findings indicate that lignocaine base in polar lipids acts as a slow-release preparation of local anaesthetic both in vitro and in vivo.


Asunto(s)
Anestésicos Locales/farmacología , Lidocaína/farmacología , Bloqueo Nervioso , Nervios Periféricos/efectos de los fármacos , Algoritmos , Anestésicos Locales/administración & dosificación , Anestésicos Locales/sangre , Animales , Antiinflamatorios/farmacología , Preparaciones de Acción Retardada , Dexametasona/farmacología , Lidocaína/administración & dosificación , Lidocaína/sangre , Masculino , Micelas , Soluciones Farmacéuticas , Ratas , Ratas Sprague-Dawley
12.
Lab Invest ; 81(1): 83-93, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11204277

RESUMEN

Although atherosclerosis progresses in an indolent state for decades, the rupture of plaques creates acute ischemic syndromes that may culminate in myocardial infarction and stroke. Mechanical forces and matrix metalloproteinase activity initiate plaque rupture, whereas tissue inhibitors of metalloproteinases have an important (albeit indirect) role in plaque stabilization. In this paper, an enzyme that could directly stabilize the plaque is described. Tissue transglutaminase (TG) catalyzes the formation of epsilon(gamma-glutamyl)lysine isopeptide bonds that are resistant to enzymatic, mechanical, and chemical degradation. We performed immunohistochemistry for TG in atherosclerotic human coronary and carotid arteries. TG was most prominent along the luminal endothelium and in the medium of the vessels with a distribution mirroring that of smooth muscle cells. Variable, often prominent, immunoreactivity for TG was also seen in the intima, especially in regions with significant neovascularization. Additionally, TG was detected in fibrous caps and near the "shoulder regions" of some plaques. A monoclonal antibody to the transglutaminase product epsilon(gamma-glutamyl)lysine isopeptide demonstrated co-localization with TG antigen. Transglutaminase activity was found in 6 of 14 coronary artery atherectomy samples. Cross-linking of TG substrates such as fibrinogen, fibronectin, vitronectin, collagen type I, and protease inhibitors stabilized the plaque. Furthermore, the activation of transforming growth factor-beta-1 by TG might be an additional mechanism for the promotion of plaque stabilization and progression by increasing the synthesis of extracellular matrix components.


Asunto(s)
Enfermedades de las Arterias Carótidas/enzimología , Enfermedades de las Arterias Carótidas/patología , Enfermedad de la Arteria Coronaria/enzimología , Enfermedad de la Arteria Coronaria/patología , Transglutaminasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Transglutaminasas/inmunología
14.
J Immunol ; 165(1): 25-33, 2000 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-10861031

RESUMEN

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1alpha (EF1alpha). EF1alpha occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1alpha peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1alpha peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1alpha isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.


Asunto(s)
Presentación de Antígeno , Supresión Clonal , Antígenos H-2/inmunología , Antígenos H-2/metabolismo , Tolerancia Inmunológica , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD8/biosíntesis , Linfocitos T CD8-positivos/inmunología , Adhesión Celular/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Células Dendríticas/metabolismo , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Oligopéptidos/biosíntesis , Oligopéptidos/inmunología , Oligopéptidos/aislamiento & purificación , Oligopéptidos/metabolismo , Factor 1 de Elongación Peptídica/biosíntesis , Factor 1 de Elongación Peptídica/inmunología , Factor 1 de Elongación Peptídica/metabolismo , Unión Proteica/inmunología , Isoformas de Proteínas/biosíntesis , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de Proteína , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas
15.
Thromb Haemost ; 84(6): 1039-44, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11154111

RESUMEN

Matrix Gla protein (MGP) is an inhibitor of calcification of the arterial wall but the mechanism of inhibition has not been resolved. Since chondrogenesis has been identified in calcified arteries from MPG null mice, we hypothesized that locally produced MGP might inhibit calcification by neutralizing the known effect of bone morphogenetic proteins (BMPs) as promotors of chondrogenesis and bone formation. As the first step to test this hypothesis, we demonstrate that MGP is a binding protein for 125I-BMP-2. Optimal binding is dependent on metals which suggests that the metal binding Gla region in MGP is involved. MGP is shown to undergo a Ca++ induced conformational change despite the presence of the gamma-carboxylase binding site being part of the mature protein sequence. The data propose that MGP matures earlier in the secretory pathway than other vitamin K-dependent proteins. Antibodies were used in an attempt to identify MGP in bovine serum. Conformational specific MGP antibodies were shown to also recognize the Gla region in prothrombin and factor X but did not identify MGP in serum. This finding is supported by electrophoresis data which demonstrate the absence of MGP among Ba-citrate absorbed vitamin K-dependent serum proteins. We conclude that MGP does not exist in normal bovine serum.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular , Factor de Crecimiento Transformador beta , Animales , Anticuerpos/metabolismo , Proteína Morfogenética Ósea 2 , Calcio/metabolismo , Calcio/farmacología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/inmunología , Bovinos , Secuencia Conservada , Epítopos/metabolismo , Factor X/inmunología , Humanos , Plasma/química , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Protrombina/inmunología , Proteína Gla de la Matriz
16.
J Biol Chem ; 274(51): 36601-8, 1999 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-10593961

RESUMEN

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935), we previously showed that p47(phox), a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22(phox), a subunit of the NADPH oxidase component flavocytochrome b(558), also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22(phox). We identified two protein kinase families capable of phosphorylating p22(phox): 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47(phox) and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47(phox). In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22(phox). These findings suggest that phosphorylation of p22(phox) by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.


Asunto(s)
Proteínas de Transporte de Membrana , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Sistema Libre de Células , Activación Enzimática , Isoenzimas/química , Isoenzimas/metabolismo , NADPH Deshidrogenasa/química , NADPH Oxidasas/química , Ácidos Fosfatidicos/química , Fosfoproteínas/química , Fosforilación , Proteína Quinasa C/química
17.
Thromb Haemost ; 82(6): 1764-7, 1999 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-10613667

RESUMEN

Matrix GLA protein (MGP) is an inhibitor of calcification in the arterial wall and its activity is dependent upon vitamin K-dependent gamma-carboxylation. This modification is carried out by a warfarin sensitive enzyme system that converts specific Glu residues to gamma-carboxyglutamic acid (GLA) residues. Recent studies have demonstrated that the gamma-carboxylation system in the arterial wall, in contrast to that in the liver, is unable to use vitamin K as an antidote to warfarin. By use of immunohistochemistry we demonstrate that MGP is expressed in the arterial wall and immunocytochemistry localized the MGP precursors to the endoplasmic reticulum in vascular smooth muscle cells. Resting smooth vascular muscle cells in the aortic wall and proliferating cells from explants of the aorta have all the enzymes needed for gamma-carboxylation of MGP. However, when compared to the liver system, expression of the enzymes of the gamma-carboxylation system in vascular smooth muscle cells is different. Of particular interest is the finding that the specific activity of the warfarin sensitive enzyme vitamin K epoxide reductase is 3-fold higher in vascular smooth muscle cells than in liver. DT-diaphorase, which catalyses the antidotal pathway for vitamin K reduction in liver, is 100-fold less active in resting vascular smooth muscle cells than in liver. Data obtained from an in vitro gamma-carboxylation system suggest that the antidotal pathway catalyzed by DT-diaphorase in the vessel wall is unable to provide the carboxylase with enough reduced vitamin K to trigger gamma-carboxylation of MGP. This finding provides an explanation to the inability of vitamin K to work as an antidote to warfarin intoxication of the arterial wall. Therefore the vitamin K dependent gamma-carboxylation system in the arterial wall share a common feature with the system in bone cells by being unable to utilize vitamin K as an antidote.


Asunto(s)
Aorta/patología , Aorta/fisiología , Proteínas de Unión al Calcio/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiología , Animales , Huesos/fisiología , Ácidos Carboxílicos , Células Cultivadas , Masculino , Ratas , Ratas Sprague-Dawley , Proteína Gla de la Matriz
18.
Biochim Biophys Acta ; 1439(2): 277-90, 1999 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-10425401

RESUMEN

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.


Asunto(s)
Proteínas de Transporte de Membrana , Ácidos Fosfatidicos/fisiología , Fosfolipasa D/metabolismo , Proteínas Quinasas/metabolismo , Sistemas de Mensajero Secundario , Animales , Línea Celular , Sistema Libre de Células , Activación Enzimática/efectos de los fármacos , Humanos , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas/biosíntesis , Neutrófilos/metabolismo , Ácidos Fosfatidicos/biosíntesis , Ácidos Fosfatidicos/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Estallido Respiratorio
19.
Acta Oncol ; 38(2): 221-8, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10227445

RESUMEN

The aim of this study was to visualize non-invasively the uptake of platinum in tumours and tissues after treatment with cisplatin. 191Pt-cisplatin was synthesized from 191PtCl4 with rigorous pharmaceutical quality control. The uptake of platinum by both tumorous and healthy tissues was studied by gamma camera imaging in 14 patients, 5 of whom showed a clear uptake of platinum in regions corresponding to known tumour sites. Maximum concentrations of platinum in the tumours were on average 4.9+/-1.0 microg/g, when normalized to an administered amount of 180 mg cisplatin. In all the patients, the liver was the organ that showed the highest uptake. Platinum uptake was also seen in the spleen, gall bladder, gastrointestinal tract, bladder, kidneys, ureter, neck and mediastinum and urogenital region. By using in-house production of 191Pt-cisplatin, it was possible to monitor the uptake of platinum in tumorous tissues and healthy organs.


Asunto(s)
Antineoplásicos/farmacocinética , Cisplatino/farmacocinética , Cámaras gamma , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Adulto , Anciano , Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/metabolismo , Platino (Metal) , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Radioisótopos , Cintigrafía , Radiofármacos
20.
J Immunol ; 161(12): 6475-9, 1998 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-9862670

RESUMEN

We have analyzed lymphocyte development in natural MHC class I chimeric mice, generated through a transgenic approach in beta2-microglobulin (beta2m)-/- mice. In these mice, MHC class I+ cells coexist with an equal proportion of MHC class I-deficient cells. These MHC class I mosaic mice had normal numbers of CD8+ T cells, which had a target cell specificity similar to that of wild-type mice. Consequently, the mice did not develop any signs of autoimmunity. They also had normal numbers of NK cells. This allowed an examination of the MHC class I influence on the expression of the Ly49C inhibitory receptor on NK cells. This receptor binds to H-2Kb. It is expressed at low levels on NK cells in wild-type mice of the H-2b haplotype, but at markedly higher levels on NK cells in beta2m-/- mice and other strains of mice lacking expression of H-2Kb. Relatively little is known about how MHC class I molecules affect expression of the Ly49 receptors. Through the analysis of the present MHC class I mosaic mice, we demonstrate that the expression levels of Ly49C on NK cells is a consequence not only of MHC class I expression in the environment, but also of the expression of MHC class I molecules by the NK cells themselves. These findings are discussed in relation to the biological role of the calibration of the Ly49 inhibitory receptor expression in relation to self-MHC class I.


Asunto(s)
Antígenos Ly , Quimera/inmunología , Regulación de la Expresión Génica , Células Asesinas Naturales/metabolismo , Glicoproteínas de Membrana/biosíntesis , Animales , Antígenos H-2/genética , Antígenos H-2/inmunología , Haplotipos , Antígeno de Histocompatibilidad H-2D , Lectinas Tipo C , Recuento de Linfocitos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores Similares a Lectina de Células NK , Regulación hacia Arriba , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética
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