RESUMEN
OBJECTIVE: Total joint replacement has been proposed as an endpoint in disease modifying osteoarthritis drug (DMOAD) randomized clinical trials (RCTs); however, disparities have generated concerns regarding this outcome. A combined Osteoarthritis Research Society International (OARSI)/Outcome Measures in Rheumatology (OMERACT) initiative was launched in 2004 to develop a composite index ['virtual total joint replacement' (VJR)] as a surrogate outcome for osteoarthritis (OA) progression in DMOAD RCTs. Our objective was to evaluate the prevalence of patients fulfilling different thresholds of sustained pain, reduced function, and X-ray change in existing DMOAD RCTs. DESIGN: Post hoc analysis of summary data from the placebo arm of eight DMOAD RCTs. RESULTS: Eight OA RCTs representing 1379 patients were included. Pain was assessed by WOMAC and/or VAS and function by WOMAC and/or Lequesne. Among six knee and two hip studies, 248 (22%) and 132 (51%) patients respectively had X-ray progression [decrease joint space width (JSW) ≥0.5 mm]. The prevalence of patients fulfilling clinical and radiographic criteria was highest (n = 163, 12%) in the least stringent scenario (pain + function ≥80 at ≥2 visits); with few patients (n = 129, 2%) in the most stringent scenario (pain + function ≥80 at ≥4 visits). Using these prevalence data, a sample size of 352-2144 per group would be needed to demonstrate a 50% difference between groups. CONCLUSIONS: The prevalence of patients with sustained symptomatic OA of at least a moderate degree with X-ray progression is low. Even using lenient criteria to define VJR, large patient numbers would be required to detect differences between groups in DMOAD RCTs. Investigation of the optimal cutoff threshold and combination of symptoms and radiographic change should be pursued.
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Antirreumáticos/uso terapéutico , Osteoartritis/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Índice de Severidad de la Enfermedad , Artroplastia de Reemplazo , Progresión de la Enfermedad , Determinación de Punto Final , Humanos , Osteoartritis/diagnóstico por imagen , Osteoartritis/fisiopatología , Dimensión del Dolor/métodos , Umbral del Dolor , Placebos , Radiografía , Resultado del TratamientoRESUMEN
OBJECTIVE: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine (1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n=29) and (2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN+CS, in patients with symptomatic knee pain (n=28). METHODS: The concentration of GlcN in the circulation was determined by established fluorophore-assisted carbohydrate electrophoresis (FACE) methods. The hydrodynamic size and disaccharide composition of CS chains in the circulation and dosage samples was determined by Superose 6 chromatography and FACE. RESULTS: We show that circulating levels of CS in human plasma are about 20 microg/ml. Most significantly, the endogenous concentration and CS disaccharide composition were not detectably altered by ingestion of CS, when the CS was taken alone or in combination with GlcN. On the other hand, the Cmax (single-dose study) and AUC values (multiple-dose study) for ingested GlcN were significantly reduced by combination dosing with CS, relative to GlcN dosing alone. CONCLUSIONS: We conclude that pain relief perceived following ingestion of CS probably does not depend on simultaneous or prior intake of GlcN. Further, such effects on joint pain, if present, probably do not result from ingested CS reaching the joint space but may result from changes in cellular activities in the gut lining or in the liver, where concentrations of ingested CS, or its breakdown products, could be substantially elevated following oral ingestion. Moreover, since combined dosing of GlcN with CS was found to reduce the plasma levels seen with GlcN dosing alone, any improved pain relief by combination dosing cannot be explained by higher circulating concentrations of GlcN.
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Artralgia/metabolismo , Sulfatos de Condroitina/farmacocinética , Glucosamina/farmacocinética , Osteoartritis/tratamiento farmacológico , Administración Oral , Adulto , Sulfatos de Condroitina/administración & dosificación , Ensayos Clínicos como Asunto , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Quimioterapia Combinada , Femenino , Glucosamina/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Dimensión del Dolor , Resultado del Tratamiento , Adulto JovenRESUMEN
To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.
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ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Calsecuestrina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico , Chaperonas Moleculares , Células de Purkinje/metabolismo , Animales , Compartimento Celular , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/ultraestructura , Pollos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Microscopía ElectrónicaRESUMEN
Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP.
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Proteínas Portadoras/genética , ADN/genética , Matriz Extracelular/análisis , Músculos/análisis , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Ácido Aspártico/análisis , Ácido Aspártico/genética , Secuencia de Bases , Proteínas de Unión al Calcio , Calsecuestrina , Proteínas Portadoras/análisis , Pollos , Clonación Molecular , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Miocardio/análisis , Hibridación de Ácido Nucleico , Señales de Clasificación de Proteína/genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.
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Calsecuestrina/fisiología , Músculos/metabolismo , Animales , Calcio/metabolismo , Diferenciación Celular , División Celular , Línea Celular , Pollos , Homeostasis , Microscopía Electrónica , Desarrollo de Músculos , Músculos/ultraestructura , RatasRESUMEN
The regulation of nerve growth factor (NGF) protein and NGF messenger RNA (mRNA) in the developing rat brain has been studied to assess the hypothesis that NGF supports the differentiation of cholinergic neurons in the basal forebrain. In the adult, the major targets of these neurons, the hippocampus and neocortex, contain the highest concentrations of NGF mRNA, but comparatively low ratios of NGF protein to its mRNA. In contrast, a high concentration of NGF protein and a low concentration of NGF mRNA were seen in the basal forebrain, consistent with retrograde transport of NGF protein into this region from the neocortex and hippocampus. In these two target regions NGF and NGF mRNA were barely detectable at birth, their concentrations increased to a peak at day 21, and then NGF mRNA, but not NGF protein, declined threefold by day 35. NGF accumulation in the basal forebrain paralleled that in the target regions and preceded an increase in choline acetyltransferase, suggesting that the differentiation of cholinergic projection neurons is indeed regulated by retrogradely transported NGF. In addition, high ratios of NGF protein to NGF mRNA, comparable to that in the basal forebrain, were seen in the olfactory bulb and cerebellum, suggesting that NGF may be transported into these regions by unidentified neurons.
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Encéfalo/crecimiento & desarrollo , Factores de Crecimiento Nervioso/biosíntesis , Animales , Encéfalo/metabolismo , Química Encefálica , Cerebelo/análisis , Corteza Cerebral/análisis , Hipocampo/análisis , Factores de Crecimiento Nervioso/análisis , Factores de Crecimiento Nervioso/genética , ARN Mensajero/análisis , Ratas , Ratas EndogámicasRESUMEN
OBJECTIVE: Evaluate long-term safety and efficacy of etanercept treatment in patients with ankylosing spondylitis (AS). METHODS: Patients with AS who previously participated in a randomised controlled trial (RCT) of etanercept were eligible to enroll in a 168-week open-label extension (OLE). Safety end points included rates of adverse events (AE), serious adverse events (SAE), infections, serious infections and death. Efficacy end points included Assessment in Ankylosing Spondylitis (ASAS20) response, ASAS 5/6 response and partial remission rates. RESULTS: A total of 257 of 277 patients (92%) enrolled in the OLE. After up to 192 weeks of treatment with etanercept, the most common AEs were injection site reactions, headaches and diarrhoea. The exposure-adjusted rate of SAEs was 0.08 per patient-year. The rate of infections was 1.1 per patient-year, and the rate for serious infections was 0.02 per patient-year. No deaths were reported. Of patients who received etanercept in both the RCT and OLE and were still in the trial, 71% were ASAS20 responders at week 96, and 81% were responders at week 192. ASAS 5/6 response rates were 61% at week 96 and 60% at week 144, and partial remission response rates were 41% at week 96 and 44% at week 192. Placebo patients who switched to etanercept in the OLE showed similar patterns of efficacy maintenance. CONCLUSIONS: Etanercept was well tolerated for up to 192 weeks in patients with AS, with no unexpected AEs or SAEs observed. No deaths were reported. Improvements in the signs and symptoms of AS were maintained for up to 192 weeks.
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Antirreumáticos/uso terapéutico , Inmunoglobulina G/uso terapéutico , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Espondilitis Anquilosante/tratamiento farmacológico , Adulto , Antirreumáticos/efectos adversos , Método Doble Ciego , Etanercept , Femenino , Estudios de Seguimiento , Humanos , Inmunoglobulina G/efectos adversos , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
The Glucosamine/chondroitin Arthritis Intervention Trial (GAIT) was a randomized double-blind placebo and active comparator (celecoxib) controlled trial of 1583 persons with symptomatic osteoarthritis (OA) of the knee(1). Patients randomized to celecoxib had significant improvement in knee pain compared to those randomized to placebo. No statistically significant improvement in knee pain compared to placebo was seen among patients randomized to the dietary supplements, although a subset of patients with moderate-to-severe knee pain at entry who were assigned to the combination of glucosamine and chondroitin sulfate did seem to experience some improvement. Additionally, patients taking chondroitin sulfate were noted to have a statistically significant improvement in knee joint swelling. An exploratory post hoc analysis of GAIT patients suggested the effect of chondroitin sulfate on joint swelling occurred more often in patients with milder pain and lower Kellgren-Lawrence Grade at entry.
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Antiinflamatorios no Esteroideos/uso terapéutico , Sulfatos de Condroitina/uso terapéutico , Glucosamina/uso terapéutico , Osteoartritis de la Rodilla/tratamiento farmacológico , Dolor/tratamiento farmacológico , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , Antiinflamatorios no Esteroideos/efectos adversos , Celecoxib , Sulfatos de Condroitina/efectos adversos , Método Doble Ciego , Femenino , Glucosamina/efectos adversos , Humanos , Articulación de la Rodilla/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/diagnóstico por imagen , Pirazoles/efectos adversos , Radiografía , Sulfonamidas/efectos adversos , Resultado del TratamientoRESUMEN
A mammalian expression vector that directs expression of murine beta-nerve growth factor (beta-NGF) from a murine sarcoma virus long terminal repeat (LTR) promoter element was constructed and characterized. The vector, designated pLTRSNGF, was stably transfected into murine L-cells, and beta-NGF mRNA and protein levels were quantified and compared to endogenous levels in control L-cells. Transfection of pLTRSNGF resulted in an approximate doubling of both beta-NGF mRNA and mature beta-NGF protein secreted into the media. Transfection of pLTRSNGF into rat PC 12 cells resulted in colonies of autocrine-differentiating cells that extended dense networks of neurites in the absence of added NGF, indicating that the beta-NGF produced from the vector is biologically active.
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Vectores Genéticos , Factores de Crecimiento Nervioso/genética , Plásmidos , Animales , Células Cultivadas , Células L , Ratones , Factores de Crecimiento Nervioso/metabolismo , Células PC12 , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Virus del Sarcoma Murino/genética , TransfecciónRESUMEN
PURPOSE: To examine the expression of integrin vitronectin receptors (integrins alphavbeta3 and alphavbeta5) in native human fetal retinal pigment epithelium (RPE) and cultured hunman fetal RPE and to examine the role of RPE VnRs in the phagocytosis of photoreceptor rod outer segments (ROS). METHODS: Monoclonal antibodies against human integrin subunit alphav and heterodimers alphavbeta3 (LM609) and alphavbeta5 (P1F6) were used to label freshly isolated human fetal RPE explant and cultured human fetal RPE and to immunoprecipitate membrane proteins from cultured human RPE. Effects of antibodies and peptides that inhibit integrin vitronectin receptors on phagocytosis of ROS by RPE were determined using cultured human fetal RPE and fluorescein-labeled bovine outer segments. RESULTS: Antibodies against integrin subunit alphav and against alphavbeta5 (P1F6) labeled the RPE apical membrane in both native tissue and cultured RPE, while anti-alphavbeta3 antibody (LM609) labeled less than 1% of cultured RPE and did not label native RPE. Antibodies against alphav and alphavbeta5 P1F6 also immunoprecipitated 2 protein bands corresponding to integrin subunits alphav and beta5 from a membrane extract of cultured human RPE. The peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP, 1 mM) inhibited the total ROS uptake (externally bound and ingested) by 48% (P < 0.001) and ingestion of ROS by 37% (P < 0.001). Antibody PIF6 (50 microg/ml) inhibited the total ROS uptake and the ingestion of ROS by 63% (P < 0.001) and 43% (P < 0.01), respectively. CONCLUSIONS: The integrin alphavbeta5 vitronectin receptor is expressed on the apical membrane of human RPE and participates in the binding of photoreceptor ROS during phagocytosis by cultured human RPE.
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Integrinas/metabolismo , Fagocitosis , Epitelio Pigmentado Ocular/fisiología , Receptores de Vitronectina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Animales , Anticuerpos Monoclonales , Bovinos , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Feto , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Oligopéptidos/farmacología , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Pruebas de PrecipitinaRESUMEN
PURPOSE: To identify integrin vitronectin receptor subunit mRNAs in the developing avian retina and to track their expression. METHODS: Reverse transcription-polymerase chain reaction was used to identify integrin vitronectin receptor subunit mRNAs expressed in embryonic chick retina. cDNA clones encoding the beta 5 subunit were isolated and sequenced. Expression patterns of mRNAs encoding alpha v, beta 3, and beta 5 subunits were analyzed using northern analysis and in situ hybridization. RESULTS: Integrin beta 1, beta 3, and beta 5 subunit mRNA were identified in embryonic day 6 chick retina. The sequence of chicken beta 5 was 77% identical to that of human beta 5, and sequences with known signaling functions were highly conserved. Integrin alpha v, beta 3, and beta 5 mRNAs were expressed throughout the development of the embryonic retina, with the highest levels per retina observed around the embryonic day 9. In situ hybridization showed that both beta 3 and beta 5 were expressed throughout the developing retina, particularly in undifferentiated neuroepithelial precursor cells. At later times, beta 3 was expressed uniformly throughout the retina, whereas beta 5 expression was highest in a band throughout the central retina. CONCLUSIONS: The strong conservation of sequences with known signaling functions in chicken beta 5 suggests that it functions in a manner similar to human beta 5. Spatial expression patterns of vitronectin receptor subunits at early times of development point to a range of possible functions beyond axon outgrowth, including retinoblast proliferation, adhesion, and migration.
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Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/metabolismo , Receptores de Vitronectina/metabolismo , Retina/embriología , Retina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Embrión de Pollo , Pollos , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Humanos , Hibridación in Situ , Integrinas/genética , Integrinas/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Receptores de Vitronectina/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética , Vitronectina/genética , Vitronectina/metabolismoRESUMEN
Methotrexate therapy has been effective in the treatment of RA with short term experience suggesting little serious adverse reactions. Our review of 168 patients receiving methotrexate has identified nine patients with probable or possible methotrexate-induced pulmonary toxicity, giving a prevalence of 5% and an incidence of 3.9 per 100 patients per year. No clinical or laboratory features showed an association that could potentially predict the development of pulmonary disease. All patients experienced complete recovery with supportive care and/or corticosteroid therapy. Clinical monitoring for this complication is warranted in all patients receiving long term methotrexate therapy for RA.
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Artritis Reumatoide/tratamiento farmacológico , Enfermedades Pulmonares/inducido químicamente , Metotrexato/efectos adversos , Adulto , Anciano , Femenino , Humanos , Enfermedades Pulmonares/complicaciones , Enfermedades Pulmonares/fisiopatología , Masculino , Metotrexato/administración & dosificación , Metotrexato/uso terapéutico , Persona de Mediana EdadRESUMEN
Our clinical experience in 28 patients receiving chlorambucil for rheumatoid arthritis (RA) and the reports on chlorambucil therapy are reviewed. Our study population and other reports generally represent patients with severe RA who had either failed to improve or developed significant toxicity during previous treatment with conventional slow acting anti-rheumatic drugs (SAARDs). Seventy-two percent of patients had a significant clinical improvement during chlorambucil therapy and reports of complete remission are given, although the incidence of remission is unknown. Hematologic complications are often reported, but appeared more frequently in our experience than previously reported. Hematologic toxicity required that chlorambucil be discontinued in the majority of our cases. Two deaths from suspected drug induced malignancies are reported. Although chlorambucil appears to be effective in the control of active RA, the potential for drug induced toxicity and malignancies may outweigh the benefit of continued use of this experimental therapy in RA.
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Artritis Reumatoide/tratamiento farmacológico , Clorambucilo/uso terapéutico , Adulto , Artritis Reumatoide/complicaciones , Artritis Reumatoide/fisiopatología , Clorambucilo/efectos adversos , Evaluación de Medicamentos , Femenino , Humanos , Leucemia Mieloide Aguda/complicaciones , Leucopenia/inducido químicamente , Enfermedades Linfáticas/complicaciones , Masculino , Persona de Mediana Edad , Mielofibrosis Primaria/complicaciones , Trombocitopenia/inducido químicamenteRESUMEN
The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.
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Técnicas Genéticas , Transfección , Animales , Fosfatos de Calcio , Diferenciación Celular/genética , Cloranfenicol O-Acetiltransferasa , ADN , Vectores Genéticos , Cinética , Liposomas , Factores de Crecimiento Nervioso/fisiología , Feocromocitoma/genética , Regiones Promotoras Genéticas , Compuestos de Amonio Cuaternario , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To study the Spondylitis Functional Index (SFI) by having two physical therapists observe patients with spondylitis perform various tasks listed on the instrument. The physical therapists' observations were compared with each other and with the self-reported abilities of the patients. METHODS: Subjects (n = 30) were recruited from a cross-section of patients participating in a prospective randomized, multicenter, double-blind, parallel clinical trial of the efficacy of sulfasalazine on ankylosing spondylitis (n = 13), psoriatic arthritis (n = 13), and Reiter's syndrome (n = 4) conducted at the Veterans Affairs Medical Center in Salt Lake City. Percents of agreement and Cohen's kappa analysis were used to assess the reliability of the observations of the therapists and patients. RESULTS: The overall percent of agreement between the observers on the SFI was 93%. The overall percent of agreement between observer 1 and patients on the SFI was 66% and between observer 2 and patients was 67%. The overall inter-observer reliability measured by the Pearson coefficient was 0.91 and by Cohen's kappa was 0.86. Between observer 1 and the patients the Pearson was r = 0.53 and kappa = 0.39. For observer 2 the Pearson was r = 0.52 and kappa 0.39. CONCLUSIONS: We consider the agreement and reliability between observers to be high. The agreement and inter-observer reliability was poor between observers and patients. The SFI, as enhanced for use in this study to assess change in functional ability of patients with spondylitis, demonstrated high reliability when used by trained observers.
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Actividades Cotidianas , Modalidades de Fisioterapia , Espondilitis/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Espondilitis/clasificaciónRESUMEN
Laminin-5 (Ln-5) is an essential component of epithelial basal laminae that is also expressed in the developing nervous system. Here we use a convenient, simple and reproducible in vitro fluorescent assay to assess the neurite outgrowth promoting activity of purified Ln-5. Embryonic chick neurons from dorsal root ganglia, ciliary ganglia, and (to a lesser extent) retina extended neurites on Ln-5, but the neurite outgrowth promoting activity was not as great as that of Ln-1 or Ln-2. Neurons from diencephalon, telencephalon, and spinal cord did not respond to Ln-5.
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Moléculas de Adhesión Celular/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Animales , Encéfalo/citología , Células Cultivadas , Embrión de Pollo , Matriz Extracelular/fisiología , Ganglios Parasimpáticos/citología , Ganglios Espinales/citología , Microscopía Fluorescente/métodos , Retina/citología , Médula Espinal/citología , KalininaRESUMEN
Ankylosing spondylitis (AS) is a common (prevalence 0.2-0.9%) chronic inflammatory disease that mainly affects young males and is characterised by inflammatory back pain with sacroiliitis and often arthritis of the peripheral joints. The disease can lead to deformities of the vertebral column, joints and extra-spinal structures, e.g. the eye (uveitis). Non-steroidal anti-inflammatory drugs (NSAIDs) and physical therapy seem to improve the long-term outcome of AS. However, the effect of disease modifying antirheumatic drugs (DMARDs) is less impressive compared with other rheumatic diseases, such as rheumatoid arthritis (RA). In placebo controlled trials, sulfasalazine showed some improvement of disease activity, especially in spondyloarthropathy patients with peripheral arthritis. Altogether the number of therapeutic options for AS is limited and other drugs, such as leflunomide or thalidomide, should be explored further in placebo-controlled trials.
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Antirreumáticos/uso terapéutico , Espondilitis Anquilosante/tratamiento farmacológico , Corticoesteroides/uso terapéutico , Azatioprina/uso terapéutico , Humanos , Mesalamina/uso terapéutico , Metotrexato/uso terapéutico , Sulfasalazina/uso terapéuticoRESUMEN
Amyloid arthropathy is said to be distinguished roentgenographically from rheumatoid arthritis by the absence of joint space narrowing and the absence of articular erosions. We present a patient with multiple myeloma with swelling, stiffness and firm synovial thickening of the wrists, metacarpophalangeal joints and proximal interphalangeal joints whose hand radiographs showed articular erosions of the carpal joints and ulnar styloid and joint space narrowing of the proximal interphalangeal joints and metacarpophalangeal joints. Synovial biopsy of the left wrist showed amyloid deposits with no inflammation. Previous reports of X-ray changes in amyloid arthropathy note preservation of joint spaces or widening. Erosions when noted are of non-articular bone rather than of the articular surface. This is the first case report of erosive articular disease in amyloid arthropathy.
Asunto(s)
Amiloidosis/complicaciones , Artritis/etiología , Anciano , Amiloidosis/diagnóstico por imagen , Artritis/diagnóstico , Artritis Reumatoide/diagnóstico , Diagnóstico Diferencial , Femenino , Mano/diagnóstico por imagen , Mano/patología , Humanos , Mieloma Múltiple/complicaciones , RadiografíaRESUMEN
The clinical utility of standard liver function tests for monitoring low dose pulse methotrexate therapy is reviewed in 163 rheumatoid arthritis patients over an eight-year period. Abnormalities of hepatic enzymes were seen in 58% of patients but led to cessation of therapy in only 5%. Moderate alcohol intake did not affect the frequency of liver test abnormalities. Abnormalities were seen more frequently in patients with longer duration of methotrexate therapy and in those with higher total dose. There was no correlation between liver test abnormalities and day of serum sampling relative to day of methotrexate dosing, nor was a correlation seen between liver test abnormalities and total weekly dose of methotrexate. Methotrexate has been demonstrated to be an effective drug in the treatment of rheumatoid arthritis. The clinical utility of standard liver tests to predict the potential for hepatotoxicity is questionable.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas , Metotrexato/efectos adversos , Adulto , Factores de Edad , Anciano , Bilirrubina/sangre , Esquema de Medicación , Femenino , Humanos , Pruebas de Función Hepática , Masculino , Metotrexato/administración & dosificación , Persona de Mediana Edad , Estudios Retrospectivos , Albúmina Sérica/metabolismo , Factores Sexuales , Transaminasas/sangreRESUMEN
Aspirin is recommended for initial therapy of RA. If aspirin is not tolerated, an NSAID is recommended. The choice of NSAID should be based on cost, convenience, safety, and the personal experience of the physician. An adequate trial of at least 2 weeks should be completed before changing to another NSAID. Therapy should be closely monitored for adverse reactions, particularly renal and gastrointestinal effects.