RESUMEN
Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1-/- and DR6-/-, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1-/- and DR6-/-, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.
Asunto(s)
Proteínas del Dominio Armadillo/fisiología , Proteínas del Citoesqueleto/fisiología , Proteínas del Tejido Nervioso/fisiología , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Receptores del Factor de Necrosis Tumoral/fisiología , Esferoides Celulares/patología , Degeneración Walleriana/fisiopatología , Animales , Axones/patología , Axotomía , Calcio/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismoRESUMEN
PURPOSE OF REVIEW: Cancer patients undergoing treatment with cytotoxic chemotherapeutic agents (CCAs) often experience a cluster of treatment-related symptoms, which include fatigue, loss of appetite, disturbed sleep, depressed mood, cognitive difficulties, and changes in body composition. This symptom cluster collectively referred to herein as cancer treatment-related symptoms (CTRSs) decrease quality of life, and physical and social functioning. The preclinical and clinical studies described in this review represent important progress in understanding potential underlying mechanisms of CTRS. RECENT FINDINGS: Recent studies support a role for CCA-induced interleukin-1ß (IL-1ß) signaling in the cause of CTRS. CCAs may share a common ability to activate intracellular stress response pathways to trigger the synthesis, processing, and release of IL-1ß from immune cells. Fatigue, sleep disturbance, and cognitive difficulties in cancer patients exposed to CCAs correlate with plasma levels of IL-6, IL-1 receptor antagonist, and soluble tumor necrosis factor receptor-I/II, surrogate markers of IL-1ß-mediated central nervous system (CNS) inflammation. Additional preclinical work suggests IL-1ß-mediated CNS inflammation may cause CTRS by altering hypothalamic and hippocampal functioning. SUMMARY: Although additional research is necessary to further establish the link between CCA exposure, IL-1ß-mediated inflammatory processes and CTRS, these data provide hints for future studies and therapeutic approaches in ameliorating these symptoms in cancer patients.
Asunto(s)
Antineoplásicos/efectos adversos , Conducta de Enfermedad/efectos de los fármacos , Inflamación/inducido químicamente , Interleucina-1beta/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Calidad de Vida , Transducción de Señal/efectos de los fármacos , Antineoplásicos/uso terapéutico , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/complicaciones , Fatiga/inducido químicamente , Fatiga/complicaciones , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Inflamación/sangre , Inflamación/complicaciones , Interleucina-1beta/sangre , Interleucina-1beta/fisiología , Interleucina-6/sangre , Interleucina-6/fisiología , Trastornos del Humor/inducido químicamente , Neoplasias/complicaciones , Receptores de Interleucina/sangre , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/fisiología , Receptores del Factor de Necrosis Tumoral/sangre , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/fisiología , Perfil de Impacto de Enfermedad , SíndromeRESUMEN
OBJECTIVE: To analyze the long-term impact of the R92Q mutation of TNFRSF1A in children with periodic fever, in comparison with children with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) with TNFRSF1A structural mutations and children with periodic fever of unknown origin fulfilling the criteria for periodic fever, aphthosis, pharyngitis, and adenitis syndrome (PFAPA). METHODS: The extracellular region of TNFRSF1A was analyzed in 720 consecutive children with periodic fever, using denaturing high-performance liquid chromatography and DNA sequencing. Followup data on 11 pediatric patients with TNFRSF1A structural mutations (cysteine or T50M), 23 pediatric patients with an R92Q substitution, and 64 pediatric patients with PFAPA were collected during routine clinic visits. The 50-item Child Health Questionnaire was used to assess health-related quality of life (HRQOL). RESULTS: The frequency of typical TRAPS-related clinical manifestations was significantly lower and the impact of the disease on HRQOL was significantly reduced in patients with the R92Q mutation compared with TRAPS patients carrying structural mutations of TNFRSF1A. Followup data on 11 TRAPS patients with TNFRSF1A structural mutations (mean followup 7.9 years), 16 patients with theR92Q substitution (mean followup 7.3 years), and 64 patients with PFAPA (mean followup 5.2 years) were available. Patients with R92Q mutations and patients with PFAPA displayed a higher rate of self-resolution or amelioration of the fever episodes than did TRAPS patients with structural mutations. CONCLUSION: Although some cases may progress to a more chronic disease course, the majority of children with an R92Q mutation of the TNFRSFA1 gene show a milder disease course than that in children with TNFRSFA1 structural mutations and have a high rate of spontaneous resolution and amelioration of the recurrent fever episodes.
Asunto(s)
Fiebre Mediterránea Familiar/genética , Fiebre/genética , Linfadenitis/genética , Mutación/genética , Faringitis/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/fisiología , Adolescente , Antirreumáticos/uso terapéutico , Terapia Biológica , Niño , Preescolar , Fiebre Mediterránea Familiar/tratamiento farmacológico , Fiebre Mediterránea Familiar/fisiopatología , Femenino , Fiebre/tratamiento farmacológico , Fiebre/fisiopatología , Estudios de Seguimiento , Genotipo , Encuestas Epidemiológicas , Humanos , Lactante , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Estudios Longitudinales , Linfadenitis/tratamiento farmacológico , Linfadenitis/fisiopatología , Masculino , Faringitis/tratamiento farmacológico , Faringitis/fisiopatología , Calidad de Vida , Recurrencia , Estudios Retrospectivos , Esteroides/uso terapéutico , SíndromeRESUMEN
A component from Emilia sonchifolia (L.) DC, γ-humulene, was investigated. Significantly decreased cell viability of human colorectal cancer HT29 cells in a dose-dependent manner with IC50 53.67±2.99 µM for 24-h treatment was found. γ-Humulene induced apoptotic cell death and apoptosis was confirmed by morphological assessment. The staining with propidium iodide (PI) and flow cytometric analysis also showed that γ-humulene significantly promoted the sub-G1 phase (an apoptotic population) in HT29 cells. Colorimetric assays indicated that pretreatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced activities of caspase-8 and caspase-3 in examined HT29 cells. γ-Humulene stimulated the death receptor 5 (DR5), DR4, Fas-associated protein with death domain (FADD), the cleavage of caspase-8 and cleavage caspase-3, but reduced the protein levels of cellular Fas-associated death-domain-like IL-1ß-converting enzyme inhibitory protein (c-FLIP) by Western blot analysis. Consequently, γ-humulene-triggered cell death was significantly attenuated by DR5 siRNA and the caspase-8 inhibitor. Based on our results, we suggest that γ-humulene induced apoptotic cell death in HT29 cells through a DR5-mediated caspase-8 and -3-dependent signaling pathway. Therefore, this agent might be useful for developing new therapeutic regimens for treatment of colorectal cancer in the future.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Receptores del Factor de Necrosis Tumoral/metabolismo , Sesquiterpenos/farmacología , Adenocarcinoma/metabolismo , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Células HT29 , Humanos , Modelos Biológicos , Sesquiterpenos Monocíclicos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVES: The otic capsule, when compared with other bones in the body, is unique in that it undergoes no significant remodeling of bone after development. We previously demonstrated that osteoprotegerin (OPG), which inhibits formation and function of osteoclasts, is produced at high levels in the inner ear of normal mice and secreted into the perilymph from where it diffuses into the surrounding otic capsule bone through a lacunocanalicular system. To test our hypothesis that the high level of OPG may be important in the inhibition of otic capsule remodeling, we studied the light microscopic histology of the otic capsule in OPG knockout mice for evidence of abnormal remodeling of bone. We also tested the hearing in OPG knockout mice to determine whether OPG and its influence on surrounding bone is important for auditory function. METHODS: Temporal bone histopathology and pathophysiology were compared in homozygous OPG knockout mice and C57BL/6 (B6) mice, the background strain for the knockouts. Auditory function in age-matched animals from each group was evaluated at approximately 4-week intervals from 8 to 21 weeks using frequency-specific auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE). After each of the last three evaluations, the cochleae from one mouse of each group were harvested, processed, and examined by light microscopy. RESULTS: Osteoprotegerin knockout mice demonstrated abnormal remodeling of bone within the otic capsule with multiple foci showing osteoclastic bone resorption and formation of new bone. Such changes were not seen in the age-matched B6 controls. The active bone remodeling process in the knockout animals showed many similarities to otosclerosis seen in human temporal bones. Over the time period that we monitored, auditory function was significantly and progressively compromised in the knockout animals relative to B6 controls. At the earliest age of test (8 wk), the loss was apparent as a mild, high-frequency reduction in sensitivity by ABR. In contrast, DPOAE losses in the knockouts were substantial even at 8 weeks, and by 21 weeks, these losses exceeded our equipment limits. Results of ABR testing showed hearing sensitivity changes in the animals of the background strain were confined largely to the high frequencies, whereas OPG knockouts demonstrated substantial low-frequency shifts in addition to those at high frequencies. CONCLUSIONS: The histopathological and pathophysiological findings in OPG knockout mice support the hypothesis that OPG is important in the inhibition of bone remodeling within the otic capsule and the maintenance of normal auditory function. This mouse may provide a valuable animal model of human otosclerosis.
Asunto(s)
Remodelación Ósea/fisiología , Glicoproteínas/fisiología , Pérdida Auditiva/fisiopatología , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Hueso Temporal/fisiopatología , Estimulación Acústica , Animales , Remodelación Ósea/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Oído Interno/fisiopatología , Potenciales Evocados Auditivos del Tronco Encefálico , Glicoproteínas/deficiencia , Glicoproteínas/genética , Pérdida Auditiva/diagnóstico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoprotegerina , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genéticaRESUMEN
PURPOSE: To investigate the roles of interaction between OX40 and OX40 ligand (OX40L) in the development of experimental allergic conjunctivitis (EC) in mice. METHODS: BALB/c mice actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0, 2, 4, 6, and 8 with agonistic anti-OX40 Ab, blocking anti-OX40L Ab, or normal rat (nr)IgG. On day 10, the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas, spleens, and blood were harvested for analyses. For examination of the effects of the Abs during the late induction (or effector) phase, actively immunized mice were treated with the Abs just before or at the same time as the challenge. In addition, splenocytes from RW-primed mice were transferred into syngeneic naïve mice, and the recipients were treated with Abs twice (on days 2 and 4). On day 4, the mice were challenged with RW and evaluated. RESULTS: When the treatments were performed during the induction phase, anti-OX40 Ab treatment significantly increased clinical EC and eosinophil infiltration into the conjunctiva, whereas anti-OX40L Ab treatment significantly reduced eosinophil infiltration. Compared with splenocytes from nrIgG-treated mice, splenocytes from anti-OX40 Ab-treated mice proliferated vigorously against RW and produced significantly higher amounts of IL-2, -4, and -5 by RW stimulation but a significantly lesser amount of IFN-gamma after Con A stimulation. In contrast, splenocytes from anti-OX40L Ab-treated mice produced significantly less IL-5 with RW stimulation and IL-2 and IL-5 with Con A stimulation, whereas significantly more IFN-gamma was induced by Con A stimulation. Treatment with anti-OX40 and anti-OX40L Abs during the late induction or effector phase of EC did not affect eosinophil infiltration. CONCLUSIONS: Blocking of the interaction between OX40 and OX40L in vivo inhibits the development of EC. In contrast, forced stimulation of OX40 in vivo significantly exacerbates EC by activating T cells, especially Th2 cells. These effects were noted only in the induction phase of EC, suggesting that the interaction between OX40 and OX40L is important in the generation of Th2 immune responses in the development of EC.
Asunto(s)
Conjuntivitis Alérgica/inmunología , Glicoproteínas de Membrana/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Factores de Necrosis Tumoral/fisiología , Traslado Adoptivo , Alérgenos/inmunología , Animales , Anticuerpos Bloqueadores/farmacología , Conjuntivitis Alérgica/etiología , Modelos Animales de Enfermedad , Eosinófilos/fisiología , Femenino , Inmunización , Inmunoglobulina E/sangre , Interferón gamma/metabolismo , Interleucinas/metabolismo , Activación de Linfocitos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ligando OX40 , Polen/inmunología , Receptores OX40 , Células Th2/inmunologíaRESUMEN
Focal bone loss around inflamed joints in patients with autoimmune disease, such as rheumatoid arthritis, remains a serious clinical problem. The recent elucidation of the RANK/RANK-ligand/OPG pathway and its role as the final effector of osteoclastogenesis and bone resorption has brought a tremendous understanding of the pathophysiology of inflammatory bone loss, and has heightened expectation of a novel intervention. Here, we review the etiology of inflammatory bone loss, the RANK/RANK-ligand/OPG pathway, and the clinical development of anti-RANK-ligand therapy.
Asunto(s)
Artritis/terapia , Enfermedades Óseas/terapia , Resorción Ósea/etiología , Proteínas Portadoras/fisiología , Inflamación/terapia , Glicoproteínas de Membrana/fisiología , Artritis/etiología , Artritis/metabolismo , Enfermedades Óseas/etiología , Resorción Ósea/inmunología , Resorción Ósea/terapia , Proteínas Portadoras/antagonistas & inhibidores , Glicoproteínas/fisiología , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Modelos Biológicos , Osteoclastos/fisiología , Osteoprotegerina , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores del Factor de Necrosis Tumoral/fisiologíaRESUMEN
Following acute injury, the liver regenerates through hepatocyte division. If this pathway is impaired, liver repair depends on the recruitment of adult liver progenitor (oval) cells. Mice fed a choline deficient, ethionine supplemented (CDE) diet possess substantial numbers of oval cells, which can be isolated, or examined in vivo. Oncostatin M (OSM) has been shown to induce maturation of murine fetal hepatoblasts into hepatocytes. We recently confirmed this in human fetal liver cultures. Here, we show that liver OSM expression increases in mice fed a CDE diet and CDE-derived oval cell isolates express OSM and its receptor (OSMR). Oval cell lines (PIL cells), as well as primary oval cell cultures, displayed STAT-3 phosphorylation following OSM stimulation. OSM had no effect on the growth of primary oval cells, but it was pro-apoptotic to PIL cells, suggesting that the two cell models are not directly comparable. Expression of PCNA and cyclin D1 was not affected by OSM treatment. No evidence was obtained to suggest an effect on oval cell maturation with OSM treatment. However, decreased albumin production, accompanied by increased expression of haptoglobin and fibrinogen, suggests that OSM induced an acute phase reaction in cultured oval cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Hígado/citología , Péptidos/farmacología , Células Madre/efectos de los fármacos , Reacción de Fase Aguda/metabolismo , Animales , Comunicación Autocrina/genética , Ciclo Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colina/metabolismo , Proteínas de Unión al ADN/metabolismo , Etionina/farmacología , Fibrinógeno/genética , Expresión Génica/efectos de los fármacos , Haptoglobinas/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Oncostatina M , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Receptores de Citocinas/genética , Receptores de Oncostatina M , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , Células Madre/citología , Células Madre/metabolismo , Transactivadores/metabolismoRESUMEN
To clarify the pathogenesis of altered bone metabolism in diabetic state and its underlying mechanisms, the bone mineral content and fasting levels of serum intact parathyroid hormone (i-PTH), intact osteocalcin (i-OC), tartrate-resistant acid phosphatase (TRAP) and osteoclastgenesis inhibitory factor/osteoprotegerin (OCIF/OPG) were measured in male type 2 diabetic patients and their age-matched controls. In addition, urine levels of osteoclastic markers, C-telopeptide of type I collagen (CTx), deoxypyridinoline (DPD), and N-telopeptide of type I collagen (NTx) were simultaneously determined. Serum levels of i-PTH and i-OC in diabetic patients were significantly lower than those in the controls. Conversely, serum concentrations of TRAP were significantly elevated in diabetic patients. However, no clear correlation was observed between serum i-OC and TRAP. It was also observed that urinary excretion of CTx, DPD, and NTx was significantly increased in the diabetics as compared with the controls. Unexpectedly, serum levels of OCIF/OPG tended to be higher in the diabetic group, and these values exhibited a significantly positive correlation with those of serum TRAP. There was found a significantly negative correlation between serum TRAP and bone mineral density (BMD) and also between serum OCIF/OPG and bone mineral density. It seems probable that OCIF/OPG has a suppressive role on the increased bone resorption to prevent further loss of the skeletal bone mass in type 2 diabetic patients.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Glicoproteínas/farmacología , Osteoclastos/fisiología , Fosfatasa Ácida/sangre , Aminoácidos/orina , Biomarcadores/sangre , Péptido C/química , Péptido C/orina , Calcitriol/sangre , Calcio/sangre , Colágeno/orina , Colágeno Tipo I , Interpretación Estadística de Datos , Diabetes Mellitus Tipo 2/orina , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicoproteínas/fisiología , Humanos , Insulina/sangre , Isoenzimas/sangre , Magnesio/sangre , Masculino , Persona de Mediana Edad , Osteocalcina/sangre , Osteoprotegerina , Hormona Paratiroidea/sangre , Péptidos/orina , Fósforo/sangre , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Factores Sexuales , Fosfatasa Ácida TartratorresistenteRESUMEN
Tumor necrosis factor-alpha (TNF-a) is produced by alveolar macrophages (AM) in response to bleomycin (BLM) exposure. This cytokine has been linked to BLM-induced pulmonary inflammation, an early drug effect, and to lung fibrosis, the ultimate toxic effect of BLM. The present study was carried out to study the time dependence of apoptotic signaling pathways and the potential roles of TNF receptors in BLM-induced AM apoptosis. Male Sprague-Dawley rats were exposed to saline or BLM (1 mg/kg) by intratracheal instillation. At 1, 3, or 7 d postexposure, AM were isolated by bronchoalveolar (BAL) lavage and evaluated for apoptosis by ELISA. The release of cytochrome c from mitochrondria, the activation of caspase-3, -8, and -9, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the expression of TNF receptors (TNF-R1/p55 and TNF-R2/p75), TNF-R-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis 1 (c-IAP1) were determined by immunoblotting. The results showed that BLM exposure induced AM apoptosis, with the highest apoptotic effect occurring at 1 d after exposure and gradually decreasing at 3 and 7 d postexposure, but still remaining significantly above the control level. The maximal translocation of cytochromec from mitochondria into the cytosol was observed at 1 d postexposure, whereas the activation of caspase-9 and caspase-3 and caspase-3-dependent cleavage of PARP was found to reach a peak level at 3 d postexposure. BLM exposure had no marked effect on AM expression of TNF-R1 or caspase-8 activation, but significantly increased the expression of TNF-R2 that was accompanied by a rise in c-IAP1 and a decrease in TRAF2. This induction of TNF-R2 by BLM was significant on d 1 and increased with greater exposure time. In vitro studies showed that pretreatment of naive AM with a TNF-R2 antibody significantly inhibited BLM-induced caspase-3 activity and apoptosis. These results suggest that BLM-induced apoptosis involves multiple pathways in a time-dependent manner. Since maximal BLM-induced AM apoptosis (1 d postexposure) preceded maximal changes in caspase-9 and -3 (3 d postexposure), it is possible that a caspase-independent mechanism is involved in this initial response. These results indicate that the sustained expression of TNF-R2 in AM by BLM exposure may sensitize these cells to TNF-a-mediated toxicity.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Bleomicina/toxicidad , Exposición a Riesgos Ambientales/efectos adversos , Macrófagos Alveolares/efectos de los fármacos , Proteínas/efectos de los fármacos , Animales , Antígenos CD/efectos de los fármacos , Antígenos CD/fisiología , Apoptosis/fisiología , Líquido del Lavado Bronquioalveolar , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/efectos de los fármacos , Citocromos c/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Inflamación , Instilación de Medicamentos , Macrófagos Alveolares/fisiología , Masculino , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Proteínas/fisiología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF , Factores de Tiempo , Translocación Genética/efectos de los fármacosRESUMEN
This research team found in previous studies, that the ginseng saponin metabolite IH901 induces apoptosis in HepG2 cells via a mitochondrial-mediated pathway, which resulted in the activation of caspase-9 and subsequently of caspase-3 and -8. Based on these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in IH901-induced apoptosis in HepG2 cells, was investigated. Levels of Fas and the Fas ligand (FasL) mRNA or protein were not increased by IH901, rather they were decreased significantly at 18 h post treatment. Soluble FasL (sFasL) was detectable by immunoprecipitation analysis in the medium of HepG2 cells treated with IH901. Increased levels of sFasL were inversely correlated with the levels of FasL. Preincubation of HepG2 cells with antagonistic anti-Fas antibody showed little protective effect, if any, on IH901-induced cell death. At a 30 microM (24 and 48 h) and 40 microM (24 h) concentration of IH901, the cytotoxic effect of IH901 was less then 50%, anti-Fas antibody prevented IH901-induced cell death. However, at a 60 microM (24 and 48 h) and 40 microM (48 h) concentration of IH901, cell death rates were about 80% or more and most of the chemopreventive and chemotherapeutic effects of IH901 were manifested. Blocking the Fas receptor did not influence IH901-induced cell death. These results indicate that the Fas/FasL system is engaged, but not required for IH901-induced cell death, at pharmacologically significant concentrations.
Asunto(s)
Apoptosis/fisiología , Glicoproteínas de Membrana/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Saponinas/farmacología , Transducción de Señal/fisiología , Receptor fas/fisiología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteína Ligando Fas , Humanos , Ligandos , Panax/metabolismo , Sapogeninas/metabolismo , Sapogeninas/farmacología , Saponinas/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor fas/metabolismoRESUMEN
Increased circulating and cardiac TNF-alpha levels during myocardial ischemia have been found in both experimental animals and patients with ischemic heart disease and advanced heart failure. Soluble TNF-alpha receptor 1 (sTNFR1) is an antagonist to TNF-alpha. In the present study, we examined whether sTNFR1 improves cardiac function in rats after myocardial infarction. Male Wistar rats were subjected to left coronary artery (LCA) ligation. Immediately after the ligation, a total of 200 microg of either the sTNFR1 or LacZ plasmid was injected into three different sites in the left ventricular wall. From 1 to 21 days after LCA ligation, TNF-alpha bioactivity in the heart was higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase. The LV diastolic dimension was significantly lower, and the fractional shortening was significantly higher in rats treated with the sTNFR1 plasmid than in those treated with the LacZ plasmid. At 21 days after LCA ligation, the LV end-diastolic pressure was also significantly lower in the rats treated with the sTNFR1 plasmid. In addition, the sTNFR1 expression plasmid had significantly reduced the infarct size. In conclusion, TNF-alpha bioactivity in the heart increased during the early stage of infarction and remained elevated. This elevation seemed partially responsible for the impairment of LV function and the increased infarct size. Suppression of TNF-alpha bioactivity from the early stage of infarction with the sTNFR1 plasmid improved cardiac function and reduced infarct size.
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Antígenos CD/fisiología , Terapia Genética , Vectores Genéticos/uso terapéutico , Infarto del Miocardio/terapia , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antígenos CD/genética , Apoptosis , Vasos Coronarios , Evaluación Preclínica de Medicamentos , Vectores Genéticos/administración & dosificación , Ventrículos Cardíacos/diagnóstico por imagen , Inyecciones Intralesiones , Ligadura , Masculino , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Transfección , Ultrasonografía , Función Ventricular IzquierdaRESUMEN
The prevailing treatment strategies for autoimmune disorders employ global immunosuppressants that have harmful side effects with long-term use. A new vision for drug development relies on the generation of therapeutics that have specific and narrow targets, such as pathogenic cell populations. The cellular processes that initiate and maintain B cell dysregulation are not well understood and autoimmune disease results, in part, from the survival and activation of self-reactive B cells. Such B cells produce tissue-damaging pathogenic autoantibodies. BAFF (B cell-activating factor belonging to the TNF family), a member of the TNF family of ligands, may play a role in B cell-mediated diseases. BAFF is a survival factor for peripheral B cells. When BAFF is overexpressed in mice, B cell number and immunoglobulin production is increased and an autoimmune-like phenotype is observed. Mouse models of lupus-nephritis have been shown to exhibit increased serum BAFF levels correlating with disease severity, and many autoimmune patients were found to have higher levels of circulating BAFF than healthy volunteers. Thus, modulating the level and activity of BAFF in these patients may alleviate symptoms associated with their disease. Several potential therapeutic inhibitors targeting BAFF are under investigation, including an anti-BAFF antibody and receptor-Fc fusion proteins.
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Enfermedades Autoinmunes/tratamiento farmacológico , Proteínas de la Membrana/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Enfermedades Autoinmunes/inmunología , Autoinmunidad/fisiología , Factor Activador de Células B , Receptor del Factor Activador de Células B , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , División Celular , Supervivencia Celular , Evaluación Preclínica de Medicamentos , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos MRL lpr , Ratones Endogámicos NZB , Ratones Noqueados , Modelos Inmunológicos , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/fisiología , Proteínas Recombinantes de Fusión/uso terapéutico , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/deficienciaRESUMEN
Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure-activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis. While current treatments exist in the form of hormone replacement therapy, antiresorptive and anabolic agents for osteoporosis, there are no disease-modifying therapies for the treatment of the most common human joint disease, osteoarthritis. A massive market potential for improved options with better safety and efficacy still remains. Therefore, the application of genomics and proteomics for both diseases should provide much needed novel therapeutic approaches to treating these major world health problems.
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Enfermedades Óseas/tratamiento farmacológico , Animales , Proteínas Portadoras , Caspasas/fisiología , Catepsina K , Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/fisiología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Proteína 1 de la Respuesta de Crecimiento Precoz , Genómica , Glicoproteínas/fisiología , Humanos , Proteínas Inmediatas-Precoces/fisiología , Glicoproteínas de Membrana , Ratones , Ratones Noqueados , Ratones Transgénicos , Estructura Molecular , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoprotegerina , Proteómica , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Factores de Transcripción/fisiología , Técnicas del Sistema de Dos Híbridos , Familia-src QuinasasRESUMEN
We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.
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Endotelio Vascular/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas/metabolismo , Acetilcisteína/farmacología , Antioxidantes/farmacología , Sitios de Unión , Clonación Molecular , ADN Complementario , Activación Enzimática , Biblioteca de Genes , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Pulmón , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Fosfoproteínas/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Factor 4 Asociado a Receptor de TNF , Transfección , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral , beta-Galactosidasa/genéticaRESUMEN
Chronic fatigue syndrome (CFS) patients show evidence of immune activation, as demonstrated by increased numbers of activated T lymphocytes, including cytotoxic T cells, as well as elevated levels of circulating cytokines. Nevertheless, immune cell function of CFS patients is poor, with low natural killer cell cytotoxicity (NKCC), poor lymphocyte response to mitogens in culture, and frequent immunoglobulin deficiencies, most often IgG1 and IgG3. Immune dysfunction in CFS, with predominance of so-called T-helper type 2 and proinflammatory cytokines, can be episodic and associated with either cause or effect of the physiological and psychological function derangement and/or activation of latent viruses or other pathogens. The interplay of these factors can account for the perpetuation of disease with remission/exacerbation cycles. A T-helper type 2 predominance has been seen among Gulf War syndrome patients and this feature may also be present in other related disorders, such as multiple chemical sensitivity. Therapeutic intervention aimed at induction of a more favorable cytokine expression pattern and immune status appears promising.
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Citocinas/fisiología , Síndrome de Fatiga Crónica/fisiopatología , Citocinas/sangre , Citocinas/metabolismo , Síndrome de Fatiga Crónica/sangre , Síndrome de Fatiga Crónica/inmunología , Humanos , Interferones/fisiología , Interleucinas/fisiología , Linfotoxina-alfa/fisiología , Neopterin/fisiología , Neuroinmunomodulación/fisiología , Psiconeuroinmunología , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/fisiología , Estrés Fisiológico/fisiopatología , Células TH1/metabolismo , Células Th2/metabolismo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.
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Eliminación de Gen , Neoplasias Hepáticas/patología , Lesiones Precancerosas/patología , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Carcinógenos/administración & dosificación , Carcinógenos/farmacología , Recuento de Células , División Celular , Células Cultivadas , Dieta , Etionina/administración & dosificación , Etionina/farmacología , Regulación de la Expresión Génica , Histocitoquímica , Interleucina-6/genética , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Noqueados , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal , Células Madre/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
In this study we show that the retinal autoantigen, S-antigen, contains a functional TNF-alpha homologous domain which stimulates maturation and differentiation of cultured dendritic cells (DC) or tissue DC via the p55 TNF-alpha receptor. Tissue DC became more dendritiform in shape, and migrated into culture supernatant. S-antigen also stimulated accumulation of cell surface MHC class II antigen with a corresponding loss of acidic intracellular vesicles, and induced IL-1beta and IL-12 mRNA expression in cultured bone marrow-derived DC. In addition, cultured splenic DC primed immune responses to S-antigen in vivo in the absence of other, exogenous cytokine sources. DC pulsed with either retinal S-antigen or another retinal autoantigen, interphotoreceptor retinoid binding protein (IRBP), were able to stimulate naive T cell proliferation in vitro, but only S-antigen-pulsed DC were able to induce an immune response in vivo and initiate antibody class switching. In contrast, IRBP-pulsed DC had no detectable in vivo priming effect and IgG antibody levels remained suppressed even after immunization with IRBP in complete Freund's adjuvant. These results indicate that DC from the same precursor population can either induce or suppress a B cell-specific response to self antigen in vivo, the outcome being dependent upon DC activation at the time of antigen uptake and presentation.
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Antígenos CD/fisiología , Arrestina/inmunología , Linfocitos B/inmunología , Células Dendríticas/fisiología , Proteínas del Ojo , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Células Cultivadas , Femenino , Inmunización , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/clasificación , FN-kappa B/fisiología , Ratas , Ratas Endogámicas Lew , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas de Unión al Retinol/inmunología , Linfocitos T/inmunologíaRESUMEN
We used the human myelomonoblastic leukemia cell line PLB-985 to study the effects of temperatures ranging from 37 degrees C to 43 degrees C for 1 h on the induction of apoptosis and cell cycle distribution in leukemia cells. The threshold temperature for the onset of apoptosis was 42 degrees C. Whereas hyperthermia exerted no effect on the expression of Bcl-2 and Bax, heat induced a >30-fold increase of tumor necrosis factor (TNF) alpha mRNA expression and a significant increase in TNF-alpha protein secretion. This endogenous production of TNF-alpha correlated directly with the temperature-induced apoptode effect. Blocking TNF-alpha expression via treatment with pyrrolidinedithiocarbamate or blocking TNF-alpha activity with neutralizing antibodies abrogated heat-provoked apoptosis. In addition, exposure of cell culture supernatant of heat-treated PLB-985 cells to untreated cells induced an apoptotic effect. These data indicate a TNF-a-mediated self eradication of the leukemia cells after heat exposure. Inducing apoptosis with wild-type TNF-alpha or p55 and p75 protein muteins demonstrated that this effect was mediated by the p55 receptor. Interestingly, the autocrine suicidal loop found in immature leukemia cells was lost after granulocytic differentiation with 0.5% N,N-dimethylformamide. These data should be of critical importance for the understanding of the biological impact of fever as well as for developing therapeutic approaches to malignant diseases
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Apoptosis/fisiología , Fiebre/fisiopatología , Calor , Leucemia Mielomonocítica Aguda/patología , Proteínas de Neoplasias/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Anticuerpos Monoclonales/farmacología , Antígenos CD/fisiología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Dimetilformamida/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Genes bcl-2 , Humanos , Hipertermia Inducida , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Pirrolidinas/farmacología , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiocarbamatos/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología , Proteína X Asociada a bcl-2RESUMEN
Costimulation of TNFR80 can strongly enhance TNFR60-induced cell death. In this study, we show that this enhancement is TNFR60 selective, as neither TNF-related apoptosis-inducing ligand/Apo2 ligand-, Apo1/Fas-, ceramide-, nor daunorubicin-mediated cell death was affected by costimulation of TNFR80. We further demonstrate that TNFR-associated factor 2 (TRAF2) is critically involved in both negative and positive regulation of TNF-induced cell death. Overexpression of TRAF2 and of a TRAF2 mutant, deficient in nuclear factor-kappaB activation, selectively desensitized and enhanced, respectively, TNFR60-induced cell death in HeLa cells. However, upon costimulation of TNFR80, which mediates activation of nuclear factor-kappaB and the c-Jun amino-terminal kinase via TRAF2, TNF-induced cell death is drastically enhanced in parental and TRAF2-transfected, but not in TRAF2 (87-501)-transfected cells. These data point to a critical role of TRAF2 in the apoptotic TNFR cross talk, whereby the TNFR80-dependent enhancement of TNFR60-induced cell death is due to TNFR80-mediated negative regulation of TRAF2 function(s). An interference with TRAF2 function was confirmed independently by analysis of c-Jun amino-terminal kinase activation via TNFR60 upon prestimulation of TNFR80. We propose that the apoptotic TNFR cross talk is based on TNFR80-mediated abrogation of antiapoptotic TRAF2-dependent signaling pathways initiated by TNFR60, but not Apo1/Fas or the apoptotic TNF-related apoptosis-inducing ligand receptors.