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Immunotherapy has reached clinical success in the last decade, with the emergence of new and effective treatments such as checkpoint blockade therapy and CAR T-cell therapy that have drastically improved patient outcomes. Still, these therapies can be improved to limit off-target effects, mitigate systemic toxicities, and increase overall efficacies. Nanoscale engineering offers strategies that enable researchers to attain these goals through the manipulation of immune cell functions, such as enhancing immunity against cancers and pathogens, controlling the site of immune response, and promoting tolerance via the delivery of small molecule drugs or biologics. By tuning the properties of the nanomaterials, such as size, shape, charge, and surface chemistry, different types of immune cells can be targeted and engineered, such as dendritic cells for immunization, or T cells for promoting adaptive immunity. Researchers have come to better understand the critical role the immune system plays in the progression of pathologies besides cancer, and developing nanoengineering approaches that seek to harness the potential of immune cell activities can lead to favorable outcomes for the treatment of injuries and diseases.
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BACKGROUND: An increasing number of patients require outpatient and interventional pain management. To help meet the rising demand for anesthesia pain subspecialty care in rural and metropolitan areas, health care providers have used telemedicine for pain management of both interventional patients and those with chronic pain. OBJECTIVE: In this study, we aimed to describe the implementation of a telemedicine program for pain management in an academic pain division in a large metropolitan area. We also aimed to estimate patient cost savings from telemedicine, before and after the California COVID-19 "Safer at Home" directive, and to estimate patient satisfaction with telemedicine for pain management care. METHODS: This was a retrospective, observational case series study of telemedicine use in a pain division at an urban academic medical center. From August 2019 to June 2020, we evaluated 1398 patients and conducted 2948 video visits for remote pain management care. We used the publicly available Internal Revenue Service's Statistics of Income data to estimate hourly earnings by zip code in order to estimate patient cost savings. We estimated median travel time and travel distance with Google Maps' Distance Matrix application programming interface, direct cost of travel with median value for regular fuel cost in California, and time-based opportunity savings from estimated hourly earnings and round-trip time. We reported patient satisfaction scores derived from a postvisit satisfaction survey containing questions with responses on a 5-point Likert scale. RESULTS: Patients who attended telemedicine visits avoided an estimated median round-trip driving distance of 26 miles and a median travel time of 69 minutes during afternoon traffic conditions. Within the sample, their median hourly earnings were US $28 (IQR US $21-$39) per hour. Patients saved a median of US $22 on gas and parking and a median total of US $52 (IQR US $36-$75) per telemedicine visit based on estimated hourly earnings and travel time. Patients who were evaluated serially with telemedicine for medication management saved a median of US $156 over a median of 3 visits. A total of 91.4% (286/313) of patients surveyed were satisfied with their telemedicine experience. CONCLUSIONS: Telemedicine use for pain management reduced travel distance, travel time, and travel and time-based opportunity costs for patients with pain. We achieved the successful implementation of telemedicine across a pain division in an urban academic medical center with high patient satisfaction and patient cost savings.
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Bullet emboli to the heart as a result of penetrating trauma are rare. We report a case of a 19-year old male who suffered a gunshot wound to the abdomen, resulting in an aorto-caval fistula and subsequent venous embolization of the bullet to the right ventricle. Successful surgical removal of the foreign body under cardiopulmonary bypass was performed.
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Traumatismos Abdominais/complicações , Aorta/lesões , Fístula Arteriovenosa/etiologia , Embolia/etiologia , Migração de Corpo Estranho/etiologia , Veia Cava Inferior/lesões , Ferimentos por Arma de Fogo/complicações , Traumatismos Abdominais/cirurgia , Aorta/cirurgia , Fístula Arteriovenosa/diagnóstico , Fístula Arteriovenosa/cirurgia , Procedimentos Cirúrgicos Cardíacos , Ponte Cardiopulmonar , Embolia/diagnóstico , Embolia/cirurgia , Migração de Corpo Estranho/diagnóstico , Migração de Corpo Estranho/cirurgia , Ventrículos do Coração , Humanos , Masculino , Esternotomia , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Veia Cava Inferior/cirurgia , Ferimentos por Arma de Fogo/cirurgia , Adulto JovemRESUMO
The innate immune system and its components play an important role in the pathogenesis of inflammatory bone destruction. Blockade of inflammatory cytokines does not completely arrest bone erosion, suggesting that other mediators also may be involved in osteolysis. Previously we showed that nucleosides promote osteoclastogenesis and bone-resorption activity in the presence of receptor activator for nuclear factor κB ligand (RANKL) in vitro. The studies described here further demonstrate that selected nucleosides and nucleoside analogues accelerate bone destruction in mice immunized with collagen II alone (CII) but also further enhance bone erosion in mice immunized by collagen II plus complete Freund's adjuvant (CII + CFA). Abundant osteoclasts are accumulated in destructive joints. These data indicate that nucleosides act as innate immune activators distinct from CFA, synergistically accelerating osteoclast formation and inflammatory osteolysis. The potential roles of the surface triggering receptor expressed on myeloid cells (TREM) and the intracellular inflammasome in nucleoside-enhanced osteoclastogenesis have been studied. These observations provide new insight into the pathogenesis and underlying mechanism of bone destruction in inflammatory autoimmune osteoarthritis.
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Imunidade Inata/efeitos dos fármacos , Inflamação/complicações , Inflamação/imunologia , Nucleosídeos/farmacologia , Osteólise/complicações , Osteólise/imunologia , Animais , Artrite Experimental/complicações , Artrite Experimental/imunologia , Artrite Experimental/patologia , Reabsorção Óssea/complicações , Reabsorção Óssea/patologia , Proteínas de Transporte/metabolismo , Fêmur/efeitos dos fármacos , Fêmur/patologia , Inflamassomos/metabolismo , Inflamação/patologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteólise/patologia , Receptores Imunológicos/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLCγ2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3-4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (>30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing microgravity-induced bone loss by targeting the molecular events occurring in microgravity-induced enhanced osteoclastogenesis.
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Osteoclastos/citologia , Ligante RANK/farmacologia , Ausência de Peso , Fosfatase Ácida/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Densidade Óssea/efeitos dos fármacos , Linhagem Celular , Elevação dos Membros Posteriores/fisiologia , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfatase Ácida Resistente a TartaratoRESUMO
We have studied excited-state proton transfer (ESPT) from chiral proton donors to chiral and achiral acceptors. The key role of the exergonicity of the reaction and the transition-state position along the reaction coordinate for the existence of an enantiomeric effect was established. This effect was observed for "super" photoacids (ΔG ⪠0) and vanished for endergonic reactions (ΔG > 0) where a "late" transition state similar to planar achiral binaphtholate anion occurs. As a result, photoracemization was observed, as confirmed by circular dichroism spectroscopy. The photoracemization effects were studied for several chiral photoacids (BINOLs and their ethers) and proton acceptors (amines, aminoalcohols, and water) using UV-vis, steady-state fluorescence, and time-resolved fluorescence spectroscopies. The nature of the solvent and the proton acceptor, as well as the chemical structure of the BINOL, played a pivotal role in the photochemical reactivity of the system. Two proposed pathways competed for photoracemization: excited-state inter- and intra-molecular proton transfer, the former being more effective. Irradiation of the dimethoxy BINOL derivative, which lacks an acidic proton and cannot undergo ESPT, produced no appreciable reaction or racemization.
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We have previously demonstrated that the antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells partially through Fas-mediated apoptosis pathways. Recently, CaM has been shown to bind to Fas, which is regulated during Fas or CaM antagonist-mediated apoptosis in Jurkat cells and osteoclasts. Accordingly, the present studies were designed to determine whether Fas interacts with CaM in cholangiocarcinoma cells and to elucidate its role in regulating Fas-mediated apoptosis. CaM bound to Fas in cholangiocarcinoma cells. CaM was identified in the Fas-mediated death inducing signaling complex (DISC). The amount of CaM recruited into the DISC was increased after Fas-stimulation, a finding confirmed by immunofluorescent analysis that demonstrated increased membrane co-localization of CaM and Fas upon Fas-stimulation. Consistently, increased Fas microaggregates in response to Fas-stimulation were found to bind to CaM. Fas-induced recruitment of CaM into the DISC was inhibited by the Ca(2+) chelator, EGTA, and the CaM antagonist, trifluoperazine (TFP). TFP decreased DISC-induced cleavage of caspase-8. Further, inhibition of actin polymerization, which has been demonstrated to abolish DISC formation, inhibited the recruitment of CaM into the DISC. These results suggest an important role of CaM in mediating DISC formation, thus regulating Fas-mediated apoptosis in cholangiocarcinoma cells. Characterization of the role of CaM in Fas-mediated DISC formation and apoptosis signaling may provide important insights in the development of novel therapeutic targets for cholangiocarcinoma.
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Apoptose , Neoplasias dos Ductos Biliares/metabolismo , Calmodulina/metabolismo , Colangiocarcinoma/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteína Ligante Fas/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/patologia , Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Caspase 8/metabolismo , Colangiocarcinoma/patologia , Humanos , Imunoprecipitação , Ligação Proteica , Transdução de Sinais , Trifluoperazina/farmacologia , Células Tumorais CultivadasRESUMO
We have previously characterized the role of Fas in tumorigenesis using two cholangiocarcinoma cell lines expressing high (Fas(H)) and low (Fas(L)) levels of Fas. Here we further characterize Fas ligand (FasL) expression and function in these two cell lines. The Fas(L) cells expressed a high level of FasL, whereas the Fas(H) cells expressed a low level of FasL showing reciprocal expression of Fas and FasL in tumor cells. FasL released from the Fas(L) cells is capable of inducing apoptosis of lymphocytes, which is blocked by neutralizing Fas antibody. To study the underlying mechanism for the reciprocal expression of Fas and FasL, we examined the activities of both the Fas and FasL promoters. The activity of the Fas promoter is suppressed and the activity of the FasL promoter is stimulated in the Fas(L) cells compared to the Fas(H) cells. The inverse activities of Fas and FasL promoter in tumor cells are regulated by NF-kappaB, which inhibits Fas expression and increases FasL expression through binding to their respective promoters. The inverse expression of Fas and FasL in tumor cells is partially reversed by an NF-kappaB inhibitor. In conclusion, human cholangiocarcinoma cells reciprocally co-express functional Fas and FasL, which are the result of the activities of the Fas and FasL promoters being regulated by NF-kappaB. These findings provide a potential unifying molecular mechanism for modulating tumorigenesis via Fas/FasL expression.
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Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/metabolismo , Proteína Ligante Fas/metabolismo , Receptor fas/metabolismo , Apoptose , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/patologia , Northern Blotting , Western Blotting , Colangiocarcinoma/imunologia , Colangiocarcinoma/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Proteína Ligante Fas/genética , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , NF-kappa B/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Receptor fas/genéticaRESUMO
BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus. METHODS: Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death. RESULTS: We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis. CONCLUSION: Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.
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Apoptose/genética , Doenças Autoimunes/genética , Proteína Ligante Fas/genética , Transtornos Linfoproliferativos/genética , Adolescente , Adulto , Apoptose/imunologia , Doenças Autoimunes/imunologia , Citotoxicidade Imunológica , Proteína Ligante Fas/imunologia , Feminino , Heterozigoto , Humanos , Células Jurkat , Transtornos Linfoproliferativos/imunologia , Masculino , Modelos Moleculares , Mutação , Subpopulações de Linfócitos T/imunologia , TransfecçãoRESUMO
Prolonged microgravity experienced by astronauts is associated with a decrease in bone mineral density. To investigate the effect of microgravity on differentiation of osteoclasts and osteoblasts, we used a NASA-recommended, ground-based, microgravity-simulating system, the Rotary Cell Culture System (RCCS). Using the RCCS, we demonstrated that modeled microgravity (MMG) inhibited osteoblastogenesis and increased adipocyte differentiation in human mesenchymal stem cells incubated under osteogenic conditions. This transformation involves reduced RhoA activity and cofilin phosphorylation, disruption of F-actin stress fibers, and decreased integrin signaling through focal adhesion kinase. We have used the system to show that MMG also stimulates osteoclastogenesis. These systems provide the opportunity to develop pharmacological agents that will stimulate osteoblastogenesis and inhibit osteoclastogenesis.
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Diferenciação Celular , Osteoblastos/citologia , Osteoclastos/citologia , Ausência de Peso , Animais , Linhagem Celular , Humanos , CamundongosRESUMO
Osteopenia is a common and debilitating side-effect of HAART, yet little is known concerning the effects of HAART on bone metabolism. We reported previously that zidovudine (AZT) stimulates osteoclastogenesis in vitro and causes osteopenia in mice. Here, we confirmed that the AZT-induced osteoclastogenesis is dependent on RANKL in that osteoclastogenesis is blocked by osteoprotegestin. Alendronate, which is used for the treatment of osteopenia and osteoporosis, failed to inhibit AZT-induced osteoclastogenesis in vitro. Osteoclastogenesis in vitro was not affected by tumor necrosis factor-alpha. Two other NRTI drugs, ddl and 3TC, also induced osteoclastogenesis in vitro and induced osteopenia in mice. The osteopenia was associated with an elevation of parameters of osteoclasts, but not with osteoblasts. Combinations of the NRTIs did not result in additive or synergistic effects in vitro or in vivo. Finally, AZT induced osteoclastogenesis of human osteoclast precursors in a RANKL-dependent manner. This in vitro osteoclastogenesis assay using human peripheral blood mononuclear cells could be useful in evaluating bone turnover and the risk of developing osteopenia in AIDS patients on HAART.
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Diferenciação Celular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Alendronato/farmacologia , Animais , Apoptose/efeitos dos fármacos , Didanosina/farmacologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoprotegerina/farmacologia , Ligante RANK/metabolismo , Zidovudina/farmacologiaRESUMO
Hypoxia produces sex dimorphic immune responses in males and proestrus females. Because Kupffer cells are the major source of proinflammatory cytokines, studies were conducted to discern IL-6 production in mouse Kupffer cells following hypoxia. Hypoxia enhances TLR4 expression in Kupffer cells irrespective of sex. However, MyD88 and Src expression in Kupffer cells decreased significantly after hypoxia in proestrus females, whereas Src protein expression and phosphorylation increased in males in concurrence with differences in IL-6 production. 17beta-estradiol administration elevated MyD88 and Src expression in males to levels in normoxic proestrus females. Administration of Src inhibitor in hypoxic males prevented increased IL-6 production. Thus, differential regulation of MyD88 and Src in males and females plays an important role in sex-specific immune response following hypoxia.
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Hipóxia/imunologia , Células de Kupffer/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Proestro/imunologia , Caracteres Sexuais , Quinases da Família src/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Estradiol/farmacologia , Feminino , Interleucina-6/análise , Interleucina-6/biossíntese , Células de Kupffer/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Fator 88 de Diferenciação Mieloide/genética , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
The advent of highly active anti-retroviral therapy (HAART) has dramatically decreased the rate of AIDS-related mortality and significantly extended the life span of patients with AIDS. A variety of metabolic side effects are associated with these therapies, one of which is metabolic bone disease. A higher prevalence of osteopenia and osteoporosis in HIV-infected patients receiving anti-retroviral therapy than in patients not on therapy has now been reported in several studies. Several factors have been demonstrated to influence HIV-associated decreases in bone mineral density (BMD), including administration of nucleoside reverse transcriptase inhibitors (NRTIs). In this article, discussion will focus on the molecular pathogenesis and treatment of HAART-associated osteopenia and osteoporosis.
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Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Doenças Ósseas Metabólicas/induzido quimicamente , Reabsorção Óssea/induzido quimicamente , Osteoporose/induzido quimicamente , Síndrome da Imunodeficiência Adquirida/imunologia , Doenças Ósseas/induzido quimicamente , Doenças Ósseas/etiologia , Doenças Ósseas Metabólicas/prevenção & controle , Infecções por HIV/complicações , Humanos , Mitocôndrias/patologia , Osteoporose/prevenção & controle , Linfócitos T/imunologiaRESUMO
One hallmark of AIDS progression is a decline in CD4+ T lymphocytes, though the mechanism is poorly defined. There is ample evidence that increased apoptosis is responsible for some, if not all, of the decline. Prior studies have shown that binding of cellular calmodulin to the envelope glycoprotein (Env) of HIV-1 increases sensitivity to fas-mediated apoptosis and that calmodulin antagonists can block this effect. We show that individual mutation of five residues in the C-terminal calmodulin-binding domain of Env is sufficient to significantly reduce fas-mediated apoptosis in transfected cells. The A835W mutation in the cytoplasmic domain of gp41 eliminated co-immunoprecipitation of Env with calmodulin in studies with stably transfected cells. Four point mutations (A835W, A838W, A838I, and I842R) and the corresponding region of HIV-1 HXB2 were cloned into the HIV-1 proviral vector pNL4-3 with no significant effect on viral production or envelope expression, although co-immunoprecipitation of calmodulin and Env was decreased in three of these mutant viruses. Only wild-type envelope-containing virus induced significantly elevated levels of spontaneous apoptosis by day 5 post-infection. Fas-mediated apoptosis levels positively correlated with the degree of calmodulin co-immunoprecipitation, with the lowest apoptosis levels occurring in cells infected with the A835W envelope mutation. While spontaneous apoptosis appears to be at least partially calmodulin-independent, the effects of HIV-1 Env on fas-mediated apoptosis are directly related to calmodulin binding.
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Apoptose , Calmodulina/metabolismo , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Mutação Puntual/genética , Replicação Viral , Linhagem Celular , Regulação Viral da Expressão Gênica , Proteína gp160 do Envelope de HIV/química , HIV-1/genética , Humanos , Ligação Proteica , Fatores de TempoRESUMO
UNLABELLED: Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown. INTRODUCTION: Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts. MATERIALS AND METHODS: RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA. RESULTS: Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated. CONCLUSION: RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.
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Apoptose/fisiologia , Reabsorção Óssea/metabolismo , Proteínas de Transporte/fisiologia , Glicoproteínas de Membrana/fisiologia , Osteoclastos/fisiologia , Receptor fas/metabolismo , Animais , Proteínas de Transporte/farmacologia , Caspase 3 , Inibidores de Caspase , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Glicoproteínas de Membrana/farmacologia , Camundongos , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligante RANK , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Regulação para Cima , Receptor fas/genéticaRESUMO
A variety of metabolic complications have been reported to be associated with highly active antiretroviral therapy (HAART), including osteopenia and osteoporosis. In this study, we determine the effects of zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, on osteoclastogenesis in a cultured mouse macrophage preosteoclast cell line (RAW264.7), in mouse primary bone marrow macrophage-monocyte precursors, and on bone mineral density in mice. The results indicate that AZT induces an increase in osteoclastogenesis in the mouse preosteoclast cell line and in mouse bone marrow osteoclast precursors in the presence of RANKL. This increased osteoclastogenesis is dependent upon the concentration of AZT. AZT increases the promoter activity of tartrate-resistant acid phosphatase (TRAP) and the binding and function of the nuclear transcription protein, NF-kappaB, in RAW264.7 cells. Therefore, the effect of AZT is mediated, at least in part, by enhancing RANKL-mediated osteoclastogenesis. Bone mineral density (BMD) in AZT-treated mice is decreased and histopathology shows marked osteopenia. These results support an important role of AZT-stimulated osteoclastogenesis in HAART-induced osteopenia.
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Fármacos Anti-HIV/farmacologia , Densidade Óssea/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Zidovudina/farmacologia , Fosfatase Ácida/genética , Animais , Fármacos Anti-HIV/uso terapêutico , Osso e Ossos/patologia , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Isoenzimas/genética , Glicoproteínas de Membrana/farmacologia , Camundongos , NF-kappa B/metabolismo , Osteoclastos/citologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Fosfatase Ácida Resistente a Tartarato , Zidovudina/uso terapêuticoRESUMO
Members of the tumour necrosis factor receptor family play a pivotal role in cell differentiation, function and apoptosis. However, signalling by many members of the family remains to be elucidated. In the present study, we developed a chimaeric receptor approach for studying signalling by receptors belonging to this family. The chimaeric receptor comprises the human Fas external domain linked to the transmembrane and cytoplasmic domains of a tumour necrosis factor receptor family member of interest. When the chimaera is expressed in mouse cells, the clustering of the chimaera induced by a human Fas-activating antibody activates the intracellular domain of the chimaera without affecting its endogenous counterpart. Since the antibody recognizes only human Fas, this approach can be used to dissect signalling by any tumour necrosis factor family member using any type of mouse cell including those endogenously expressing Fas. Moreover, we also showed that the chimaeric receptor approach can be used to study signalling at any stage of cell differentiation or function.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Camundongos , Família Multigênica , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoprotegerina , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral/classificação , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Receptor fas/química , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Calmodulin (CaM) antagonists have been shown to inhibit tumor cell invasion and metastasis and to induce apoptosis in various tumor models, but the molecular mechanism of CaM antagonist-mediated apoptosis is poorly understood. Here, we demonstrate that interferon (IFN)-gamma induces susceptibility to CaM antagonist-mediated apoptosis in human cholangiocarcinoma cells weakly expressing Fas (Fas-low cells). During CaM antagonist-mediated apoptosis in IFN-gamma-pretreated Fas-low cells, cleavage of caspases-8, -9, and -3 and Bid, release of cytochrome c from the mitochondria and an increase in the free cytosolic calcium concentration were observed. CaM antagonists also caused depolarization of the mitochondrial membrane independent of caspase activation. Although a broad-range caspase inhibitor partially blocked CaM antagonist-mediated apoptosis, the neutralizing Fas antibody had no effect, suggesting that CaM antagonist-mediated apoptosis does not require interaction between CaM antagonists and surface Fas. CaM antagonists induce apoptosis via mechanisms other than inhibition of CaM-dependent protein kinase II and calcineurin, as their inhibitors, KN93 and cyclosporine A, had no effect on apoptosis. Taken together, these results indicate that CaM antagonists induce apoptosis in both caspase-dependent and -independent manners, and that susceptibility to CaM antagonists is modulated by IFN-gamma. The combination of IFN-gamma and CaM antagonists, including tamoxifen, may be a potential therapeutic modality for cholangiocarcinoma and possibly other malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Calmodulina/antagonistas & inibidores , Caspases/metabolismo , Interferon gama/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Western Blotting , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Inibidores de Caspase , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Grupo dos Citocromos c/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptor fas/genéticaRESUMO
Human cholangiocarcinoma is a malignancy with no effective therapy and a poor prognosis. Previously, we demonstrated that cultured human cholangiocarcinoma cell lines heterogeneously express Fas on their surface, resulting in 2 subpopulations, Fas-high and Fas-low cells. Fas-low cells are resistant to apoptosis induced by Fas antibody and the calmodulin antagonists tamoxifen and trifluoperazine and are tumorigenic in nude mice (Pan et al., Am J Pathol 1999;155:193-203). Here, we show that IFN-gamma enhances apoptosis in both Fas-high and Fas-low cells. IFN-gamma upregulates many apoptosis-related molecules, including Fas, caspase-3, caspase-4, caspase-7, caspase-8 and Bak, in both cell lines. Pretreatment with IFN-gamma facilitated Fas-mediated caspase cleavage, cytochrome c release and Bax translocation. The ability of IFN-gamma to inhibit tumorigenesis of Fas-low cells was demonstrated in nude mice. Intratumoral injection of IFN-gamma decreased tumor volumes by 78%. These findings indicate that IFN-gamma modulates the apoptotic pathway by upregulating apoptosis-related genes. This renders tumorigenic Fas-low cholangiocarcinoma cells nontumorigenic and sensitive to Fas apoptosis, thus representing a possible therapeutic modality.
Assuntos
Apoptose/fisiologia , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/patologia , Interferon gama/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Receptor fas/fisiologia , Proteínas Reguladoras de Apoptose , Transporte Biológico , Testes de Carcinogenicidade , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Variação Genética , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Receptor fas/efeitos dos fármacosRESUMO
OBJECTIVES: To evaluate the response of human cholangoicarcinoma cells to TMX treatment through the Fas pathway by pretreatment with IFN-gamma. SUMMARY BACKGROUND DATA: Cholangiocarcinoma remains one of the most difficult tumors to treat in clinical medicine. Currently, there are no effective chemotherapy treatments for this disease. Surgery offers the only opportunity for a cure, with the majority of patients failing to qualify for such treatment. This study seeks to evaluate a potential new modality for treatment of this disease. METHODS: Human cholangiocarcinoma cells were treated with anti Fas mab and sorted to two populations (Fas-positive and Fas-negative) by FAC analysis. In vitro individual cell populations were pretreated with IFN-gamma 250 units/mL x 18hs. The treated cells assayed for caspase 3, 7, 8, Bak, and for apoptosis with Annexin V after treatment with or without TMX. In Vivo 2 x 106 5 SK-ChA-1 Fas-negative cells were injected into nude mice for development of tumor xenografts. Mice received either no treatment or intra tumor IFN-gamma and/or intra peritoneal TMX. RESULTS: More than 90% (90% +/- 3.5%) of Fas-positive and 70% (71 +/- 2.3%) of Fas-negative cells underwent apoptosis after TMX treatment when pretreated with IFN-gamma. In contrast, TMX alone and IFN-gamma alone stimulated apoptosis by only 22% (22 +/- 3%) P <.00013, and 17% (17 +/- 2%) P <.0001 in Fas-ve cells respectively. In vivo human cholangiocarcinomas xenograft growth was significantly inhibited by a combination of TMX + IFN-gamma compared to controls P <.0007. CONCLUSION: TMX exposure to human cholangiocarcinoma after pretreatment with IFN-gamma allows for induction of apoptosis in vitro and significant inhibition tumor xenograft growth. The combination of these two compounds may provide novel treatment regimen for cholangiocarcinoma.