RESUMO
PURPOSE: Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities, including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. METHODS: We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. RESULTS: We established a flow cytometry gating strategy for identifying and isolating FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines, and expression increased when exposed to Tregs but not CD4 + helper T-cells. CONCLUSION: Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.
Assuntos
Neoplasias Encefálicas , Quimiocina CCL2 , Glioma , Receptores CCR4 , Linfócitos T Reguladores , Cães , Animais , Receptores CCR4/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Glioma/metabolismo , Glioma/imunologia , Glioma/patologia , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , HumanosRESUMO
Purpose: Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high grade glioma and human glioblastomas share many molecular similarities, including accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford targeting the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic model of glioma. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. Methods: We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. Results: We established a flow cytometry gating strategy for identification and isolation of FOXP3+ Tregs in dogs. The canine CD4 + CD25high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines and expression increased when exposed to Tregs but not to CD4 + helper T-cells. Conclusion: Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.
RESUMO
The purpose of the study was to determine the long-term safety and effectiveness of high-dose immunosuppressive therapy (HDIT) followed by autologous hematopoietic cell transplantation (AHCT) in advanced multiple sclerosis (MS). TBI, CY and antithymocyte globulin were followed by transplantation of autologous, CD34-selected PBSCs. Neurological examinations, brain magnetic resonance imaging and cerebrospinal fluid (CSF) for oligoclonal bands (OCB) were serially evaluated. Patients (n=26, mean Expanded Disability Status Scale (EDSS)=7.0, 17 secondary progressive, 8 primary progressive, 1 relapsing/remitting) were followed for a median of 48 months after HDIT followed by AHCT. The 72-month probability of worsening ≥1.0 EDSS point was 0.52 (95% confidence interval, 0.30-0.75). Five patients had an EDSS at baseline of ≤6.0; four of them had not failed treatment at last study visit. OCB in CSF persisted with minor changes in the banding pattern. Four new or enhancing lesions were seen on MRI, all within 13 months of treatment. In this population with high baseline EDSS, a significant proportion of patients with advanced MS remained stable for as long as 7 years after transplant. Non-inflammatory events may have contributed to neurological worsening after treatment. HDIT/AHCT may be more effective in patients with less advanced relapsing/remitting MS.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Terapia de Imunossupressão/métodos , Esclerose Múltipla/terapia , Adulto , Soro Antilinfocitário/uso terapêutico , Feminino , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Esclerose Múltipla/cirurgia , Transplante Autólogo , Resultado do Tratamento , Irradiação Corporal TotalRESUMO
BACKGROUND: Patients treated with chemoradiotherapy (CRT) for head and neck cancers often require feeding tubes (FTs) due to toxicity. We sought to identify factors associated with a prolonged FT requirement. PATIENTS AND METHODS: We retrospectively reviewed 80 patients treated with CRT for head and neck cancers. The pharyngeal constrictors (PCs), supraglottic larynx (SGL), and glottic larynx (GL) were contoured and the mean radiation doses and the volumes of each receiving >40, 50, 60, and 70 Gy (V40, V50, V60, and V70) were determined. RESULTS: A total of 33 of 80 patients required a FT either before or during the course of CRT. Fifteen patients required the FT for > or = 6 months. On univariate analysis, significant factors associated with a prolonged FT requirement were mean PC dose, PC-V60, PC-V70, SGL dose, SGL-V70, and advanced T3-T4 disease. Multivariate analyses found both PC-V70 and T3-T4 disease as significant factors .The proportions of patients requiring a FT > or = 6 months were 8% and 28% for treatment plans with PC-V70 <30% and > or = 30%, respectively. CONCLUSIONS: Increased radiation dose to the PCs is associated with a higher risk of a prolonged FT need. Dose sparing of the PC muscles may reduce this risk.
Assuntos
Nutrição Enteral , Neoplasias de Cabeça e Pescoço/radioterapia , Faringe/efeitos da radiação , Lesões por Radiação/complicações , Radioterapia/efeitos adversos , Adulto , Idoso , Antineoplásicos/efeitos adversos , Terapia Combinada , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/efeitos da radiação , Estadiamento de Neoplasias , Dosagem Radioterapêutica , Estudos Retrospectivos , TempoRESUMO
Caspases are a family of cysteine proteases that are expressed as inactive zymogens and undergo proteolytic maturation in a sequential manner in which initiator caspases cleave and activate the effector caspases 3, 6 and 7. Effector caspases cleave structural proteins, signaling molecules, DNA repair enzymes and proteins which inhibit apoptosis. Activation of effector, or executioner, caspases has historically been viewed as a terminal event in the process of programmed cell death. Emerging evidence now suggests a broader role for activated caspases in cellular maturation, differentiation and other non-lethal events. The importance of activated caspases in normal cell development and signaling has recently been extended to the CNS where these proteases have been shown to contribute to axon guidance, synaptic plasticity and neuroprotection. This review will focus on the adaptive roles activated caspases in maintaining viability, the mechanisms by which caspases are held in check so as not produce apoptotic cell death and the ramifications of these observations in the treatment of neurological disorders.
Assuntos
Caspases/metabolismo , Sistema Nervoso Central/metabolismo , Animais , Encéfalo/irrigação sanguínea , Isquemia Encefálica/metabolismo , Calpaína/metabolismo , Inibidores de Caspase , Inibidores Enzimáticos/farmacologia , Humanos , Doenças do Sistema Nervoso/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
To determine if the macrophage mannose receptor transcript is present in mouse, rat, pig, and human retinal pigment epithelium (RPE), primary cultures and/or freshly dissected retinal pigment epithelium from four different species were used to isolate total RNA. RT-PCR was used to amplify segments of the macrophage mannose receptor from each sample. Amplified products were sequenced and compared with known sequences of the macrophage mannose receptor. Macrophage mannose receptor transcripts were identified in all RPE samples. Comparison between sequences identified in RPE with macrophage sequences from the same species revealed 100% identity. Sequence homology between the different species was 74% or greater. These data are consistent with the transcription of a single mannose receptor gene by these two phagocytic cell types.
Assuntos
Lectinas Tipo C/genética , Macrófagos/metabolismo , Lectinas de Ligação a Manose/genética , Epitélio Pigmentado Ocular/metabolismo , Receptores de Superfície Celular/genética , Transcrição Gênica , Animais , Sequência de Bases , Células Cultivadas , Humanos , Receptor de Manose , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA/isolamento & purificação , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2'-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 microm DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDP-induced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.
Assuntos
Caspases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Oxidantes/farmacologia , Canais de Potássio/metabolismo , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Benzamidas/farmacologia , Inibidores de Caspase , Células Cultivadas , Dissulfetos/antagonistas & inibidores , Dissulfetos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neurônios/citologia , Neurônios/efeitos dos fármacos , Niacinamida/farmacologia , Oxidantes/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/antagonistas & inibidores , Reagentes de Sulfidrila/farmacologia , Zinco/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: The purpose of this study was to identify the profile of gene expression during retinal pigment epithelial (RPE) wound repair. METHODS: ARPE-19 cells derived from a human RPE cell line were grown for 4 weeks and injured by creating multiple concentric wounds. Unwounded cultures served as controls. During the proliferative phase of wound repair, total RNA was extracted from control and wounded cultures, and a [32P]dATP-labeled cDNA probe was synthesized and hybridized to Atlas Arrays (Clontech, Palo Alto, Calif.) containing 588 cDNAs. The autoradiograms obtained were then analyzed using the Molecular Dynamics software program. Semiquantitative PCR was carried out to confirm up-regulation of four genes associated with wound repair. ELISA was performed to quantitate the secreted MCP-1. RESULTS: In wounded cultures prominent up-regulation (greater than fivefold) was seen for genes encoding DNA synthesis and DNA repair proteins. A greater than threefold increase was seen for genes encoding mitogen-activated protein kinase, CD44, MCP-1 (monocyte chemotactic protein), thymosin beta-10, and HDGF (hepatoma-derived growth factor), among others. Genes encoding tumor suppressors were downregulated three- to five-fold in the wounded compared with the unwounded cultures. Semiquantitative PCR confirmed up-regulation of transcripts for thymosin beta-10, HDGF, CD44, and MCP-1. ELISA showed a 20% increase in secreted MCP-1. CONCLUSIONS: Gene array analysis revealed a differentiation program that included increased expression of genes involved in wound repair (adhesion molecules, cytokines, signal transducers), along with increased MCP-1 secretion. The RPE may be an early participant in the inflammatory response that occurs with proliferative vitreoretinopathy.
Assuntos
Proteínas do Olho/genética , Peptídeos e Proteínas de Sinalização Intercelular , Epitélio Pigmentado Ocular/metabolismo , Cicatrização , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , DNA/biossíntese , Sondas de DNA , Reparo do DNA , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/biossíntese , Expressão Gênica , Perfilação da Expressão Gênica , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Humanos , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/lesões , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Timosina/biossíntese , Timosina/genética , Regulação para CimaRESUMO
The membrane-permeant oxidizing agent 2,2'-dithiodipyridine (DTDP) can induce Zn(2+) release from metalloproteins in cell-free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad-spectrum cysteine protease inhibitor butoxy-carbonyl-aspartate-fluoromethylketone. N,N,N', N'-Tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and other cell-permeant metal chelators also effectively blocked DTDP-induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK-801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc-selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura-2- and magfura-2-loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP-mediated increase in intracellular free Zn(2+) concentrations. Our studies suggest that under conditions of oxidative stress, Zn(2+) released from intracellular stores may contribute to the initiation of neuronal apoptosis.
Assuntos
2,2'-Dipiridil/análogos & derivados , Apoptose , Líquido Intracelular/metabolismo , Neurônios/metabolismo , Compostos de Sulfidrila/metabolismo , Zinco/metabolismo , 2,2'-Dipiridil/toxicidade , Animais , Células Cultivadas , Quelantes/farmacologia , Técnicas de Cocultura , Fragmentação do DNA , Dissulfetos/antagonistas & inibidores , Dissulfetos/toxicidade , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , L-Lactato Desidrogenase/metabolismo , N-Metilaspartato/toxicidade , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Potássio/metabolismo , Potássio/farmacologia , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Reagentes de Sulfidrila/antagonistas & inibidores , Reagentes de Sulfidrila/toxicidade , Tetraetilamônio/farmacologiaRESUMO
In central neurons, glutamate receptor activation causes massive calcium influx and induces a mitochondrial depolarization, which is partially blocked by cyclosporin A, suggesting a possible activation of the mitochondrial permeability transition pore (PTP) as a mechanism. It has been recently reported that tamoxifen (an antiestrogen chemotherapeutic agent) blocks the PTP in isolated liver mitochondria, similar to cyclosporin A. In this study, we tested whether tamoxifen inhibits the mitochondrial depolarization induced by glutamate receptor activation in intact cultured neurons loaded with the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazolylcarbocyanine iodide. This dye reports disruptions in mitochondrial membrane potential, which can be caused by PTP activation. We found that glutamate (100 microM for 10 min) causes a robust mitochondrial depolarization that is partially inhibited by tamoxifen. The maximum inhibitory concentration of tamoxifen was 0.3 microM, with concentrations higher and lower than 0.3 microM being less effective. However, although tamoxifen (0.3 microM) blocked glutamate-induced mitochondrial depolarization, it did not inhibit glutamate-induced neuronal death, in contrast to the PTP inhibitor cyclosporin A. A relatively high concentration of tamoxifen (100 microM) caused mitochondrial depolarization itself and was neurotoxic. These data suggest that tamoxifen may be an inhibitor of the PTP in intact neurons. However, the lack of specificity of most PTP inhibitors, and the difficulty in measuring PTP in intact cells, preclude definite conclusions about the role of PTP in excitotoxic injury.
Assuntos
Antineoplásicos Hormonais/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Tamoxifeno/farmacologia , Animais , Antineoplásicos Hormonais/toxicidade , Benzimidazóis , Carbocianinas , Morte Celular/efeitos dos fármacos , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Antagonistas de Aminoácidos Excitatórios/toxicidade , Corantes Fluorescentes , Histocitoquímica , Masculino , Malonatos/toxicidade , Potenciais da Membrana/efeitos dos fármacos , Membranas/efeitos dos fármacos , Neostriado/citologia , Neostriado/efeitos dos fármacos , Neurônios/ultraestrutura , Permeabilidade/efeitos dos fármacos , Prosencéfalo/citologia , Prosencéfalo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tamoxifeno/toxicidadeRESUMO
Neovascularization appears to play an early and important part in the evolution of psoriatic plaques. We studied the distribution and production of two known angiogenesis factors, endothelial cell stimulating angiogenesis factor (ESAF) and vascular endothelial growth factor (VEGF), in the skin of patients with chronic plaque psoriasis and normal control subjects. Our results showed that tissue levels of ESAF and VEGF were significantly elevated in involved as compared with normal control skin (P = 0.006 and P < 0. 0001, respectively). Tissue levels of ESAF and VEGF were also raised in involved skin as compared with uninvolved skin in patients with psoriasis (P = 0.001 and P < 0.0001, respectively). Tissue levels of ESAF and VEGF in plaques of psoriasis correlated closely with the clinical severity of psoriasis (r = 0.6 and r = 0.9, respectively). Serum levels of ESAF and VEGF were significantly raised in patients with psoriasis as compared with control subjects (P = 0.001 and P = 0.02, respectively). In vitro culture studies revealed that ESAF is produced by both keratinocytes and fibroblasts in approximately equal quantities in normal skin, whereas VEGF is secreted predominately by keratinocytes. A similar pattern is seen in both involved and uninvolved skin of patients with psoriasis. However, there is increased secretion of both factors in keratinocytes and fibroblasts from involved and uninvolved skin as compared with normal control skin (P < 0.001). The increased levels and secretion in plaques of psoriasis of two molecules, ESAF and VEGF, known to promote new blood vessel formation, suggest a pathogenetic role for them in this disease.
Assuntos
Indutores da Angiogênese/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Psoríase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Indutores da Angiogênese/sangue , Técnicas de Cultura de Células , Doença Crônica , Fatores de Crescimento Endotelial/sangue , Feminino , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Linfocinas/sangue , Masculino , Pessoa de Meia-Idade , Psoríase/sangue , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Several inhibitors of mitochondrial complex II cause neuronal death in vivo and in vitro. The goal of the present work was to characterize in vitro the effects of malonate (a competitive blocker of the complex) which induces neuronal death in a pattern similar to that seen in striatum in Huntington's disease. Exposure of striatal and cortical cultures from embryonic rat brain for 24 h to methylmalonate, a compound which produces malonate intracellularly, led to a dose-dependent cell death. Methylmalonate (10 mM) caused >90% mortality of neurons although cortical cells were unexpectedly more vulnerable. Cell death was attenuated in a medium containing antioxidants. Further characterization revealed that DNA laddering could be detected after 3 h of treatment. Morphological observations (videomicroscopy and Hoechst staining) showed that both necrotic and apoptotic cell death occurred in parallel; apoptosis was more prevalent. A decrease in the ATP/ADP ratio was observed after 3 h of treatment with 10 mM methylmalonate. In striatal cultures it occurred concomitantly with a decline in GABA and a rise in aspartate content and the aspartate/glutamate ratio. Changes in ion concentrations were measured in similar cortical cultures from mouse brain. Neuronal [Na+]i increased while [K+]i and membrane potential decreased after 20 min of continuous incubation in 10 mM methylmalonate. These changes progressed with time, and a rise in [Ca2+]i was also observed after 1 h. The results demonstrate that malonate collapses cellular ion gradients, restoration of which imposes an additional load on the already compromised ATP-generation machinery. An early elevation in [Ca2+]i may trigger an increase in activity of proteases, lipases and endonucleases and production of free radicals and DNA damage which, ultimately, leads to cells death. The data also suggest that maturational and/or extrinsic factors are likely to be critical for the increased vulnerability of striatal neurons to mitochondrial inhibition in vivo.
Assuntos
Apoptose , Encéfalo/citologia , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Ácido Metilmalônico/toxicidade , Neurônios/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Complexo II de Transporte de Elétrons , Feto , Ácido Glutâmico/metabolismo , Cinética , Camundongos , Microscopia de Vídeo , Complexos Multienzimáticos/antagonistas & inibidores , Neurônios/metabolismo , Neurônios/patologia , Oxirredutases/antagonistas & inibidores , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Succinato Desidrogenase/antagonistas & inibidores , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
Endothelial cell stimulating angiogenesis factor (ESAF) is a small (> 1000 Da) dialysable non-peptide molecule with potent angiogenic activity. ESAF activates the major pro-matrix metalloproteinases and also uniquely reactivates the complex of these active enzymes with their tissue inhibitors resulting in both active enzyme and inhibitor. These actions may be pivotal in its role as an angiogenic factor. ESAF is primarily involved in angiogenic conditions where inflammatory cells are not evident such as foetal bone growth and electrically stimulated skeletal muscles and proliferative retinopathy. However, high levels also occur in actively growing human intracranial tumours but it is not noticeably elevated in rheumatoid arthritic synovial fluid. Its extreme potency and low molecular mass make its structural determination difficult. Possible therapeutic applications would be in the treatment of ischaemic ulcers, acceleration of fracture repair, infertility and more modestly in the correction of baldness. Analogues of ESAF could be of value in treating angiogenic diseases such as psoriasis and proliferative retinopathy.
Assuntos
Indutores da Angiogênese/fisiologia , Endotélio Vascular/fisiologia , Neovascularização Patológica , Neovascularização Fisiológica , Indutores da Angiogênese/farmacologia , Endotélio Vascular/patologia , HumanosRESUMO
Intrastriatal injections of the mitochondrial toxins malonate and 3-nitropropionic acid produce selective cell death similar to that seen in transient ischemia and Huntington's disease. The extent of cell death can be attenuated by pharmacological or surgical blockade of cortical glutamatergic input. It is not known, however, if dopamine contributes to toxicity caused by inhibition of mitochondrial function. Exposure of primary striatal cultures to dopamine resulted in dose-dependent death of neurons. Addition of medium supplement containing free radical scavengers and antioxidants decreased neuronal loss. At high concentrations of the amine, cell death was predominantly apoptotic. Methyl malonate was used to inhibit activity of the mitochondrial respiratory chain. Neither methyl malonate (50 microM) nor dopamine (2.5 microM) caused significant toxicity when added individually to cultures, whereas simultaneous addition of both compounds killed 60% of neurons. Addition of antioxidants and free radical scavengers to the incubation medium prevented this cell death. Dopamine (up to 250 microM) did not alter the ATP/ADP ratio after a 6-h incubation. Methyl malonate, at 500 microM, reduced the ATP/ADP ratio by approximately 30% after 6 h; this decrease was not augmented by coincubation with 25 microM dopamine. Our results suggest that dopamine causes primarily apoptotic death of striatal neurons in culture without damaging cells by an early adverse action on oxidative phosphorylation. However, when combined with minimal inhibition of mitochondrial function, dopamine neurotoxicity is markedly enhanced.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/toxicidade , Malonatos/toxicidade , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Trifosfato de Adenosina/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Benzazepinas/farmacologia , Células Cultivadas , Corpo Estriado/metabolismo , Corpo Estriado/ultraestrutura , DNA/análise , Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Sinergismo Farmacológico , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Nomifensina/farmacologia , Norepinefrina/farmacologia , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem , Fatores de Tempo , Gravação em VídeoRESUMO
Endothelial-cell-stimulating angiogenesis factor (ESAF) has been shown to activate procollagenase and reactivate complexes of collagenase and gelatinase A with tissue inhibitor of metallo-proteinase (TIMP)-1. In the present paper we show a purification protocol for bovine pineal ESAF and that purified ESAF activates progelatinase A and prostromelysin-1. Unlike the activation of procollagenase by plasmin/plasminogen activator, which requires the presence of stromelysin for full activation, ESAF is able to activate fully all three proenzymes. Purified ESAF is also shown to reactivate the complexes of gelatinase A, collagenase and stromelysin-1 with TIMP-2. Once separated, both enzyme and inhibitor are active; however, ESAF binds to the enzyme in a manner preventing it from further inhibition by TIMP. ESAF is the only physiological molecule able to reactivate the TIMP/enzyme complex.
Assuntos
Indutores da Angiogênese/fisiologia , Precursores Enzimáticos/metabolismo , Glicoproteínas/metabolismo , Neovascularização Fisiológica , Proteínas/metabolismo , Animais , Bovinos , Colagenases/química , Colagenases/metabolismo , Ativação Enzimática , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Gelatinases/antagonistas & inibidores , Gelatinases/química , Gelatinases/metabolismo , Humanos , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Peso Molecular , Ligação Proteica , Inibidor Tecidual de Metaloproteinase-2 , Inibidores Teciduais de MetaloproteinasesRESUMO
Penetrating keratoplasty is currently the only treatment for corneal endothelial dysfunction. Although corneal transplantation has a high success rate, a few problems still remain, such as the limited availability of donor grafts, the change in refraction after penetrating keratoplasty, and the higher chance of immune rejection. In this study, a coated hydrogel lens (Chiron Ophthalmics Inc., Irvine CA, U.S.A.) has been used as a carrier to transplant cultured homologous kitten and rabbit corneal endothelial cells into adult cats and rabbits. The transplantation procedure was the same in both species. Corneal endothelial cells from homologous rabbits or cats were seeded on coated hydrogel lenses and cultured until they reached a complete monolayer with an average cell density of 2,500 cells/mm2. Five weeks before transplantation surgery, corneal endothelial cells were scraped to induce corneal edema. The cell carrier device was then transplanted as follows: a trephine cut (7.7 mm) was made into the stroma, producing an outer corneal plug. The inner cornea was then cut by using a 5.5-mm trephine, and this inner plug was discarded. The implant was inserted and the outer corneal plug was sutured back into place. Corneas cleared completely within 3 days in both rabbits and cats, and stayed clear for an average of 40 days in rabbits and 50 days in cats. The histopathological evaluation of the rejected grafts showed vascularized retrocorneal membrane formation in cats, whereas in rabbits severe cellular infiltration of the stroma with neovascularization occurred without retrocorneal membrane formation.
Assuntos
Transplante de Células , Lentes de Contato Hidrofílicas , Endotélio Corneano/citologia , Polietilenoglicóis , Animais , Gatos , Contagem de Células , Divisão Celular , Células Cultivadas , Córnea/ultraestrutura , DNA/biossíntese , Endotélio Corneano/ultraestrutura , Hidrogel de Polietilenoglicol-Dimetacrilato , CoelhosRESUMO
Nitric oxide (NO) is important in many physiological, pharmacological, and pathological processes. According to current concepts, guanylyl cyclase is considered to be a receptor for NO in vascular and nonvascular smooth muscle and other tissues. Since there are no suitable radioisotopes of oxygen and nitrogen available for conventional radioligand-receptor binding studies for NO, a novel method was developed to identify NO binding site(s). A chemiluminescence-headspace gas assay was utilized to measure the sequestration of NO in biological systems, and this was used as an index of NO binding. In the present report, myoglobin (a hemoprotein, Mb) was used as a prototype macromolecule to develop the binding assay for subsequent application to studies of putative NO receptors. Solutions containing various concentrations of Mb were incubated with NO in sealed micro-Fernbach flasks at 37 degrees C in an argon atmosphere for 30 min; NO remaining in the headspace gas was analyzed by means of the chemiluminescence assay. The magnitude of NO sequestration was dependent on Mb concentration, and 5 nM Mb was the lowest Mb concentration for which NO sequestration was measurable. Application of the method to the measurement of NO sequestration by bovine serum albumin (BSA) and pulmonary artery medial layer homogenate (BPA-M) revealed that the lowest BSA concentration at which NO sequestration was measurable was 1.6 microM, which was 320 times greater than that for Mb. Applicability of the method to address the question of putative NO receptors was indicated by significant NO sequestration after incubation with 20% (w/v) homogenate of BPA-M, which is responsive to NO and putative NO prodrugs.
Assuntos
Óxido Nítrico/metabolismo , Animais , Sítios de Ligação , Bovinos , Técnicas de Química Analítica/métodos , Gases/análise , Cavalos , Medições Luminescentes , Mioglobina/metabolismo , Óxido Nítrico/análise , Artéria Pulmonar/química , Soroalbumina Bovina/metabolismoRESUMO
Isolation of Fc receptor-dependent phagosomes from a macrophage cell line, J774 A.1, was accomplished using antibody-opsonized, paramagnetic beads. Following binding and ingestion of these beads, cells were homogenized in a standard membrane isolation buffer. Phagosomes containing the trapped paramagnetic beads were isolated by subjecting the whole cell homogenate to a magnetic field. The method is extremely simple and the preparation of an enriched phagosome fraction from a whole cell homogenate is rapid and highly selective. The method should provide useful starting material for investigators interested in cytoskeletal involvement in phagocytosis, in kinetic studies of ingestion, in phagosome-lysosome fusion, and in the ability of a particular ligand to initiate phagocytosis.
Assuntos
Macrófagos/fisiologia , Organelas/ultraestrutura , Fagocitose , 5'-Nucleotidase/análise , 5'-Nucleotidase/metabolismo , Fosfatase Ácida/análise , Fosfatase Ácida/metabolismo , Animais , Biomarcadores/análise , Fracionamento Celular/métodos , Linhagem Celular , Proteínas do Citoesqueleto/análise , Eletroforese em Gel de Poliacrilamida/métodos , Macrófagos/ultraestrutura , Magnetismo , Proteínas de Membrana/análise , Microscopia Eletrônica , Peso Molecular , Organelas/fisiologia , Receptores Fc/metabolismoRESUMO
Endothelial cell stimulating angiogenesis factor (ESAF) is important in the neovascularisation that precedes new bone formation, and raised levels are found in association with healing fractures and osteoarthritis. We investigated its relevance to the new bone growth that is found at inflammatory sites in ankylosing spondylitis (AS). Forty-one patients with AS were studied clinically and radiographically and had their serum ESAF levels measured. In comparison to age-matched controls the AS patients had significantly raised ESAF levels (p < 0.0001). Within the AS group, patients with relatively higher ESAF levels had no characteristic clinical or radiological features.