Assuntos
Antineoplásicos/uso terapêutico , Indóis/uso terapêutico , Neoplasias Meníngeas/tratamento farmacológico , Meningioma/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Pirróis/uso terapêutico , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patologia , Meningioma/metabolismo , Meningioma/patologia , Pessoa de Meia-Idade , Proteômica/métodos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/análise , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Espectrometria de Massas por Ionização por Electrospray , SunitinibeAssuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Fragmentos de Peptídeos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Knockout , RatosRESUMO
Evidence is mounting that proinflammatory and proapoptotic thioredoxin-interacting protein (TXNIP) has a causative role in the development of diabetes. However, there are no studies investigating the role of TXNIP in diabetic retinopathy (DR). Here, we show that, in diabetic rats, TXNIP expression and hexosamine biosynthesis pathway (HBP) flux, which regulates TXNIP, are elevated in the retina and correlates well with the induction of inflammatory cyclooxygenase 2 (Cox-2) and sclerotic fibronectin (FN). We blocked the expression of TXNIP in diabetic rat retinas by: (i) inhibiting HBP flux; (ii) inducing post-transcriptional gene silencing (PTGS) for TXNIP mRNA; and (iii) performing an in vivo transcriptional gene silencing (TGS) approach for TXNIP knockdown by promoter-targeted small interfering RNAs and cell-penetrating peptides as RNA interference (RNAi) transducers. Each of these methods is efficient in downregulating TXNIP expression, resulting in blockade of its target genes, Cox-2 and FN, demonstrating that TXNIP has a causative role in aberrant gene induction in early DR. RNAi TGS of TXNIP abolishes diabetes-induced retinal gliosis and ganglion injury. Thus, TXNIP has a critical role in inflammation and retinal injury in early stages of DR. The successful employment of TXNIP TGS and amelioration of its pathological effects open the way for novel therapeutic strategies aimed to block disease onset and progression of DR.
Assuntos
Proteínas de Transporte/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Animais , Vias Biossintéticas/efeitos dos fármacos , Proteínas de Ciclo Celular , Células Cultivadas , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Retinopatia Diabética/complicações , Retinopatia Diabética/enzimologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Fibronectinas/metabolismo , Fibrose , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Gliose/complicações , Gliose/patologia , Glucose/farmacologia , Hexosaminas/biossíntese , Hexosaminas/farmacologia , Humanos , Inflamação/patologia , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Retina/efeitos dos fármacos , Retina/enzimologia , Retina/patologiaRESUMO
PURPOSE: The two most widely investigated animal models for diabetic retinopathy (DR) are the rat and dog. In dogs, aldose reductase (AR) is present only in retinal capillary pericytes and their destruction has been linked to polyol accumulation and resulting apoptosis. Since both rat capillary pericytes and endothelial cells have been reported to contain AR, the role of polyol pathway activity in capillary cell destruction has been investigated in rat retinal capillary pericyte (TR-rPCT) and endothelial (TR-iBRB) cells. METHODS: TR-rPCT and TR-iBRB cell lines were recloned and their identities were reconfirmed by characteristic immunostaining. Cells were cultured up to 72 h in media containing 50 mM glucose or galactose with/without the AR inhibitors or a sorbitol dehydrogenase inhibitor (SDI) or with 30 mM 3-fluoro-3-deoxyglucose. Polyol levels were determined by HPLC or (19)F-NMR. Apoptosis was detected with TUNEL/DAPI staining. RESULTS: Smooth muscle actin is present only in pericytes while only endothelial cells stain for von Willebrand factor and accumulate acetylated low-density lipoprotein. AR is present in both cells but AR levels are lower in endothelial cells. Aldehyde reductase is also present in both cells. Cells cultured in 50 mM glucose or galactose show significant polyol accumulation in pericytes but endothelial cells show little accumulation of galactitol and no accumulation of sorbitol. Sorbitol accumulation in pericytes resulted in increased cellular permeability and increased TUNEL staining, which was reduced by AR inhibition. CONCLUSIONS: Although both rat retinal pericytes and endothelial cells contain AR, sorbitol accumulation and TUNEL staining primarily occur in pericytes and are inhibited by AR inhibitors.
Assuntos
Aldeído Redutase/metabolismo , Glucose/toxicidade , Pericitos/metabolismo , Álcoois Açúcares/metabolismo , Actinas/metabolismo , Aldeído Redutase/antagonistas & inibidores , Animais , Apoptose , Capilares/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Galactitol/metabolismo , Galactose/metabolismo , Galactose/toxicidade , Glucose/metabolismo , Marcação In Situ das Extremidades Cortadas , Indóis , L-Iditol 2-Desidrogenase/antagonistas & inibidores , L-Iditol 2-Desidrogenase/metabolismo , Pericitos/enzimologia , Ratos , Ratos Transgênicos , Retina/metabolismo , Vasos Retinianos/metabolismo , Sorbitol/metabolismo , Coloração e Rotulagem , Fator de von Willebrand/metabolismoRESUMO
PURPOSE: To characterize the uptake mechanism of zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, in syncytiotrophoblast cells using the TR-TBT 18d-1 cell line previously established by our group. MATERIALS AND METHODS: The effects of several transporter inhibitors on the initial and steady-state apical uptake of AZT by TR-TBT 18d-1 were characterized, in order to identify the transporter(s) involved. RESULTS: Initial uptake of AZT was sodium-independent and saturable; the K(m) value was about 16 microM. Nitrobenzylthioinosine (NBMPR), probenecid and cimetidine each had little effect on the saturable AZT uptake, indicating that well characterized transporters, such as organic anion transporters (OATs and OATPs), organic cation transporters (OCTs) and equilibrative nucleoside transporters (ENTs), are not involved. However, thymidine and 2'-deoxyuridine strongly inhibited AZT uptake. These results suggest that an unidentified nucleoside uptake transporter is responsible for the uptake of AZT. Cyclosporin A, Ko143 and probenecid had little effect on AZT accumulation by TR-TBT 18d-1 cells, suggesting that transporter-mediated efflux of AZT is not substantial. CONCLUSION: Our results indicate that saturable AZT uptake into TR-TBT 18d-1 is mediated by a so-far-unidentified transporter.
Assuntos
Fármacos Anti-HIV/metabolismo , Trofoblastos/metabolismo , Zidovudina/metabolismo , Algoritmos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Interpretação Estatística de Dados , Interações Medicamentosas , Células Gigantes/citologia , Células Gigantes/metabolismo , Proteínas de Transporte de Nucleosídeos/antagonistas & inibidores , Proteínas de Transporte de Nucleosídeos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , RatosRESUMO
Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens, such as isoprenoid pyrophosphomonoester intermediates, alkylamine compounds, and bisphosphonate drugs, as well as some tumor cells. Although attempts have been made to derive novel cancer immunotherapies based on the discovery of these unconventional antigens, effective therapies remain to be developed. Here, we synthesized a series of pyrophosphate-containing compounds and examined the chemical requirements for the recognition of pyrophosphomonoester antigens by gammadelta T cells. The structural analysis clearly demonstrated that a proximal methylene moiety plays a crucial role in the stimulatory activity of the antigens. For optimal gammadelta T cell proliferation, we find that the use of human serum albumin was preferred and that pyrophosphomonoesters were superior to nitrogen-containing bisphosphonate compounds. Using these techniques, we have successfully expanded gammadelta T cells from healthy donors as well as from cancer patients using one of the most active compounds, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). The resulting expanded gammadelta T cells exhibited potent, cytotoxic activity against a wide variety of tumor cell lines. Even gammadelta T cells from a patient with advanced liver carcinoma efficiently responded to 2M3B1PP and exhibited strong cytotoxic activity against tumor cells. The pretreatment of tumor cells with nonpeptide antigens was essential for efficient cytotoxicity via TCR-gammadelta. The present study suggests a novel strategy for cancer immunotherapy using synthetic small pyrophosphate-containing compounds and nitrogen-containing bisphosphonates.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Difosfatos/síntese química , Difosfatos/farmacologia , Imunoterapia , Neoplasias/terapia , Especificidade de Anticorpos , Antígenos de Neoplasias/química , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Clonais , Difosfonatos/farmacologia , Citometria de Fluxo , Humanos , Interleucina-2/metabolismo , Células Jurkat , Modelos Moleculares , Monócitos/efeitos dos fármacos , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologiaRESUMO
BACKGROUND: Postoperative respiratory failure is often encountered in patients suffering from acute aortic dissection (AAD) and is believed to be influenced by release of neutrophil elastase after cardiopulmonary bypass. Sivelestat is a specific neutrophil elastase inhibitor, and this study aims to evaluate the effects of sivelestat on postoperative respiratory failure due to AAD. METHODS AND RESULTS: Patients who were operated for AAD from January 2000 to April 2005 and who had less than 300 mmHg initial postoperative PaO (2)/FiO (2) were investigated retrospectively and divided into two groups. Group 1 (n = 9) received intravenous administration of sivelestat immediately after the operation, while Group II (n = 9) received no sivelestat. There were no significant differences between Group I and II with respect to patients' characteristics or background (age, body weight, operating time, cardiopulmonary bypass time, amount of bleeding, preoperative WBC number and initial PaO (2)/FiO (2)). Though patients in Group I showed a subtle improvement in certain parameters such as PaO (2)/FiO (2), A-aDO (2) and respiratory index (RI) over a 3-day observation period compared to those of Group II, there were no significant differences. Neither postoperative mechanical ventilation time nor ICU stay differed between Group I and II. However, Group I showed a significantly greater improvement in the ratio of RI to initial RI on the 3POD compared to that of Group II (61.6 +/- 44.2 % vs. 111.9 +/- 40.9 %, P = 0.02). CONCLUSION: Inhibiting the activity of the neutrophil elastase may attenuate the postoperative respiratory complications of patients with AAD.
Assuntos
Aneurisma Aórtico/cirurgia , Dissecção Aórtica/cirurgia , Glicina/análogos & derivados , Proteínas Secretadas Inibidoras de Proteinases/uso terapêutico , Insuficiência Respiratória/prevenção & controle , Inibidores de Serina Proteinase/uso terapêutico , Sulfonamidas/uso terapêutico , Doença Aguda , Idoso , Ponte Cardiopulmonar/efeitos adversos , Feminino , Glicina/uso terapêutico , Humanos , Masculino , Cuidados Pós-Operatórios , Estudos RetrospectivosRESUMO
The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.
Assuntos
Barreira Hematoencefálica/citologia , Células Cultivadas/metabolismo , Endotélio Vascular/citologia , Modelos Biológicos , Xenobióticos/farmacocinética , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Bovinos , Cães , Endotélio Vascular/metabolismo , Humanos , Camundongos , Permeabilidade , Ratos , Reprodutibilidade dos TestesRESUMO
To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Aminoácidos/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Linhagem Celular Transformada , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/genética , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Glutamato-Amônia Ligase/metabolismo , Imuno-Histoquímica , Cinética , Masculino , Ratos , Sódio/metabolismo , TemperaturaRESUMO
In this study, the gamma-aminobutyric acid (GABA) transporter at the blood-brain barrier (BBB) was identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis, and the transport mechanism was characterized using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB) as an in vitro model of the BBB. gamma-Aminobutyric acid transport was studied by the cellular uptake of [ 3 H]GABA. [3H]GABA uptake by TM-BBB cells was Na (+)-, Cl(-)-, and concentration-dependent. The corresponding Michaelis-Menten constant was 679 +/- 80 micromol/L and the maximal uptake rate was 4,790 +/- 494 pmol/(mg protein x 5 minutes). [3H]GABA uptake by TM-BBB cells was significantly inhibited by betaine, beta-alanine, nipecotic acid, taurine, and quinidine, whereas probenecid, L-proline, creatine, and glycine had no effect. This type of inhibition is consistent with the predominant involvement of the GAT2/BGT-1 transporter in TM-BBB cells. RT-PCR analysis showed that GAT2/BGT-1 mRNA was expressed in TM-BBB cells, whereas Western blot analysis showed that TM-BBB cells and mouse brain capillaries express GAT2/BGT-1 protein. Moreover, confocal immunofluorescent microscopy of dual-labeled mouse brain sections demonstrated the colocalization of GAT2/BGT-1 and P-glycoprotein, a BBB-specific marker, on brain capillaries labeled with anti-GAT2/BGT-1 antibody and anti-P-glycoprotein antibody, respectively. These results are evidence that GAT2/BGT-1 is expressed at the BBB and is involved in GABA transport across the BBB.
Assuntos
Barreira Hematoencefálica/fisiologia , Proteínas de Membrana Transportadoras , Transportadores de Ânions Orgânicos , Ácido gama-Aminobutírico/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Células Cultivadas , Proteínas da Membrana Plasmática de Transporte de GABA , Glucana Endo-1,3-beta-D-Glucosidase/genética , Rim/metabolismo , Cinética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Urotélio/metabolismoRESUMO
In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.
Assuntos
Barreira Hematoencefálica , Proteínas de Membrana Transportadoras , Transportadores de Ânions Orgânicos , Ácido gama-Aminobutírico/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Capilares , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , Endotélio Vascular/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA , Meia-Vida , Cinética , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Microinjeções , Ácidos Nipecóticos/farmacologia , Lobo Parietal/metabolismo , Probenecid/farmacologia , Ratos , Ratos Sprague-Dawley , Taurina/farmacologia , Trítio , beta-Alanina/farmacologia , Ácido gama-Aminobutírico/administração & dosagem , Ácido gama-Aminobutírico/farmacologiaRESUMO
Three hundred eight patients with childhood and adolescent epilepsy were examined to clarify the incidence of epileptic discharges on initial and follow-up electroencephalograms. Epileptic discharges were found in 75.6% patients on the initial electroencephalogram, which is higher than figures previously reported for adults. The cumulative incidence of epileptic discharges was 92.3% by the third electroencephalogram recording. However, in 17.1% patients with nonspecific idiopathic generalized epilepsy, no epileptic discharges were found even after three electroencephalogram recordings. The incidence of epileptic discharges in patients with generalized epilepsy (84.3%) was significantly higher than in patients with localization-related epilepsy (71.6%). The incidence of epileptic discharges in patients with partial seizures was lower than those in patients with generalized seizures. The incidence of epileptic discharges was low in the 0- to 3-year-old and 15- to 20-year-old groups, and high in the 3- to 12-year-old groups. In the positive epileptic discharge patients, 38.8% of electroencephalograms were abnormal only during the waking or sleeping portion of the recordings. Knowing the incidence of epileptic discharges for each type of epilepsy will be useful in planning further electroencephalogram research and performing electroencephalograms in the clinical setting.
Assuntos
Encéfalo/fisiopatologia , Epilepsia/fisiopatologia , Adolescente , Fatores Etários , Criança , Pré-Escolar , Ritmo Circadiano , Diagnóstico Diferencial , Eletroencefalografia/estatística & dados numéricos , Epilepsias Parciais/fisiopatologia , Epilepsia/diagnóstico , Epilepsia Generalizada/fisiopatologia , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Estudos ProspectivosAssuntos
Barreira Hematoencefálica/fisiologia , Barreira Hematorretiniana/fisiologia , Cistina/metabolismo , Ácido 2-Aminoadípico/farmacologia , Animais , Ácido Aspártico/farmacologia , Transporte Biológico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematorretiniana/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Masculino , Maleatos/farmacologia , Ratos , Ratos WistarRESUMO
This is the first case report of Hodgkin's disease (HD) which showed both remission and exacerbation of neurological signs before a confirmed diagnosis of HD. The episodes occurred three times and multiple lesions were involved. Immunoabsorption plasmapheresis and double filtration plasmapheresis were effective for the first episode, whereas, corticosteroids partly improved the second and third episodes. Fever and lymph node swelling were apparent afterward and she was diagnosed as having HD from a supraclavicular lymph node biopsy. The remaining neurologic deficits responded to chemotherapy and radiotherapy. The neurological symptoms were considered as a paraneoplastic syndrome of HD.
Assuntos
Doença de Hodgkin/diagnóstico , Síndromes Paraneoplásicas do Sistema Nervoso/diagnóstico , Adulto , Terapia Combinada , Diagnóstico Diferencial , Feminino , Doença de Hodgkin/terapia , Humanos , Síndromes Paraneoplásicas do Sistema Nervoso/terapia , Plasmaferese , Biópsia de Linfonodo Sentinela , Resultado do TratamentoRESUMO
PURPOSE: To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). METHODS: Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. RESULTS: Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. CONCLUSIONS: Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.
Assuntos
Aminoácidos/metabolismo , Barreira Hematoencefálica/fisiologia , Linhagem Celular Transformada/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/metabolismo , Transporte Biológico/fisiologia , Ácido Glutâmico/metabolismo , Masculino , Pré-Albumina/metabolismo , Prolina/metabolismo , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
PURPOSE: Postneonatal neovascularization is thought to result exclusively from the proliferation, migration, and remodeling of fully differentiated endothelial cells (ECs). Recently, it has been reported that bone marrow contains cells which can differentiate into ECs and contribute to neoangiogenesis in adult species. In this study, we tried to establish conditionally immortalized endothelial cell lines (TR-BME) derived from rat bone marrow. METHODS: Mononuclear cells were isolated and differentiated into ECs at 37 degrees C from the bone marrow of a transgenic rat harboring temperature-sensitive SV40 large T-antigen (ts T-Ag) gene. Then, the cells were transferred and incubated at 33 degrees C, a permissive temperature for ts T-Ag. Expression of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1, 2, Tie-1, 2 and von Willebrand factor (VWF) were assayed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR). RESULTS: We have established three cell lines incorporating 1,1'-dioctadecyl-3,3,3',3-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL) with a spindle shape. One of these, clone 2, strongly expressed VEGFR-2, and weakly expressed VEGFR-1 and VWF. In contrast, clone 8 showed strong expression of Tie-1, 2, and VWF, and weak expression of VEGFR-1,2. All markers were expressed strongly in clone 3. CONCLUSIONS: These data confirm that the above three TR-BME cells are novel ECs derived from bone marrow progenitors.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular/citologia , Linhagem Celular/metabolismo , Endotélio/citologia , Endotélio/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/metabolismo , Técnicas de Cultura de Células/métodos , Feminino , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de TIE-1 , Receptor TIE-2 , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de TIE , Receptores de Fatores de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fator de von Willebrand/metabolismoRESUMO
We report here two patients with recurrent paradoxical brain embolism through the patent foramen ovale(PFO). The TIAs, which occurred frequently under antiplatelet therapy, resolved soon after commencing the anticoagulation therapy. Case 1, a 57-year-old woman, was diagnosed as having lacunar brain infarction and was treated with ticlopidine hydrochloride. Eleven months later, she suffered from frequent TIAs. Anticoagulation therapy was started after the presence of PFO was documented on transesophageal echocardiography(TEE). Thereafter, her TIA disappeared. Case 2 was a 67-year-old man with a history of lacunar brain infarction. Although he was treated with aspirin for 9 months, he showed transient monoplegia in his right leg. His TEE study also revealed the presence of PFO. After the anticoagulation therapy reached the proper level(PT-NR = 2), he never experienced the recurrence of TIA. If the antiplatelet therapy failed to prevent the recurrence of TIA, it is recommended to find out the presence of PFO which is a potential source of paradoxical brain embolism. In such a case, anticoagulation therapy should be instituted.
Assuntos
Anticoagulantes/uso terapêutico , Comunicação Interatrial/complicações , Embolia Intracraniana/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Idoso , Ecocardiografia Transesofagiana , Feminino , Comunicação Interatrial/diagnóstico por imagem , Humanos , Embolia Intracraniana/etiologia , Masculino , Pessoa de Meia-Idade , RecidivaRESUMO
The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 approximately 9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33 degrees C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degrees C due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3- O -methyl- D -glucose (3-OMG) uptake activity. This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 +/- 0.51 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.
Assuntos
Barreira Hematorretiniana , Endotélio Vascular/patologia , Células Tumorais Cultivadas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Animais , Animais Geneticamente Modificados , Antígenos Virais de Tumores/análise , Antígenos Virais de Tumores/genética , Western Blotting , Capilares , Divisão Celular , Separação Celular , Genes MDR , Transportador de Glucose Tipo 1 , Temperatura Alta , Modelos Animais , Proteínas de Transporte de Monossacarídeos/análise , Ratos , Receptores Proteína Tirosina Quinases/análise , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento do Endotélio Vascular , Vasos Retinianos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.
Assuntos
Linhagem Celular Transformada , Endotélio Vascular/citologia , Modelos Biológicos , Animais , Endotélio Vascular/metabolismo , Técnicas In Vitro , Camundongos , Farmacocinética , RatosRESUMO
PURPOSE: The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB. METHODS: TR-iBRB2 cells were cultured at 33 degrees C, and L-lactic acid uptake was monitored by measuring [14C]L-lactic acid at 37 degrees C. The expression and mRNA level of monocarboxylate transporter (MCT)1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively. RESULTS: The [14C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [14C]L-lactic acid uptake at pH 6.0 was both temperature- and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxyphenylhydrazone (protonophore), alpha-cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H+-coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H+ gradient. [14C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT2 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [14C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na+/H+ exchanger I mRNA was expressed in TR-iBRB2 cells. CONCLUSION: L-Lactic acid transport at the iBRB is an H-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.