RESUMO
In rats, iron deficiency produces an alteration in myelin formation. However, there is limited information on the effects of this condition on oligodendroglial cell (OLGc) proliferation and maturation. In the present study, we further analyzed the hypomyelination associated with iron deficiency by studying the dynamics of oligodendrogenesis. Rats were fed control (40 mg Fe/kg) or iron-deficient (4 mg Fe/kg) diets from gestation day 5 until postnatal day 3 (P3) or 11 (P11). OLGc proliferation, migration and differentiation were investigated before and after an intracranial injection of apotransferrin at 3 days of age (P3). The proliferating cell population was evaluated at P3. Iron-deficient (ID) animals showed an increase in the oligodendrocyte precursors cell (OPC) population in comparison with controls. The overall pattern of migration of cells labeled with BrdU was investigated at P11. Iron deficiency increased the amount of BrdU(+) cells in the corpus callosum (CC) and decreased OLGc maturation and myelin formation. Changes in nerve conduction were analyzed by measuring visual evoked potentials. Latency and amplitude were significantly disturbed in ID rats compared with controls. Both parameters were substantially normalized when animals were treated with a single intracranial injection of 350 ng apotransferrin (aTf). The current results give support to the idea that iron deficiency increases the number of proliferating and undifferentiated cells in the CC compared with the control. Treatment with aTf almost completely reverted the effects of iron deficiency, both changing the migration pattern and increasing the number of mature cells in the CC and myelin formation.
Assuntos
Apoproteínas/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/patologia , Deficiências de Ferro , Oligodendroglia/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Transferrina/uso terapêutico , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Apoproteínas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Peso Corporal/fisiologia , Encéfalo , Bromodesoxiuridina/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/fisiopatologia , Eletroencefalografia/métodos , Potenciais Evocados Visuais/efeitos dos fármacos , Potenciais Evocados Visuais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hematócrito/métodos , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Oligodendroglia/fisiologia , Estimulação Luminosa/métodos , Gravidez , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ácidos Siálicos/metabolismo , Transferrina/metabolismoRESUMO
Mechanisms that regulate oligodendroglial cell (OLGc) differentiation are the focus of intensive research in the field of cellular and molecular neurobiology. We have previously shown that the addition of apotransferrin (aTf) to primary OLGc cultures accelerates their differentiation and induces an increase in the expression of different components of the myelin cytoskeleton (CSK) such as actin, tubulin, and some of the microtubule-associated proteins, particularly the stable tubulin only peptide (STOP). Fyn protein-tyrosine kinase (Fyn kinase), a member of the Src family, participates in signalling pathways that regulate OLGs/myelin cytoskeletal reorganization. It is essential for myelin development in the central nervous system (CNS), and its absence results in hypomyelination. In the present study, we used both primary cell and N19 cell line cultures to investigate further the mechanisms of action involved in the accelerated differentiation of OLGcs induced by aTf. In particular, we were interested in studying the participation of Fyn kinase in the different pathways involved in the reorganization of the OLGc/myelin cytoskeleton. In agreement with results already published, we found that in OLGcs, Fyn kinase is associated with Tau and tubulin. Using a dominant-negative of Tau in which the Fyn-Tau-microtubules (MTs) interaction is blocked, we found that aTf was unable to induce OLGc morphological differentiation. It was also observed that aTf decreases the activated RhoA content in coincidence with a redistribution of actin immunoreactivity. These results give support to our hypothesis that Fyn kinase plays a key role in the differentiation process of OLGcs promoted by aTf.
Assuntos
Apoproteínas/farmacologia , Citoesqueleto/metabolismo , Oligodendroglia/enzimologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Células-Tronco/enzimologia , Transferrina/farmacologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Apoproteínas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/enzimologia , Bainha de Mielina/ultraestrutura , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Transferrina/metabolismo , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Anemia Ferropriva/patologia , Apoproteínas/uso terapêutico , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/patologia , Fibras Nervosas Mielinizadas/patologia , Transferrina/uso terapêutico , Anemia Ferropriva/sangue , Animais , Animais Recém-Nascidos , Apoproteínas/farmacologia , Células Cultivadas , Doenças Desmielinizantes/sangue , Modelos Animais de Doenças , Feminino , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Transferrina/farmacologiaRESUMO
Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.
Assuntos
Encéfalo/metabolismo , Proteínas do Citoesqueleto/biossíntese , Bainha de Mielina/metabolismo , Transferrina/genética , Animais , Encéfalo/efeitos dos fármacos , Proteínas do Citoesqueleto/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/genética , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/genética , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Transferrina/farmacologiaRESUMO
A phase II trial was performed to assess the efficacy and toxicity of a combination of ifosfamide (IFX), cisplatin (CDDP), and vinorelbine (VNB) as neoadjuvant chemotherapy (NAC) for untreated advanced cervical carcinoma (ACC). Between October 1995 and February 1998, 40 patients were entered in this study. Their median age was 43 years (range: 23-74 years). International Federation of Gynecology and Obstetrics stages were: IIB, 23; IIIB, 13; and IVA, 4. Therapy consisted of: IFX 2,000 mg/m2 1-hour (H) IV infusion days 1 to 3; 2-mercaptoethanesulfonic acid sodium salt (mesna) 400 mg/m2 IV bolus H 0 and 4, and 800 mg/m2 by mouth H 8, days 1 to 3; VNB 25 mg/m2 20-minute IV infusion days 1 and 8; and CDDP 75 mg/m2 IV day 3. Cycles were repeated every 28 days for a total of three courses. Both staging and response (R) assessment were performed by a multidisciplinary team. An objective response (OR) was observed in 24 of 40 patients (60%; 95% confidence interval, 45-75%). Four patients achieved complete response (CR) (10%); 20 partial response (50%); 12 patients stable disease (30%); and 4 progressive disease (10%). Eight of 24 patients (33%) with OR underwent radical surgery, and histologic CRs were recorded in 2 of them. The remaining patients received definitive radiotherapy after NAC. The dose-limiting toxicity was myelosuppression. Leukopenia occurred in 32 patients (80%) and was grade III or IV in 14 patients (36%). Peripheral neuropathy occurred in 9 patients (22%), whereas myalgias occurred in 10 (25%). Constipation was observed in 9 patients (23%); emesis occurred in 35 patients (88%). There were no therapy-related deaths. These results indicate that IFX/CDDP/VNB is an active combination for ACC with moderate toxicity. Implementation of this regimen in a multimodal therapy protocol deserves further study.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma de Células Escamosas/patologia , Cisplatino/administração & dosagem , Feminino , Humanos , Ifosfamida/administração & dosagem , Mesna/administração & dosagem , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Análise de Sobrevida , Neoplasias do Colo do Útero/patologia , Vimblastina/administração & dosagemRESUMO
A phase II trial was conducted to evaluate the efficacy and toxicity of a modulation of 5-fluorouracil (5-FU) by methotrexate (MTX) (with leucovorin (LV) rescue) as first-line chemotherapy in patients with locally advanced (inoperable) or metastatic gastric carcinoma. From July 1993 through August 1996, 36 patients with advanced gastric carcinoma received a regimen that consisted of: MTX 200 mg/m2 diluted in 250 ml normal saline by intravenous infusion over 20 minutes at hour 0; 5-FU 1,200 mg/m2 intravenous push injection at hour 20. Beginning 24 hours after MTX administration all patients received LV 15 mg/m2 intramuscularly every 6 hours for six doses. Cycles were repeated every 15 days. One patient was not assessable for response. Objective regression was observed in 15 of 37 patients (43%; 95% confidence interval, 26%-60%). One patient (3%) achieved complete response and 14 (40%) achieved partial response. No change was recorded in 14 patients (40%) and progressive disease was noted in six patients (17%). The median time to treatment failure was 7 months and the median survival was 12 months. Toxicity was within acceptable limits but one therapy-related death resulting from severe leukopenia occurred. The dose-limiting toxicity was mucositis. Five episodes of grade 3 or 4 stomatitis were observed and caused dosage modifications of MTX and 5-FU. Biochemical modulation of 5-FU by MTX appears as an attractive modality in patients with advanced gastric cancer. Further investigation both in experimental and clinical fields is needed to clearly define its role and to design the best modulatory strategy.