RESUMO
Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.
Assuntos
Antivirais/metabolismo , Benzimidazóis/metabolismo , Fígado , Quimeras de Transplante/metabolismo , Animais , Antivirais/sangue , Antivirais/farmacocinética , Antivirais/uso terapêutico , Benzimidazóis/sangue , Benzimidazóis/farmacocinética , Benzimidazóis/uso terapêutico , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Meia-Vida , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , Hepatite C/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Valor Preditivo dos Testes , Ratos , Ritonavir/metabolismo , Ritonavir/farmacocinética , Ritonavir/farmacologia , Especificidade da Espécie , Replicação Viral/efeitos dos fármacosRESUMO
Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.
Assuntos
Fator de Crescimento de Hepatócito/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Callithrix , Imagem de Tensor de Difusão , Dedos/fisiopatologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Indóis/metabolismo , Movimento (Física) , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Tamanho do Órgão/efeitos dos fármacos , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/fisiopatologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Reprodutibilidade dos Testes , Medula Espinal/enzimologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Coloração e Rotulagem , Fatores de Tempo , Punho/fisiopatologiaRESUMO
The inhibitory effects of yogurt consisting of milk fermented by Lactobacillus delbrueckii subsp. bulgaricus strain 2038 and Streptococcus salivarius subsp. thermophilus strain 1131 on formation of colonic aberrant crypt foci (ACF) in rats and also on development of colorectal tumors in transgenic mice harboring human prototype c-Ha-ras genes (rasH2 mice) were examined. F344 rats and rasH2 mice were fed commercial diet containing freeze-dried yogurt or starter medium (non-fermented milk). Rats were inoculated orally with heterocyclic amine 2-amino-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP) for two weeks. The rats were necropsied 14 days after the PhIP treatment, and ACF in the colon and rectum were counted. RasH2 mice were injected with 1,2-dimethylhydrazine dihydrochloride (DMH) for 20 weeks. Three weeks after the last injection of DMH, rasH2 mice were necropsied to determine the number and the size of colorectal tumors. Yogurt supplementation in diet significantly reduced the number of ACF and aberrant crypts (ACs) in rats fed control diet (P<0.01), but not in rats fed non-fermented milk diet. On the other hand, rasH2 mice receiving the yogurt-supplemented diet had significantly reduced numbers of tumors induced by DMH compared with those fed the non-fermented milk-supplemented diet (P<0.05). These results demonstrate that the yogurt used in this study appears to have tumor-suppressing properties, and rasH2 mice are a useful model for the evaluation of antitumor activities of foods.
Assuntos
Anticarcinógenos/administração & dosagem , Colo/efeitos dos fármacos , Neoplasias Colorretais/prevenção & controle , Mucosa Intestinal/efeitos dos fármacos , Lesões Pré-Cancerosas/prevenção & controle , Iogurte , Animais , Carcinógenos/toxicidade , Colo/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Feminino , Genes ras/genética , Humanos , Imidazóis/toxicidade , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Endogâmicos F344 , Reto/efeitos dos fármacos , Reto/patologia , Proteínas ras/genéticaRESUMO
Delayed transplantation of neural stem/progenitor cells (NS/PCs) into the injured spinal cord can promote functional recovery in adult rats and monkeys. To enhance the functional recovery after NS/PC transplantation, we focused on galectin-1, a carbohydrate-binding protein with pleiotropic roles in cell growth, differentiation, apoptosis, and neurite outgrowth. Here, to determine the combined therapeutic effect of NS/PC transplantation and galectin-1 on spinal cord injury (SCI), human NS/PCs were transfected by lentivirus with galectin-1 and green fluorescent protein (GFP), (Gal-NS/PCs) or GFP alone (GFP-NS/PCs), expanded in vitro, and then transplanted into the spinal cord of adult common marmosets, 9 days after contusive cervical SCI. The animals' motor function was evaluated by their spontaneous motor activity, bar grip power, and performance on a treadmill test. Histological analyses revealed that the grafted human NS/PCs survived and differentiated into neurons, astrocytes, and oligodendrocytes. There were significant differences in the myelinated area, corticospinal fibers, and serotonergic fibers among the Gal-NS/PC, GFP-NS/PC, vehicle-control, and sham-operated groups. The Gal-NS/PC-grafted animals showed a better performance on all the behavioral tests compared with the other groups. These findings suggest that Gal-NS/PCs have better therapeutic potential than NS/PCs for SCI in nonhuman primates and that human Gal-NS/PC transplantation might be a feasible treatment for human SCI.
Assuntos
Callithrix/cirurgia , Galectina 1/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/cirurgia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Galectina 1/genética , Sobrevivência de Enxerto/fisiologia , Proteínas de Fluorescência Verde/genética , Regeneração Nervosa/fisiologia , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Paralisia/metabolismo , Paralisia/fisiopatologia , Paralisia/cirurgia , Tratos Piramidais/citologia , Tratos Piramidais/lesões , Tratos Piramidais/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Transfecção/métodos , Resultado do TratamentoRESUMO
The somatic cell nuclear transfer technique has been applied to various mammals to produce cloned animals; however, a standardized method is not applicable to all species. We aimed here to develop optimum procedures for somatic cell cloning in nonhuman primates, using common marmosets. First, we confirmed that parthenogenetic activation of in vitro matured oocytes was successfully induced by electrical stimulation (three cycles of 150 V/mm, 50 microsec x 2, 20 min intervals), and this condition was applied to the egg activation procedure in the subsequent experiments. Next, nuclear transfer to recipient enucleated oocytes was performed 1 h before, immediately after, or 1 h after egg activation treatment. The highest developmental rate was observed when nuclear transfer was performed 1 h before activation, but none of the cloned embryos developed beyond the eight-cell stage. To investigate the causes of the low developmental potential of cloned embryos, a study was performed to determine whether the presence of metaphase II (MII) chromosome in recipient ooplasm has an effect on developmental potential. As a result, only tetraploid cloned embryos produced by transferring a donor cell into a recipient bearing the MII chromosome developed into blastocysts (66.7%). In contrast, neither parthenogenetic embryos nor cloned embryos (whether diploid or tetraploid) produced using enucleated oocytes developed past the eight-cell stage. These results suggest that MII chromosome, or cytoplasm proximal to the MII chromosome, plays a major role in the development of cloned embryos in common marmosets.
Assuntos
Células da Medula Óssea/citologia , Cromossomos de Mamíferos/fisiologia , Citoplasma/fisiologia , Metáfase/fisiologia , Oócitos/citologia , Animais , Células da Medula Óssea/fisiologia , Callithrix , Núcleo Celular/fisiologia , Clonagem de Organismos/métodos , Implantação do Embrião , Desenvolvimento Embrionário , Feminino , Masculino , Técnicas de Transferência Nuclear , Oócitos/fisiologia , PartenogêneseRESUMO
We previously reported that implantation of dendritic cells (DCs) into the injured site activates neural stem/progenitor cells (NSPCs) and promotes functional recovery after spinal cord injury (SCI) in mice. Working toward clinical application of DC therapy for SCI, we analyzed whether DCs promote functional recovery after SCI in a non-human primate, the common marmoset (CM). CMs are usually born as dizygotic twins. They are thus natural bone marrow and peripheral blood chimeras due to sharing of the placental circulation between dizygotic twins, leading to functional immune tolerance. In this study, to identify adequate CM donor-and-host pairs, mixed leukocyte reaction (MLR) assays were performed. Then, CM-DCs were generated from the bone marrow of the twin selected to be donor and transplanted into the injured site of the spinal cord of the other twin selected to be host, 7 days after injury. Histological analyses revealed fewer areas of demyelination around the injured site in DC-treated CMs than in controls. Immunohistochemical analysis showed that more motor neurons and corticospinal tracts were preserved after SCI in DC-treated CMs. Motor functions were evaluated using three different behavior tests and earlier functional recovery was observed in DC-treated CMs. These results suggest DC therapy to possibly be beneficial in primates with SCI and that this treatment has potential for clinical application.
Assuntos
Células Dendríticas/transplante , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/cirurgia , Animais , Callithrix , Citometria de Fluxo , Imageamento por Ressonância Magnética , Microscopia Eletrônica de Transmissão , Atividade Motora/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.
Assuntos
Animais Geneticamente Modificados/genética , Callithrix/genética , Modelos Animais de Doenças , Células Germinativas/metabolismo , Hereditariedade/genética , Transgenes/genética , Animais , Animais Recém-Nascidos , Callithrix/embriologia , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Transcrição GênicaRESUMO
CByB6F1-Tg(HRAS)2Jic mice (brand name: rasH2 mouse) are produced by two breeding facilities, CLEA Japan, Inc. (Fuji, Shizuoka, Japan) and Taconic (Germantown, NY, USA), and supplied world wide. To confirm carcinogenic conformity of both mice, a 26-week carcinogenicity test was performed on a total of 120 mice obtained from both facilities under the same protocol and same timing in our facility. All mice were divided into a vehicle (citrate buffer at pH 4.5, 10 ml/kg, single intraperitoneal injection) group and a MNU (N-methyl-N-nitrosourea, 75 mg/kg, single intraperitoneal injection) group. Fifteen mice of each sex were assigned to each group. The survival rate of the vehicle group was maintained at 100% for mice from both facilities at completion of the test. In the MNU group, MNU-induced tumor death occurred from 9 to 12 weeks after administration, and the final survival rate for both facilities was 6.7%. In the pathological examination, only benign tumors of lungs, spleen, forestomach and skin were observed in a few mice in the vehicle group of both facilities. In the MNU group, the incidence of forestomach papilloma/squamous cell carcinoma in mice from both facilities was 100%. The incidences of malignant lymphoma in CLEA Japan mice and Taconic mice were 86.7% and 93.3%, respectively, and no significant difference was observed (Fisher's exact probability test). Although lung adenoma and skin papilloma/keratoacanthoma, which are major MNU induced tumors in this strain, were observed in several mice from both facilities, no significant differences were found. Consequently, carcinogenic conformity of rasH2 mice derived from two breeding facilities was confirmed by the present study.
Assuntos
Testes de Carcinogenicidade/métodos , Metilnitrosoureia/toxicidade , Animais , Cruzamento , Carcinógenos/toxicidade , Modelos Animais de Doenças , Feminino , Genes ras , Masculino , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais/induzido quimicamenteRESUMO
Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).
Assuntos
Transferência Embrionária/métodos , Fertilização in vitro/métodos , Camundongos Endogâmicos C57BL/genética , Animais , Diabetes Mellitus Tipo 2/genética , Feminino , Masculino , CamundongosRESUMO
Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c(+) cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.
Assuntos
Callithrix/imunologia , Células Dendríticas/imunologia , Animais , Células da Medula Óssea/imunologia , Relação Dose-Resposta Imunológica , Endocitose/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-4/imunologia , Teste de Cultura Mista de Linfócitos , Neurotrofina 3/biossíntese , Proteínas Recombinantes/imunologia , Baço/imunologia , Células-Tronco/imunologiaRESUMO
We have investigated novel vaccine strategies against severe acute respiratory syndrome (SARS) CoV using cDNA constructs encoding the structural antigens: (S), (M), (E), or (N) protein, derived from SARS CoV. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain disrupted SCID mice, and SCID-PBL/hu mice were constructed. These mice can be used to analyze the human immune response in vivo. SARS M DNA vaccine and N DNA vaccine induced human CTL specific for SARS CoV antigens. Alternatively, SARS M DNA vaccines inducing human neutralizing antibodies and human monoclonal antibodies against SARS CoV are now being developed. These results show that these vaccines can induce virus-specific immune responses and should provide a useful tool for development of protective and therapeutic vaccines.
Assuntos
Imunização Passiva/métodos , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinas Virais/administração & dosagem , Animais , Modelos Animais de Doenças , Imunoterapia , Camundongos , Camundongos SCID , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Human uterine endometrium exhibits unique properties of cyclical regeneration and remodeling throughout reproductive life and also is subject to endometriosis through ectopic implantation of retrogradely shed endometrial fragments during menstruation. Here we show that functional endometrium can be regenerated from singly dispersed human endometrial cells transplanted beneath the kidney capsule of NOD/SCID/gamma(c)(null) immunodeficient mice. In addition to the endometrium-like structure, hormone-dependent changes, including proliferation, differentiation, and tissue breakdown and shedding (menstruation), can be reproduced in the reconstructed endometrium, the blood to which is supplied predominantly by human vessels invading into the mouse kidney parenchyma. Furthermore, the hormone-dependent behavior of the endometrium regenerated from lentivirally engineered endometrial cells expressing a variant luciferase can be assessed noninvasively and quantitatively by in vivo bioluminescence imaging. These results indicate that singly dispersed endometrial cells have potential applications for tissue reconstitution, angiogenesis, and human-mouse chimeric vessel formation, providing implications for mechanisms underlying the physiological endometrial regeneration during the menstrual cycle and the establishment of endometriotic lesions. This animal system can be applied as the unique model of endometriosis or for other various types of neoplastic diseases with the capacity of noninvasive and real-time evaluation of the effect of therapeutic agents and gene targeting when the relevant cells are transplanted beneath the kidney capsule.
Assuntos
Endométrio/fisiologia , Endométrio/transplante , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/cirurgia , Adulto , Animais , Endométrio/patologia , Feminino , Humanos , Rim/irrigação sanguínea , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Imunodeficiência Combinada Severa/patologia , Transplante Heterólogo/instrumentação , Transplante Heterólogo/métodos , Gravação em VídeoRESUMO
We have investigated novel vaccines strategies against severe acute respiratory syndrome (SARS) CoV infection using cDNA constructs encoding the structural antigens; spike (S), membrane (M), envelope (E), or nucleocapsid (N) protein, derived from SARS CoV (strain HKU39849, TW1, or FFM-1). As SARS-CoV is thought to infect the alveolar epithelial cell of the lung,in the present study, a type II alveolar epithelial cell clone, T7, was used to analyze the mechanism of CTL against SARS CoV membrane antigens. Mice vaccinated with SARS CoV (N) DNA or (M) DNA using pcDNA 3.1 (+) plasmid vector showed T-cell immune responses (CTL induction and proliferation) against type II alveolar epithelial cells (T7) transfected with SARS (N) or (M) DNA, respectively. To determine whether these DNA vaccines could induce T-cell immune responses in humans as well as in mice, SCID-PBL/hu mice were immunized with these DNA vaccines. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain-disrupted NOD-SCID mice [IL-2R(-/-) NOD-SCID]. SCID-PBL/hu mice thus constructed can be used to analyze the human immune response in vivo. The SCID-PBL/hu mice were immunized with SARS (N) DNA or (M) DNA and analyzed for a human T-cell immune response. The M DNA vaccine enhanced CTL activity and proliferation in the presence of M peptide in SCID-PBL/hu mice. Furthermore, the SARS N DNA vaccine induced CTL activity (IFN-gamma production by recombinant N protein or N protein-pulsed autologous B blast cells) and proliferation of spleen cells in SCID-PBL/hu mice. These results, demonstrate that SARS M and N DNA vaccines induced human CTL and human T-cell proliferative responses. On the other hand, we have developed SARS DNA vaccines that induce human neutralizing antibodies and human monoclonal antibodies against SARS CoV. Transgenic mice expressing SARS-CoV receptor (angiotensin converting enzyme 2) are also under development. These vaccines are expected to induce immune responses specific for SARS CoV in human and should provide useful tool for development of protective vaccines.
Assuntos
Imunização Passiva/métodos , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Vacinas Virais , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos TransgênicosRESUMO
A 9-week in vivo rasH2/butylhydroxytoluene (BHT) model for the detection of genotoxic lung carcinogens was validated, using six potent positive test compounds, dimethylnitrosamine (DMN; 15 mg/kg, i.p.), diethylnitrosamine (DEN; 100 mg/kg, i.p.), ethylnitrosourea (ENU; 120 mg/kg, i.p.), 3-methylcholanthrene (MC; 100 mg/kg, i.p.), 7,12-dimethylbenz(a)anthracene (DMBA; 5 mg/kg, i.g.) and benzo(a)pyrene (B(a)P; 80 mg/kg, i.p.), each given to rasH2 mice of both genders by single administration for initiation followed by promoter BHT treatment. Statistically significant increase in the incidence and multiplicity of lung tumors was observed in rasH2 mice treated with BHT following exposure to all of the carcinogens tested. The data overall suggest the rasH2/BHT model to be a powerful screening tool for genotoxic lung carcinogens.
Assuntos
Antioxidantes/farmacologia , Hidroxitolueno Butilado/farmacologia , Testes de Carcinogenicidade/métodos , Carcinógenos/farmacologia , Modelos Animais de Doenças , Neoplasias Pulmonares/induzido quimicamente , Proteína Oncogênica p21(ras)/fisiologia , 9,10-Dimetil-1,2-benzantraceno/farmacologia , Alquilantes/farmacologia , Animais , Benzo(a)pireno/farmacologia , Dietilnitrosamina/farmacologia , Dimetilnitrosamina/farmacologia , Etilnitrosoureia/farmacologia , Feminino , Humanos , Incidência , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Metilcolantreno/farmacologia , Camundongos , Camundongos Transgênicos , Proteína Oncogênica p21(ras)/genéticaRESUMO
Leptin is detected in the sera, and leptin receptors are expressed in the cerebrum of mouse embryos, suggesting that leptin plays a role in cerebral development. Compared with the wild type, leptin-deficient (ob/ob) mice had fewer cells at embryonic day (E) 16 and E18 and had fewer 5-bromo-2'-deoxyuridine(+) cells at E14 and E16 in the neuroepithelium. Intracerebroventricular leptin injection in E14 ob/ob embryos increased the number of neuroepithelium cells at E16. In cultured neurosphere cells, leptin treatment increased Hes1 mRNA expression and maintained neural progenitors. Astrocyte differentiation was induced by low-dose (0.1 microg/ml) but not high-dose (1 microg/ml) leptin. High-dose leptin decreased Id mRNA and increased Ngn1 mRNA in neurosphere cells. The neuropeptide Y mRNA level in the cortical plate was lower in ob/ob than the wild type at E16 and E18. These results suggest that leptin maintains neural progenitors and is related to glial and neuronal development in embryos.
Assuntos
Astrócitos/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Leptina/metabolismo , Células-Tronco Multipotentes/citologia , Neurônios/citologia , Animais , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/anatomia & histologia , Diferenciação Celular , Células Cultivadas , Córtex Cerebral/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leptina/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Células-Tronco Multipotentes/fisiologia , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neurônios/metabolismo , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Tamanho do Órgão , RNA Mensageiro/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Fatores de Transcrição HES-1RESUMO
We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.
Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinas Virais/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Feminino , Humanos , Técnicas In Vitro , Ativação Linfocitária , Transfusão de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Testes de Neutralização , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Linfócitos T Citotóxicos/imunologia , Transplante Heterólogo , Vacinas de DNA/genética , Vacinas de DNA/farmacologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/genéticaRESUMO
OBJECTIVE: Extensive WHO collaborative studies were performed to evaluate the suitability of transgenic mice susceptible to poliovirus (TgPVR mice, strain 21, bred and provided by the Central Institute for Experimental Animals, Japan) as an alternative to monkeys in the neurovirulence test (NVT) of oral poliovirus vaccine (OPV). METHODS: Nine laboratories participated in the collaborative study on testing neurovirulence of 94 preparations of OPV and vaccine derivatives of all three serotypes in TgPVR21 mice. FINDINGS: Statistical analysis of the data demonstrated that the TgPVR21 mouse NVT was of comparable sensitivity and reproducibility to the conventional WHO NVT in simians. A statistical model for acceptance/rejection of OPV lots in the mouse test was developed, validated, and shown to be suitable for all three vaccine types. The assessment of the transgenic mouse NVT is based on clinical evaluation of paralysed mice. Unlike the monkey NVT, histological examination of central nervous system tissue of each mouse offered no advantage over careful and detailed clinical observation. CONCLUSIONS: Based on data from the collaborative studies the WHO Expert Committee for Biological Standardization approved the mouse NVT as an alternative to the monkey test for all three OPV types and defined a standard implementation process for laboratories that wish to use the test. This represents the first successful introduction of transgenic animals into control of biologicals.
Assuntos
Sistema Nervoso Central/virologia , Vacina Antipólio Oral/toxicidade , Poliovirus/patogenicidade , Animais , Comportamento Cooperativo , Feminino , Laboratórios , Macaca mulatta , Masculino , Camundongos , Camundongos Transgênicos , Poliovirus/imunologia , Sensibilidade e Especificidade , Virulência , Organização Mundial da SaúdeRESUMO
Neuroepithelial stem cells derived from the swine mesencephalic neural tube were examined regarding their eligibility for neural xenografting as a donor material, with the aim of evaluating myelinated axon formation and both types of synaptic formation with xenogeneic host neurons as part of possible neural circuit reconstruction. The mesencephalic neural tube tissues were dissected out from swine embryos at embryonic days 17 and 18 and were implanted immediately into the striatum of the Parkinsonian model rat. The swine-derived grafts had many nestin-positive rosette-forming, neurofilament-positive, and tyrosine hydroxylase-positive cells in the rat striatum. Electron microscopic study revealed both efferent and afferent synaptic formations in the donor-derived immature neurons or tyrosine hydroxylase-positive donor cells in the grafts. Myelinated axons, both positive and negative for swine-specific neurofilament antibody, were mingled together in the graft. These results indicated that implanted neuroepithelial stem cells could survive well and divide asymmetrically into both nestin-expressing precursors and differentiated neurochemical marker-expressing neurons in the xenogeneic rat striatum, with the help of an immunosuppressant. Donor-derived immature neurons formed both efferent and afferent synapses with xenogeneic host neurons, and donor-derived axons were myelinated, which suggests that implanted swine neuroepithelial stem cells could possibly restore damaged neuronal circuitry in the diseased brain.
Assuntos
Axônios/transplante , Transplante de Tecido Encefálico/métodos , Fibras Nervosas Mielinizadas/transplante , Neurônios Aferentes/transplante , Neurônios Eferentes/transplante , Transplante de Células-Tronco/métodos , Sinapses/fisiologia , Transplante Heterólogo/métodos , Animais , Axônios/ultraestrutura , Diferenciação Celular/genética , Feminino , Masculino , Fibras Nervosas Mielinizadas/ultraestrutura , Neurônios Aferentes/citologia , Neurônios Eferentes/citologia , Transtornos Parkinsonianos/terapia , Gravidez , Ratos , Ratos Wistar , SuínosRESUMO
There is increasing evidence that human hematopoietic stem cells can develop into lymphocytes expressing T cell surface markers in the organ culture of murine embryonic thymic lobes. If human T cells with functional maturity are inducible from human stem cells in the mouse, it may be a useful model to investigate human T cell development and the human immune response in vivo. To approach this, we produced a hybrid cluster of murine fetal thymic epithelial cells and human cord blood-derived CD34(+) cells (hu/m cluster) using reaggregate thymic organ culture, and subsequently implanted it under the kidney capsule of NOD/SCID mice. The implanted hu/m cluster grew in volume under the kidney capsule and contained increased numbers of CD4(+)CD8(+)cells as well as CD4 or CD8 single-positive cells with low CD1a expression. These lymphocytes were also shown to possess activity for producing IL-2 and IL-4. Characteristics similar to human T cells also developed in the thymus of newly established mice lacking NK activity from NOD/SCID mice. These results indicate that functionally mature T cells can develop in vivo from human hematopoietic progenitors in the murine environment composed of thymic epithelial cells.