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Intravascularly transplanted bone marrow cells, including bone marrow mononuclear cells (BM-MNC) and mesenchymal stem cells, transfer water-soluble molecules to cerebral endothelial cells via gap junctions. After transplantation of BM-MNC, this fosters hippocampal neurogenesis and enhancement of neuronal function. Herein, we report the impact of transplanted BM-MNC on neural stem cells (NSC) in the brain. Surprisingly, direct transfer of water-soluble molecules from transplanted BM-MNC and peripheral mononuclear cells to NSC in the hippocampus was observed already 10 min after cell transplantation, and transfer from BM-MNC to GFAP-positive cortical astrocytes was also observed. In vitro investigations revealed that BM-MNC abolish the expression of hypoxia-inducible factor-1α in astrocytes. We suggest that the transient and direct transfer of water-soluble molecules between cells in circulation and NSC in the brain may be one of the biological mechanisms underlying the repair of brain function.
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Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. ß-cyclodextrin polyrotaxane (ßCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with ßCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Modelos Animais de Doenças , Cirrose Hepática , Macrófagos , Colesterol , LisossomosRESUMO
X-ray microscopes adopting computed tomography enable nondestructive 3D visualization of biological specimens at micron-level resolution without conventional 2D serial sectioning that is a destructive/laborious method and is routinely used for analyzing renal biopsy in clinical diagnosis of kidney diseases. Here we applied a compact commercial system of laboratory-based X-ray microscope to observe a resin-embedded osmium-stained 1-mm strip of a mouse kidney piece as a model of renal biopsy, toward a more efficient diagnosis of kidney diseases. A reconstructed computed tomography image from several hours of data collection using CCD detector allowed us to unambiguously segment a single nephron connected to a renal corpuscle, which was consistent with previous reports using serial sectioning. Histogram analysis on the segmented nephron confirmed that the proximal and distal tubules were distinguishable on the basis of their X-ray opacities. A 3D rendering model of the segmented nephron visualized a convoluted structure of renal tubules neighboring the renal corpuscle and a branched structure of efferent arterioles. Furthermore, another data collection using scientific complementary metal-oxide semiconductor detector with a much shorter data acquisition time of 15 min provided similar results from the same samples. These results suggest a potential application of the compact laboratory-based X-ray microscope to analyze mouse renal biopsy.
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Nefropatias , Microscopia , Camundongos , Animais , Raios XRESUMO
Background and Purpose- Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis. Methods- We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue. Results- The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed. Conclusions- Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy. Visual Overview- An online visual overview is available for this article.
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Transplante de Medula Óssea/métodos , Comunicação Celular/fisiologia , Junções Comunicantes/fisiologia , Neovascularização Fisiológica/fisiologia , Acidente Vascular Cerebral/terapia , Animais , Células da Medula Óssea/fisiologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Acidente Vascular Cerebral/patologiaRESUMO
Mucopolysaccharidosis type II (MPS II) is a rare lysosomal storage disease (LSD) involving a genetic error in iduronic acid-2-sulfatase (IDS) metabolism that leads to accumulation of glycosaminoglycans within intracellular lysosomes. The primary treatment for MPS II, enzyme replacement therapy, is not effective for central nervous system (CNS) symptoms, such as intellectual disability, because the drugs do not cross the blood-brain barrier. Recently, autophagy has been associated with LSDs. In this study, we examined the morphologic relationship between neuronal damage and autophagy in IDS knockout mice using antibodies against subunit c of mitochondrial adenosine triphosphate (ATP) synthetase and p62. Immunohistological changes suggesting autophagy, such as vacuolation, were observed in neurons, microglia, and pericytes throughout the CNS, and the numbers increased over postnatal development. Oral administration of chloroquine, which inhibits autophagy, did not suppress damage to microglia and pericytes, but greatly reduced neuronal vacuolation and eliminated neuronal cells with abnormal inclusions. Thus, decreasing autophagy appears to prevent neuronal degeneration. These results suggest that an autophagy modulator could be used in addition to conventional enzyme replacement therapy to preserve the CNS in patients with MPS II.
Assuntos
Autofagia , Mucopolissacaridose II/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cloroquina/farmacologia , Iduronato Sulfatase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mucopolissacaridose II/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.
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Vetores Genéticos/genética , Vírus da Parainfluenza 2 Humana/genética , Vacinas Sintéticas/genética , Vacinologia/métodos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Epitopos/genética , Epitopos/imunologia , Ordem dos Genes , Engenharia Genética , Humanos , Camundongos , Testes de Neutralização , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células VeroRESUMO
The mechanisms underlying neuropathic pain are poorly understood. However, several studies have implied a role for reactive microglia located in the dorsal horn in neuropathic pain. To clarify the roles of activated microglia in neuropathic pain, we investigated the interactions among microglia and other neural components in the dorsal horn using electron microscopy. Microglia were more abundantly localized in layers II-III of the dorsal horn than in other areas, and some of them adhered to and engulfed both injured and uninjured myelinated axons. This microglial engulfment was rarely observed in the normal dorsal horn, and the number of microglia attached to myelinated axons was markedly increased on postoperative day 7 on the operated side. However, after blocking the P2Y12 ATP receptor in microglia by intrathecal administration of its antagonist, AR-C69931MX, the increase in the number of microglia attached to myelinated axons, as well as the development of tactile allodynia, were markedly suppressed, although the number of activated microglia did not change remarkably. These results indicate that engulfment of myelinated axons by activated microglia via P2Y12 signaling in the dorsal horn may be a critical event in the pathogenesis of neuropathic pain.
Assuntos
Microglia/patologia , Fibras Nervosas Mielinizadas/patologia , Neuralgia/patologia , Células do Corno Posterior/patologia , Receptores Purinérgicos P2Y12/metabolismo , Transdução de Sinais/fisiologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Masculino , Proteínas dos Microfilamentos , Microglia/ultraestrutura , Microscopia Imunoeletrônica/métodos , Fibras Nervosas Mielinizadas/ultraestrutura , Células do Corno Posterior/ultraestrutura , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y12/genética , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/patologia , Nervos Espinhais/patologiaRESUMO
We performed a cell transplantation study to treat the brain involvement in lysosomal storage diseases. We used acid beta-galactosidase knock-out mice (BKO) from C57BL/6 as recipients. To minimize immune responses, we used cells derived from transgenic mice of C57BL/6 overexpressing the normal human beta-galactosidase. Fetal brain cells (FBC), bone marrow-derived mesenchymal stem cells (MSC), and mixed FBC and MSC cells were prepared and injected into the ventricle of newborn BKO mouse brain. The mice were examined at 1, 2, 4, and 8 weeks and 6 months after injection. In each experiment, the injected cells migrated into the whole brain effectively and survived for at least 8 weeks. Decrease in ganglioside GM1 level was also observed. FBC could survive for 6 months in recipient brain. However, the number of transplanted FBC decreased. In the brains of MSC- or mixed cell-treated mice, no grafted cells could be found at 6 months. To achieve sufficient long-term effects on the brain, a method of steering the immune response away from cytotoxic responses or of inducing tolerance to the products of therapeutic genes must be developed.
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Córtex Cerebral/transplante , Gangliosidose GM1/terapia , Transplante de Células-Tronco Mesenquimais , Animais , Western Blotting , Transplante de Tecido Encefálico , Células Cultivadas , Transplante de Tecido Fetal , Gangliosidose GM1/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
PURPOSE: We examined the expression profile of the members of the pancreatitis associated proteins/regenerating gene family in the bladder and in the primary afferent neurons of dorsal root ganglia using an animal model of cystitis. MATERIALS AND METHODS: We examined the expression of pancreatitis-associated protein-I and pancreatitis-associated protein-III in the bladder and the dorsal root ganglia of female rats 4 hours, 48 hours or 10 days after cyclophosphamide (Sigma) injection using immunohistochemistry and reverse transcriptase-polymerase chain reaction. RESULTS: No pancreatitis-associated protein-III immunoreactivity was identified in control bladders but prominent expression was observed in the urothelium of animals with chronic cystitis. Cells expressing pancreatitis-associated protein-I were seen in the dorsal root ganglia but not in the bladder. In normal dorsal root ganglia pancreatitis-associated protein-I was expressed in a minor population of small diameter neurons (2.4%) that were also positive for isolectin-B4. However, by 10 days following the onset of cystitis the number of pancreatitis-associated protein-I positive neurons was increased (7.6%) and pancreatitis-associated protein-I immunoreactivity was further observed in a slightly larger group of neurons and tyrosine kinase A positive small neurons. CONCLUSIONS: The current results suggest that pancreatitis-associated protein-III is associated with bladder inflammation and they implicate pancreatitis-associated protein-I in the abnormal sensation in cystitis.
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Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Cistite/genética , Cistite/fisiopatologia , Lectinas Tipo C/genética , Animais , Ciclofosfamida/efeitos adversos , Cistite/induzido quimicamente , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Perfilação da Expressão Gênica , Neurônios Aferentes/metabolismo , Noxas/efeitos adversos , Proteínas Associadas a Pancreatite , Ratos , Ratos Sprague-Dawley , Urotélio/metabolismoRESUMO
There is no animal model of ganglion. We describe a simple and reproducible animal model of pseudocystic diseases. First, we experimented to establish a pseudocystic model. We used cylindrical glass implants (6 mm diameter, 30 mm long) to create fibrous capsules in rats. The implants were inserted in the subcutaneous tissue in the dorsum of rats. Sixty implants were carried out (two implants per rat). Twelve weeks after implantation, the glass implants were removed and 0.5 mL sodium hyaluronate solution was injected into each cavity. Next, we tested the model by histological examination after OK-432 administration. Microscopic examination revealed that the wall was composed of a layer of collagenous fibers similar to those noted in ganglia; the lumen was retained for 3 weeks. Histopathological changes after OK-432 administration showed nonspecific inflammatory response induced by OK-432, resulting in in vivo activation of many inflammatory cells and then fast and reliable closure of cavities. No harmful reactions to OK-432 were observed histopathologically. These data suggest that our experimental cyst is a suitable model for studying pseudocystic diseases. This model can be used for research evaluating safe drug doses, conducting therapeutic comparison of several agents, and histopathological time course studies of the affected tissues. OK-432 administration on this model showed the potential of one of the ideal agents to treat pseudocystic lesions like ganglion.
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Antineoplásicos/uso terapêutico , Cistos Glanglionares/tratamento farmacológico , Picibanil/uso terapêutico , Animais , Modelos Animais de Doenças , Cistos Glanglionares/etiologia , Cistos Glanglionares/patologia , Injeções Intralesionais , Masculino , Ratos , Ratos WistarRESUMO
PURPOSE: To establish a new therapeutic method to treat bladder carcinoma, we investigated the therapeutic potential of doxorubicin hydrochloride (DXR) combined with hemagglutinating virus of Japan-envelope vector (HVJ-E) in an orthotropic mouse bladder cancer model. METHODS: DXR and/or HVJ-E were instilled into the bladder after implantation of MB49 cells. Antitumor effects of combination therapy were evaluated by histological analysis of the bladder on day 14 after tumor implantation. The survival rate of MB49-disseminated mice was examined for 60 days after single or double administration of DXR alone or DXR/HVJ-E. The surviving mice were re-challenged with intravesical injection of MB49 cells, and the bladder was observed after 3 weeks. RESULTS: Combined intravesical instillation of HVJ-E and DXR resulted in a significantly higher rate of tumor-free mice (11/21) compared with mice treated using DXR alone (3/19, P<0.05). Median survival was >60 days for intravesical instillation of HVJ-E and DXR, compared with the 29 days for DXR instillation alone (P<0.05). After combination therapy, surviving mice formed no tumors in the bladder following intravesical re-instillation of MB49. CONCLUSIONS: HVJ-E increased antitumor effects in combination with chemotherapeutic agent (DXR). Antitumor immunity appeared to be enhanced using HVJ-E.
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Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Carcinoma de Células de Transição/tratamento farmacológico , Doxorrubicina/química , Doxorrubicina/farmacologia , Vírus Sendai/química , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteínas do Envelope Viral/química , Animais , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Análise de Sobrevida , Sais de Tetrazólio/farmacologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
The aim of this study was to examine the expression profile of the vesicular acetylcholine transporter (VAChT), which is a cholinergic pre-synaptic marker, in the lower neural tract following spinal cord injury (SCI) and its effect on coordination of micturition. In adult female Sprague-Dawley rats, SCI was induced by complete transection of the spinal cord at T9. At various time points, 3, 7, 14 and 28 days, after SCI, cystometry was performed on conscious rats. Bladder areflexia was observed during the first week. Twenty-eight days after SCI the rats showed reflex contractions and voiding. The expression of VAChT was examined with immunohistochemistry. The number of VAChT-positive nerve terminals, which were surrounding neuronal soma, was transiently decreased in pelvic ganglion and spinal cord (L1, L2, L6 and S1). In particular VAChT terminals surrounding motor neurons in the ventral horn and autonomic pre-ganglion cells were dramatically decreased from 3 to 14 days after SCI. Similarly, and the number of VAChT-positive fibers in the bladder wall was also decreased. The intensity of VAChT terminals recovered in all above regions in conjunction with recovery of bladder function. These observations indicate that the transient decrease of the VAChT-positive nerve might cause a failure of cholinergic neuronal transmission along the urinary bladder tract after SCI. As the cholinergic system was recovered at least in rat, the functional recovery of neurogenic bladder syndrome in SCI patients may become possible by further understanding the mechanism underlying the recovery of cholinergic system in rat.
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Vias Aferentes/metabolismo , Traumatismos da Medula Espinal/patologia , Bexiga Urinária/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Vias Aferentes/patologia , Animais , Cordotomia/métodos , Feminino , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/fisiologia , Plexo Hipogástrico/metabolismo , Imuno-Histoquímica/métodos , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Periferinas , Ratos , Ratos Sprague-Dawley , Reflexo Anormal , Traumatismos da Medula Espinal/metabolismo , Fatores de Tempo , Bexiga Urinária/fisiopatologiaRESUMO
PURPOSE: The aim of this study was to determine the potential use of high-resolution FDG-microPET for predicting the primary effects of radiotherapy and/or hyperthermia on tumor-bearing rabbits. METHODS: Twenty-eight VX2 xenografts in the thighs of rabbits were divided into the following 5 treatment groups: radiotherapy at a single dose of 10, 20 or 30 Gy, hyperthermia (43 degrees Celsius, 1 hour), and the combination of radiotherapy and hyperthermia (10 Gy + 43 degrees Celsius, 1 hour). FDG-microPET images were obtained by using a microPET P4 system at pretreatment and at 24 hours and 7 days after treatment. For the evaluation by FDG-microPET, tumor/muscle (T/M) ratios, retention index [RI = (T/M ratio at 120 min - T/M ratio at 60 min) / T/M ratio at 60 min], and time activity curve (TAC) were acquired. RESULTS: We divided the xenografts into a responder group (partial response + stable disease, n=14) and a non-responder group (progressive disease, n = 14). The T/M ratio at 24 hours after the treatment in the responder group was decreased remarkably with that at pre-treatment (p < 0.05), while in the non-responder group it showed no significant change between the time points. The RI and TAC patterns were comparable to T/M ratios in each treatment group. T/M ratios, RI, and TAC indicated marked changes at the time point of 24 hours in the responder group, although the tumors did not show any significant hange in volume at that time. Photomicrographs of sections showed that the number of viable tumor cells in the responder group decreased at 24 hours after treatment and that inflammatory cell infiltration was marked and almost all viable tumor cells had disappeared by day 7 after treatment. CONCLUSION: These results suggest that early evaluation by FDG-microPET, especially 24 hours after treatment, is useful to predict the primary effects of the treatment. Histological analysis showed that inflammatory cell infiltration at 7 days after treatment was considered to be a cause of accumulation of FDG in the tumors that showed a significant decrease in tumor cell number. This false-positive should be noted when predicting tumor response by FDG accumulation.
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Carcinoma/diagnóstico por imagem , Carcinoma/terapia , Fluordesoxiglucose F18 , Hipertermia Induzida/métodos , Tomografia por Emissão de Pósitrons/métodos , Radioterapia/métodos , Animais , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/terapia , Tomografia por Emissão de Pósitrons/veterinária , Prognóstico , Coelhos , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.
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Astrocitoma/terapia , Proteínas da Matriz Extracelular/uso terapêutico , Terapia Genética , Integrases/uso terapêutico , Proteína-Lisina 6-Oxidase/uso terapêutico , Proteínas Virais/uso terapêutico , Adenoviridae/genética , Animais , Astrocitoma/patologia , Western Blotting/métodos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virologia , Relação Dose-Resposta a Droga , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Imuno-Histoquímica/métodos , Integrases/biossíntese , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias/métodos , Proteína-Lisina 6-Oxidase/biossíntese , Proteína-Lisina 6-Oxidase/genética , Ratos , Ratos Wistar , Fatores de Tempo , Células Tumorais Cultivadas , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Cyclin G1 (CG1) was identified as a p53-transactivated target gene, and yet its physiological and pathological roles have been unclear. Here, we demonstrate that CG1 is translocated from cytoplasm to the nuclei of neurons in response to variety of injuries. In the normal matured rodent brain, CG1 immunoreactivity was hardly observed; however, some brain injuries exhibited intense CG1 immunoreactivity in the nuclei of the damaged neurons. Transient common carotid artery occlusion (CCAO) in the gerbil showed strong CG1-like immunoreactivity in the hippocampal CA1 neurons, and permanent middle cerebral artery occlusion (MCAO) in the mouse showed strong CG1-like immunoreactivity in the nuclei of neurons located in the ischemic brain regions. TUNEL staining did not exactly overlap with the CG1-positive cells, but overlapped highly with Fluoro-Jade B staining, a degeneration marker. Brain trauma caused by knife cut, cold injury, and kinate injection also showed CG1 accumulation in the neuronal nuclei located near the injury site. These observations were obtained in p53-deficient mice as well, suggesting that the accumulation of CG1 in the injured neurons is p53-independent. A similar nuclear translocation of endogenous CG1 was confirmed in a primary culture of cortical neurons when a toxic level of N-methyl-D-aspartate (NMDA) was applied. These results demonstrate that nuclear translocation of CG1 from cytoplasmic region occurs in damaged and degenerating neurons in a p53-independent manner, and the CG1 nuclear staining could be a good marker for the neurons received fatal damages.
Assuntos
Lesões Encefálicas/complicações , Isquemia Encefálica/metabolismo , Ciclinas/metabolismo , Degeneração Neural/etiologia , Neurônios/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Lesões Encefálicas/metabolismo , Isquemia Encefálica/complicações , Morte Celular/fisiologia , Células Cultivadas , Ciclina G , Ciclina G1 , Ciclinas/genética , Cicloeximida/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/toxicidade , Fluoresceínas , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Gerbillinae , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Ácido Caínico/toxicidade , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica/métodos , N-Metilaspartato/farmacologia , Degeneração Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Compostos Orgânicos , Fosfopiruvato Hidratase/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , Fatores de Tempo , Proteína Supressora de Tumor p53/deficiênciaRESUMO
PURPOSE: To determine the association of the KL-6 epitope (sugar moiety) with MUC1 and its distribution on the ocular surface of human non-dry and dry eyes. METHODS: The human ocular surface was examined immunohistochemically and immunoelectron microscopically using monoclonal antibody (mAb) KL-6, which recognize a carbohydrate epitope of MUC1. The expression patterns of KL-6 epitope in corneal and conjunctival cells were examined by impression cytology from 24 non-dry eye volunteers and 43 dry-eye patients. RESULTS: In the cornea, bulbar conjunctiva, and limbus epithelium, mAb KL-6 reacted to the apical cell membrane of superficial cells and the intercellular space of superficial and wing cells. No immune reactivity of mAb KL-6 was observed in the basal plasma membrane of basal epithelial cells. Results of impression cytology indicated that the corneal epithelium of 13 of 24 non-dry eyes was weakly stained by mAb KL-6, whereas 42 of 43 dry eyes showed a mosaic pattern. In non-dry eyes, 19 of 24 bulbar conjunctival epithelia expressed the KL-6 epitope in a honeycomb pattern. In mild (17/19) and moderate (17/17) dry eye conjunctiva, the KL-6 epitope showed a mosaic pattern. However, the expression of KL-6 epitope decreased in severe dry eyes, showing a mosaic pattern in three of seven patients and labeling a few cells weakly in four of seven patients. CONCLUSIONS: These findings suggest that there is an upregulation of the sialylated KL-6 epitope of MUC1 by apical corneal and conjunctival cells in mild and moderate dry eyes. This upregulation may in part alleviate the consequences caused by goblet cell mucin dysfunction in dry eyes. It is noteworthy that the KL-6 epitope is downregulated in the conjunctiva of severe dry eyes, a phenomenon that may be explained in part by the malfunction of conjunctival epithelial cells.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/metabolismo , Túnica Conjuntiva/metabolismo , Síndromes do Olho Seco/metabolismo , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Glicoproteínas/metabolismo , Antígenos/imunologia , Antígenos de Neoplasias , Biomarcadores , Túnica Conjuntiva/patologia , Síndromes do Olho Seco/patologia , Células Epiteliais/patologia , Epitélio Corneano/patologia , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Glicoproteínas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mucina-1 , Mucinas/imunologia , Mucinas/metabolismoRESUMO
Transgenic mouse lines expressing the active form Akt gene under the control of the damage-induced neuronal endopeptidase (DINE) promoter were made from three different founder mice, and its neuroprotective potential against ischemic brain damage was investigated. Twenty-four hours after middle cerebral artery occlusion, two DINE-Akt-transgenic mouse lines displayed reductions of the infarcted area by 35% compared to the wild-type littermate. RT-PCR assays showed a high level of transgene in response to ischemic brain damage in these lines. These results suggest that the DINE promoter is a useful promoter, which responds to neuronal insults and that the Akt-induced neuroprotective effect against ischemic damage is potent in vivo.
Assuntos
Infarto Cerebral/genética , Infarto Cerebral/metabolismo , Melhoramento Genético/métodos , Terapia Genética/métodos , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Animais , Infarto Cerebral/patologia , Infarto Cerebral/terapia , Estudos de Viabilidade , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/terapia , Camundongos/genética , Camundongos/metabolismo , Camundongos Transgênicos/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/metabolismo , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.
Assuntos
Proteínas Aviárias , Proteínas de Transporte/fisiologia , Moléculas de Adesão Celular/fisiologia , Colículos Superiores/embriologia , Animais , Anticorpos/metabolismo , Western Blotting/métodos , Bromodesoxiuridina/metabolismo , Antígeno CD146 , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Agregação Celular/fisiologia , Movimento Celular/fisiologia , Embrião de Galinha , Humanos , Imuno-Histoquímica/métodos , Microscopia Eletrônica/métodos , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Técnicas de Cultura de Órgãos , Colículos Superiores/citologia , Colículos Superiores/metabolismo , Colículos Superiores/ultraestruturaRESUMO
It has been demonstrated that some of immediate early genes such as c-Jun are induced immediately and transiently following focal cerebral ischemia. Here we newly characterize the activating transcription factor (ATF)-3 as a focal ischemia associated immediate early gene. Using in situ hybridization and immunohistochemistry, we compared the expression profile of ATF-3 with those of ATF-2 and c-Jun after middle cerebral artery (MCA) occlusion. Focal cerebral ischemia induced two temporal and spatial patterns of ATF-3 expression. Early and transient induction of ATF-3 mRNA was observed in the core and margins of the cortex immediately after MCA occlusion. Late-onset and prolonged expression of ATF-3 mRNA and its protein were specifically identified in the peri-infarct cortex and thalamus where neurons survive at least 1 month. The expression profiles of ATF-3 and c-Jun were virtually similar, but c-Jun expression was also observed in other regions of the brain in control rats. Expression of ATF-2 was ubiquitously seen in neuronal cells throughout the brain in normal rats, but was suppressed in ischemic regions. Double immunohistochemical labeling revealed concurrent expression of ATF-3 and phospho-c-Jun in neurons. We conclude that the transcription factor ATF-3 is a suitable marker of neurons subjected to ischemic insult directly and indirectly, and that cooperative works of ATF-3 and c-Jun may be crucial triggers of various transcriptional responses to the ischemic insult.
Assuntos
Infarto Cerebral/patologia , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Fator 2 Ativador da Transcrição , Fator 3 Ativador da Transcrição , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Infarto Cerebral/etiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Infarto da Artéria Cerebral Média/complicações , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.