RESUMO
The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-ß-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.
Assuntos
Adjuvantes Imunológicos/metabolismo , Aloenxertos/fisiologia , Antígenos CD40/metabolismo , Transplante de Coração , Células Mieloides/metabolismo , RNA Interferente Pequeno/metabolismo , Sizofirano/metabolismo , Adjuvantes Imunológicos/química , Aloenxertos/citologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Sizofirano/química , Subpopulações de Linfócitos T/imunologia , TransfecçãoRESUMO
BACKGROUND: Sodium hyaluronate/carboxymethylcellulose (HA/CMC) is difficult to use in a moist environment because of its susceptibility to moisture. METHODS: We developed the three-layered nDM-14R membrane. The surface layers are composed of 1-lactide, glycolide and e-caprolactone copolymers. HA/CMC and nDM-14R were used in all these studies. (1) The central region of 1 × 10 cm specimens (n = 5) was moistened for 0, 5, 10, 20, 30 or 60 s, after which the tensile strength was determined; (2) one side of specimens of 1 × 10 cm (n = 5) was moistened with agar gel for 5, 10, 15 or 30 s, after which the adhesion strength was determined, and (3) Rat cecum (n = 10) was scratched, 3 × 3 cm specimens were placed on the scratched area, and adhesions were evaluated on postoperative day 14. RESULTS AND CONCLUSION: (1) The tensile strength of nDM-14R after contact for 10-30 s was greater than that of HA/CMC. (2) The adhesive strength of HA/CMC after contact for 5-10 s was greater than that of nDM-14R. (3) Adhesion scores in treatment groups were significantly lower than in the control group. The results suggest that nDM-14R has the same antiadhesive effect and allows easier placement under moist conditions than HA/CMC.
Assuntos
Membranas Artificiais , Complicações Pós-Operatórias/prevenção & controle , Aderências Teciduais/prevenção & controle , Animais , Teste de Materiais , Ratos , Solubilidade , Resistência à TraçãoRESUMO
The programmed death-1 (PD-1)/B7-H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD-1/B7-H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7-H1 is highly expressed on the donor-derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD-1/B7-H1 pathway via anti-B7-H1mAb or using B7-H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody-treated group in comparison to the controls. Taken together, these data revealed that the B7-H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.
Assuntos
Antígenos de Superfície/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Antígeno B7-1/imunologia , Transplante de Fígado/imunologia , Glicoproteínas de Membrana/imunologia , Peptídeos/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Antígeno B7-1/genética , Antígeno B7-H1 , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica , Transplante de Fígado/patologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/antagonistas & inibidores , Peptídeos/deficiência , Peptídeos/genética , Receptor de Morte Celular Programada 1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Linfócitos T/patologia , Quimeras de Transplante/imunologia , Transplante HomólogoRESUMO
Intestinal transplant-associated microangiopathy (i-TAM) is an important complication after allogeneic hematopoietic SCT. From 1997 to 2006, 87 of 886 patients with diarrhea after transplantation received colonoscopic biopsy. i-TAM, GVHD and CMV colitis were diagnosed histopathologically. The median duration from transplantation to the onset of diarrhea was 32 days (range: 9-130 days) and that from the onset of diarrhea to biopsy was 12 days (range: 0-74 days). The median maximal amount of diarrhea was 2 l/day (range: 130-5600 ml/day). Histopathological diagnosis included i-TAM (n=80), GVHD (n=26), CMV colitis (n=17) and nonspecific findings (n=2) with overlapping. Among 80 patients with i-TAM, abdominal pain was a major symptom, and only 11 patients fulfilled the proposed criteria for systemic TAM. Non-relapse mortality (NRM) among patients without resolution of diarrhea was 72% and i-TAM comprised 57% of NRM. NRM was 25% among patients without intensified immunosuppression, but was 52, 79 and 100% among those with intensified immunosuppression before diarrhea, after diarrhea, and before and after diarrhea, respectively. In conclusion, i-TAM is a major complication presenting massive refractory diarrhea and abdominal pain, which causes NRM. Avoiding intensified immunosuppression that damages vascular endothelium until the resolution of i-TAM may improve transplant outcome.
Assuntos
Colite/terapia , Infecções por Citomegalovirus/terapia , Diarreia/terapia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas , Terapia de Imunossupressão/métodos , Adolescente , Adulto , Colite/etiologia , Colite/mortalidade , Colite/patologia , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/mortalidade , Infecções por Citomegalovirus/patologia , Diarreia/etiologia , Diarreia/mortalidade , Diarreia/patologia , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/mortalidade , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Terapia de Imunossupressão/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.
Assuntos
Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/terapia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular/genética , Senescência Celular/fisiologia , Feminino , Terapia Genética/métodos , Células HeLa , Papillomavirus Humano 16/crescimento & desenvolvimento , Humanos , Immunoblotting , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas E7 de Papillomavirus , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Carga Tumoral , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thrombotic microangiopathy (TMA) is one of the severe complications after stem cell transplantation (SCT) and is associated with graft-versus-host disease (GVHD) prophylaxis including FK506. In this study, we experimented on rats using FK506 to demonstrate the occurrence of intestinal TMA. FK506 was administrated into Wistar/ST rats intraperitoneally for 7 days. Rats were examined histopathologically after FK506 injection using light and electron microscopy and immunohistochemistry. FK506 concentrations in whole blood were measured by enzyme immunoassay. In the acute phase, hemorrhagic lesions with multifocal erosions and crypt loss were found in the small intestines of all treated rats. Capillary vessels were dilated, and a few platelet thrombi were found. Electron microscopy demonstrated degenerative swelling of endothelial cells and platelet aggregates adhering to the vessel walls. In the later phase, epithelial regenerative failure, characterized by crypt ghosts, was found in the affected mucosa. Apoptotic epithelial cells were increased in number. The extent of intestinal injury was proportional to the whole blood levels of FK506. The intestinal lesions in rats were consistent with TMA and induced by the injection of FK506 alone. Apoptotic enteropathy was also observed and similar to intestinal GVHD. In this study, we established an intestinal TMA model induced by immunosuppressant (Tacrolimus) only without irradiation.
Assuntos
Capilares/efeitos dos fármacos , Imunossupressores/efeitos adversos , Mucosa Intestinal/irrigação sanguínea , Doenças Vasculares Periféricas/induzido quimicamente , Tacrolimo/efeitos adversos , Trombose/induzido quimicamente , Animais , Apoptose , Capilares/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Histocitoquímica , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Masculino , Ratos , Ratos WistarRESUMO
Modulation of donor organs by transfection of a gene encoding immmunosuppresive molecules has been recognized as a less toxic approach to prevent allograft rejection. Fas-ligand (FasL) plays a critical role in activation-induced cell death of activated cytotoxic lymphocytes. This may provide a potential for induction of "immune privileged sites" to escape the host immune surveillance system. Cytokine response modifier A (CrmA), a gene product of cowpox virus, blocks caspase as well as perforin/granzyme-mediated apoptotic pathways. Therefore, it may suppress intragraft apoptosis. The aim of the present study was to investigate whether transfection of FasL or CrmA genes prolonged the survival of rat liver allografts. Using the high responder rat combination of DA (RT-1(a)) donor to LEW (RT-1(1)) recipient, we performed orthotopic liver transplantation with subsequent delivery of adenoviral vectors containing FasL, CrmA, or LacZ, at a dose of 1 x 10(9) pfu via a recipient tail vein using a Cre-mediated gene expression system. Recipient survival was assessed as well as immunohistochemical examination of the grafts for anti-CD2, TUNEL, and H&E staining. Statistical analysis was performed with the Mann-Whitney U test. The therapeutic groups showed significantly prolonged recipient survival compared with the LacZ-treated control group. Histologic analysis revealed reduced hepatocyte apoptosis in the CrmA-treated group and increased apoptosis of infiltrating mononuclear cells in the FasL-treated group. These data suggested that FasL and CrmA may be potent genes to prolong rat liver allograft survival.
Assuntos
Proteína Ligante Fas/genética , Sobrevivência de Enxerto/fisiologia , Transplante de Fígado/fisiologia , Animais , Vetores Genéticos , Rejeição de Enxerto , Modelos Animais , Ratos , Ratos Endogâmicos Lew , Serpinas/genética , Transfecção , Transplante Homólogo , Proteínas Virais/genética , beta-Galactosidase/genéticaRESUMO
Activated cytotoxic T-cell-mediated hepatocyte apoptosis via Fas/Fas-ligand and perforin/granzyme pathways are believed to involve the model of concanavalin A (ConA)-induced hepatitis. The purpose of the present study is to investigate whether the cytokine response modifier A (crmA) gene effectively inhibits the hepatocyte apoptosis of ConA-induced hepatitis. We examined survival rates, liver pathology, immune histological changes, and cytokine profiles from mice receiving the recombinant adenovirus vectors containing cre and/or crmA genes, transferred to the liver 3 days before ConA injection, and a crmA gene nonexpression control group. Injection of ConA into mice rapidly led to massive hepatocyte apoptosis, and infiltration of leukocytes, especially CD11b(+) inflammatory cells. In contrast, liver damage was dramatically reduced in the mice that expressed the crmA gene. However, infiltration by CD4(+) cells was not affected. The survival of the mice increased significantly to 100% in the treated group versus the control group. Furthermore, we demonstrated that interleukin (IL)-18 plays an important role in ConA-induced hepatitis, and that crmA expression significantly inhibited IL-18 secretion. Our results showed that the crmA gene effectively inhibits apoptosis induced by ConA hepatitis. This indicates a potential therapeutic usage of crmA for protection from cellular damage due to hepatitis.
Assuntos
Apoptose , Terapia Genética/métodos , Hepatite/prevenção & controle , Interleucina-18/metabolismo , Serpinas/genética , Proteínas Virais , Adenoviridae/genética , Animais , Concanavalina A , Expressão Gênica , Vetores Genéticos/administração & dosagem , Hepatite/imunologia , Hepatite/patologia , Hepatócitos/patologia , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND AND AIMS: Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), is an appetite stimulatory signal from the stomach with structural resemblance to motilin. We examined the effects of the gastric peptide ghrelin and GHS-R antagonists on energy balance and glycaemic control in mice. MATERIALS AND METHODS: Body weight, fat mass, glucose, insulin, and gene expression of leptin, adiponectin, and resistin in white adipose tissue (WAT) were measured after repeated administrations of ghrelin under a high fat diet. Gastric ghrelin gene expression was assessed by northern blot analysis. Energy intake and gastric emptying were measured after administration of GHS-R antagonists. Repeated administration of GHS-R antagonist was continued for six days in ob/ob obese mice. RESULTS: Ghrelin induced remarkable adiposity and worsened glycaemic control under a high fat diet. Pair feeding inhibited this effect. Ghrelin elevated leptin mRNA expression and reduced resistin mRNA expression. Gastric ghrelin mRNA expression during fasting was increased by a high fat diet. GHS-R antagonists decreased energy intake in lean mice, in mice with diet induced obesity, and in ob/ob obese mice; it also reduced the rate of gastric emptying. Repeated administration of GHS-R antagonist decreased body weight gain and improved glycaemic control in ob/ob obese mice. CONCLUSIONS: Ghrelin appears to be closely related to excess weight gain, adiposity, and insulin resistance, particularly under a high fat diet and in the dynamic stage. Gastric peptide ghrelin and GHS-R may be promising therapeutic targets not only for anorexia-cachexia but also for obesity and type 2 diabetes, which are becoming increasingly prevalent worldwide.
Assuntos
Ingestão de Alimentos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Hormônios Peptídicos/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Receptores Acoplados a Proteínas G , Aumento de Peso/fisiologia , Adiponectina , Tecido Adiposo/metabolismo , Animais , Northern Blotting , Gorduras na Dieta/administração & dosagem , Metabolismo Energético/fisiologia , Esvaziamento Gástrico/fisiologia , Expressão Gênica , Grelina , Glucose/análise , Hormônios Ectópicos/análise , Insulina/análise , Leptina/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Fator de Crescimento Neural , Hormônios Peptídicos/metabolismo , Proteínas/análise , RNA Mensageiro/análise , Receptores de Grelina , ResistinaRESUMO
AIM: This study was designed to investigate the effect of orexin on anorexia induced by cholecystokinin (CCK),a peripheral satiety signal. METHODS: We administered orexin A (0.01-1 nmol/mouse) and CCK-8 (3 nmol/mouse) to mice. Food intake was measured at different time-points: 20 min, 1, 2 and 4 h post-intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) administrations. RESULTS: Intracerebroventricular-administered orexin significantly increased food intake in a dose-dependent manner. The inhibitory effect of i.p.-administered CCK-8 on food intake was significantly negated by the simultaneous i.c.v. injection of orexin in a dose-dependent manner. CONCLUSIONS: Orexin reversed the CCK-induced loss of appetite. Our results indicate that orexin might be a promising target for pharmacological intervention in the treatment of anorexia and cachexia induced by various diseases.
Assuntos
Anorexia/induzido quimicamente , Proteínas de Transporte/farmacologia , Colecistocinina/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Neuropeptídeos/farmacologia , Análise de Variância , Animais , Proteínas de Transporte/administração & dosagem , Relação Dose-Resposta a Droga , Camundongos , Neuropeptídeos/administração & dosagem , Orexinas , Redução de PesoAssuntos
Eritroblastos/citologia , Síndromes Mielodisplásicas/enzimologia , Óxido Nítrico Sintase/biossíntese , Idoso , Idoso de 80 Anos ou mais , Feminino , Hemoglobina Fetal/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Óxido Nítrico Sintase Tipo II , Regulação para CimaRESUMO
To investigate the relationship between the fetal hemoglobin-containing erythroblasts (F blasts) and apoptosis in myelodysplastic syndromes (MDS), we immunohistochemically assessed F blasts, F cells, and apoptosis in 137 patients with MDS. A marked increase in the number of F blasts in the bone marrow was identified in 116 of 137 patients (84.7%), and the number of F cells was elevated in 54 patients (39.4%). Among the erythroblasts stained by anti-glycophorin C antibody, the mean percentage of F blasts was 14.63 +/- 9.17% in MDS, which was significantly higher than that in non-MDS patients with stress erythropoiesis (4.82 +/- 3.35%, P < 0.01), although there were no significant differences in the number of F cells between these groups. In particular, 62 of the 137 MDS patients (45.3%) had an apparent increase in F blasts but no elevation of F cells. The apoptotic rate was significantly higher in the patients with a F blast/F cell (Fb/Fc) ratio >or=5.0 than in those with a Fb/Fc ratio <1.0 (P < 0.01). The results indicate that F cell precursors are incapable of maturing into functioning end-stage F cells, presumably owing to apoptotic cell death. The measurement of F blasts in the bone marrow is needed for the precise evaluation of fetal-type erythropoiesis in MDS.
Assuntos
Células Precursoras Eritroides/química , Hemoglobina Fetal/análise , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Células da Medula Óssea/patologia , Contagem de Células , Eritroblastos/química , Eritroblastos/patologia , Células Precursoras Eritroides/patologia , Feminino , Glicoforinas/análise , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The role of insulin-like growth factor I (IGF-I) in the release of luteinizing hormone (LH) is unclear in ruminants. In the present study, the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) were examined in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17beta (E(2)) and IGF-I was characterized. GnRH(100nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36h after addition of IGF-I (250ng/ml), with a maximum at 12h (48.4ng/ml media versus 35.4ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500ng/ml increased the release by 18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2ng/ml media). E(2) (10nM), IGF-I (250ng/ml) and combined treatment of E(2) plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E(2) alone was 37.3% greater than with IGF-I alone (39.0ng/ml media versus 28.4ng/ml media) (P<0.05). When E(2) and IGF-I were added together (45.6ng/ml media), the release of LH was significantly greater than with either E(2) alone or IGF-I alone (P<0.05). E(2) (10nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250ng/ml) failed to increase GnRH binding. These results show that IGF-I enhances GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interacts with E(2) to increase the response to GnRH.
Assuntos
Bovinos/fisiologia , Hormônio Liberador de Gonadotropina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Hormônio Luteinizante/metabolismo , Adeno-Hipófise/metabolismo , Animais , Células Cultivadas , Interações Medicamentosas , Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Receptores LHRH/metabolismoAssuntos
Apoptose/efeitos dos fármacos , Imunossupressores/farmacologia , Mitocôndrias/fisiologia , Propilenoglicóis/farmacologia , Linfócitos T/imunologia , Cloridrato de Fingolimode , Genes bcl-2 , Humanos , Células Jurkat , Mitocôndrias/efeitos dos fármacos , Esfingosina/análogos & derivados , Linfócitos T/efeitos dos fármacos , TransfecçãoRESUMO
A recently identified ribonucleotide reductase (RR), p53R2, is directly regulated by p53 for supplying nucleotides to repair damaged DNA. We examined the role of this p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint, comparing it to R2-dependent DNA synthesis. The elevation of DNA synthesis activity through RR in response to gamma-irradiation was closely correlated with the level of expression of p53R2 but not of R2. The p53R2 product accumulated in nuclei, whereas R2 levels in cytoplasm decreased. We found a point mutation of p53R2 in cancer cell line HCT116, which resulted in loss of RR activity. In those cells, DNA damage-inducible apoptotic cell death was enhanced through transcriptional activation of p53AIP1. The results suggest that p53R2-dependent DNA synthesis plays a pivotal role in cell survival by repairing damaged DNA in the nucleus and that dysfunction of this pathway might result in activation of p53-dependent apoptosis to eliminate dangerous cells.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , DNA/biossíntese , Ribonucleotídeo Redutases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Linhagem Celular , Dano ao DNA , Reparo do DNA , Fibroblastos/citologia , Fibroblastos/fisiologia , Inativação Gênica , Genes p53/genética , Humanos , Mutação Puntual , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Transdução de Sinais/fisiologia , Frações Subcelulares/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
To date there are few antibodies available for the detection of fetal hemoglobin (HbF)-containing erythroblasts (F-blasts) in paraffin-embedded hematopoietic tissues. Recently, we developed a new polyclonal antibody specific to F-blasts by immunization of a rabbit with synthetic peptides of human HbF. Specificity and reactivity of the antibody were confirmed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. ELISA confirmed that the antibody showed strong immunoreactivity to fetal hemoglobin but no reaction to adult hemoglobin. The antibody could detect the presence of fetal blood hemolysates (10 microg/mL) at a dilution of 10(-5). According to immunohistochemical analysis, there were strong positive reactions to fetal erythroblasts in the liver and the spleen at 29 weeks of gestation and to erythroblasts from patients with myelodysplastic syndromes and erythroleukemia but no reactions to normal adult erythroblasts in bone marrow. Fetal erythrocytes in fetal blood vessels of placental tissues were strictly distinguished from maternal erythrocytes in the same sections by their positive reactions, indicating the presence of HbF. This new antibody will be a useful tool for studying fetal hemoglobin synthesis and for detecting F-blasts in archival specimens of various hematological diseases.
Assuntos
Anticorpos , Eritroblastos/imunologia , Hemoglobina Fetal/imunologia , Sequência de Aminoácidos , Anticorpos/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Células da Medula Óssea/citologia , Epitopos/química , Eritroblastos/química , Eritroblastos/citologia , Hemoglobina Fetal/análise , Humanos , Imuno-Histoquímica , Leucemia Eritroblástica Aguda/patologia , Fígado/química , Fígado/citologia , Fígado/embriologia , Dados de Sequência Molecular , Síndromes Mielodisplásicas/patologia , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Baço/química , Baço/citologia , Baço/embriologiaRESUMO
Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 x 10(6) cells in female MRL-+/+ (wild-type mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.
Assuntos
Hepatócitos/metabolismo , Hepatócitos/transplante , Regeneração Hepática , Fígado/fisiologia , Receptor fas/fisiologia , Animais , Anticorpos/metabolismo , Apoptose , Aspartato Aminotransferases/sangue , Sequência de Bases , Feminino , Genes sry , Humanos , Marcação In Situ das Extremidades Cortadas , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Quimeras de Transplante , Receptor fas/genéticaRESUMO
Cell-mediated therapy for mucopolysaccharidosis type VII (MPSVII) was studied using monkey amniotic epithelial cells (mAEC). The cells were transduced with a recombinant adenovirus expressing human beta-glucuronidase (GUSB), and cells overexpressing GUSB were generated. The cells expressed 2000-fold higher activities than the endogenous GUSB activities of nontransduced mAEC, demonstrating that mAEC were successfully transduced with adenoviral vectors. These cells also secreted high levels of GUSB. To clarify the cross-correction of GUSB secreted from mAEC, the conditioned medium containing high levels of GUSB was added into the medium for culturing human or murine fibroblasts established from an MPSVII patient or a mouse model of the disease. Dramatic increases in GUSB activities were observed in both fibroblasts. We then transplanted the cells transduced with an adenovirus expressing LacZ into the caudate-putamen of monkey brain. Survival and distribution of the transplanted cells 1 month after the treatment were evaluated. Histochemical analysis showed that LacZ-positive cells were widely distributed in the brain, suggesting that the transplanted cells had migrated and were distributed even at regions far from the implantation site. These findings suggest that local intracerebral engraftment of genetically engineered amniotic epithelial cells is favorable for the treatment of lysosome storage disorders, whose pathological abnormalities are not restricted to specific regions of the brain.
Assuntos
Adenoviridae/genética , Âmnio/citologia , Encéfalo/citologia , Células Epiteliais/fisiologia , Células Epiteliais/transplante , Adenoviridae/metabolismo , Animais , Encéfalo/metabolismo , Transplante de Células , Células Cultivadas , Ventrículos Cerebrais/citologia , Meios de Cultivo Condicionados , Células Epiteliais/virologia , Fibroblastos/fisiologia , Técnicas de Transferência de Genes , Vetores Genéticos , Glucuronidase/genética , Glucuronidase/metabolismo , Humanos , Óperon Lac/genética , Macaca fascicularis , Camundongos , Mucopolissacaridose VII/terapia , Receptor IGF Tipo 2/metabolismo , Transdução Genética , Transplante HomólogoRESUMO
We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.
Assuntos
Proteínas/farmacologia , Receptores de Superfície Celular/química , Receptores de Neuropeptídeos , Animais , Antineoplásicos/farmacologia , Células CHO , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Cricetinae , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Kisspeptinas , Ligantes , Peptídeos/química , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor , CicatrizaçãoRESUMO
Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of beta-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into beta-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24-76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.