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1.
Xenotransplantation ; 31(1): e12838, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38112053

RESUMO

BACKGROUND: αGal-deficient xenografts are protected from hyperacute rejection during xenotransplantation but are still rejected more rapidly than allografts. Despite studies showing the roles of non-Gal antibodies and αß T cells in xenograft rejection, the involvement of γδ T cells in xenograft rejection has been limitedly investigated. METHODS: Six male cynomolgus monkeys were transplanted with porcine vessel xenografts from wild-type (n = 3) or GGTA1 knockout (n = 3) pigs. We measured the proportions and T cell receptor (TCR) repertoires of blood γδ T cells before and after xenotransplant. Grafted porcine vessel-infiltrating immune cells were visualized at the end of experiments. RESULTS: Blood γδ T cells expanded and infiltrated into the graft vessel adventitia following xenotransplantation of α-Gal-deficient pig blood vessels. Pre- and post-transplant analysis of γδ TCR repertoire revealed a transition in δ chain usage post-transplantation, with the expansion of several clonotypes of δ1, δ3, or δ7 chains. Furthermore, the distinctions between pre- and post-transplant δ chain usages were more prominent than those observed for γ chain usages. CONCLUSION: γδ TCR repertoire was significantly altered by xenotransplantation, suggesting the role of γδ T cells in sustained xenoreactive immune responses.


Assuntos
Primatas , Subpopulações de Linfócitos T , Animais , Masculino , Xenoenxertos , Receptores de Antígenos de Linfócitos T , Suínos , Transplante Heterólogo , Macaca fascicularis
2.
Curr Issues Mol Biol ; 43(1): 226-239, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071865

RESUMO

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, "exhausted" T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.


Assuntos
Complexo CD3/imunologia , Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexo CD3/genética , Complexo CD3/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , RNA-Seq/métodos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
3.
Xenotransplantation ; 23(4): 279-92, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27373998

RESUMO

BACKGROUND: Despite the development of α1,3-galactosyl transferase-knockout (GTKO) pigs, acute humoral xenograft rejection caused by antibodies against non-Gal antigens, along with complement activation, are hurdles that need to be overcome. Among non-Gal antigens, N-glycolylneuraminic acid (Neu5Gc) is considered to play an important role in xenograft rejection in human. METHODS: We generated human embryonic kidney 293 (HEK293) cells that expressed xenogeneic Neu5Gc (HEK293-pCMAH) or α1,3Gal (HEK293-pGT) antigen and investigated the degree of human antibody binding and complement-dependent cytotoxicity (CDC) against these antigens using 100 individual human sera. RESULTS: Both IgM and IgG bound to α1,3Gal, while only IgG bound to Neu5Gc. Of the ABO blood groups, the degree of IgG binding to α1,3Gal was highest for blood group A. The degree of CDC against HEK293-pCMAH cells was significantly lower than that against HEK293-pGT cells. However, CDC against HEK293-pCMAH cells was significantly higher than that against control HEK293 cells. In addition, the severity of CDC against HEK293-pCMAH cells positively correlated with that against GTKO pig aortic endothelial cells (PAECs), suggesting that Neu5Gc is the main antigen in GTKO PAECs. Similar to antibody-binding activity, only IgG binding correlated with CDC against HEK293-pCMAH cells. The most common subclass of IgGs against Neu5Gc was IgG1, which typically induces strong complement activation. CONCLUSIONS: We showed that IgG-mediated CDC was detected in Neu5Gc-overexpressed HEK293 cells incubated with human sera; however, this antibody reactivity to Neu5Gc was highly variable among individuals. Our results suggest that additional modifications to the CMAH gene should be considered for widespread use of pig organs for human transplants.


Assuntos
Galactose/imunologia , Rejeição de Enxerto/imunologia , Imunoglobulina G/imunologia , Ácidos Neuramínicos/imunologia , Transplante Heterólogo , Animais , Animais Geneticamente Modificados , Ativação do Complemento/imunologia , Células HEK293 , Humanos , Imunoglobulina G/sangue , Ácidos Neuramínicos/metabolismo , Suínos , Transplante Heterólogo/métodos
4.
Clin Immunol ; 161(2): 308-15, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26360254

RESUMO

We previously generated humanized TB34N mice that received human fetal thymus (T), bone tissue (B) and fetal liver-derived (FL)-CD34(+) cells (34) in immunodeficient, NOD/SCID IL2Rγ(null) (N) mice. Although humanized TB34N mice had excellent hematopoiesis, here, we sought to further improve this model by additional transplantation of human spleen tissue (S) as a secondary hematopoietic tissue (TBS34N). The human spleen grafts were enlarged and differentiated into a similar morphology of adult humans, including follicular lymphoid structures with T- and B-cells. The TBS34N mice mimicked mature human immune system (HIS): mature T- and B-cells and follicular dendritic cells; activated germinal center B-cells expressing CD71, BR3(+) cells, memory B-cells and activation-induced cytidine deaminase(+) B-cells; CD138(+) plasma cells were enriched in the mouse spleen. HBsAg-specific hIgG antibodies were secreted into the sera of all TBS34N mice upon immunization with HBsAg. Taken together, the humanized TBS34N mice improved mature HIS and achieved adaptive antibody responses.


Assuntos
Linfócitos B/imunologia , Transplante de Tecido Fetal/métodos , Baço/imunologia , Baço/transplante , Imunidade Adaptativa/imunologia , Animais , Formação de Anticorpos/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linfócitos B/metabolismo , Células Dendríticas Foliculares/imunologia , Citometria de Fluxo , Hematopoese/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Animais , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismo , Linfócitos T/imunologia , Transplante Heterólogo
5.
Clin Immunol ; 157(2): 156-65, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25725428

RESUMO

Both the thymus (T) and bone (B) are necessary hematopoietic niches in adult humans. We previously showed that co-transplantation of human fetal T and B tissues into neonatal immunodeficient NOD/SCID IL2Rγ(null) (NSG, N) mice facilitated hematopoiesis. However, transplantation into neonatal mice resulted in high frequency of early death, making it unrealistic for repetitive experiments. In this study, young adult N mice were pre-engrafted with T and B, T alone, B alone or no tissues. The animals were irradiated and injected with autologous fetal liver (FL)-derived CD34(+) cells (34). The resultant mice were TB34N, T34N, B34N and 34N, respectively, and challenged with T cell dependent antigens (Ags). The humanized TB34N mice showed best performance of these mouse models in many aspects resembling the adult human Ag-experienced spleen. The TB34N mice exhibited better hematopoietic reconstitution; balanced development of T- and B-cell, and common progenitor cells; follicular lymphoid structures with a functional germinal center (GC) enriched with follicular dendritic cells (FDCs) and plasma cells (PCs); secretion of hIgG in the sera in response to Ags at comparable levels to those of human; derivations of hIgG mAb-secreting hybridoma clones. Collectively, the humanized TB34N mice could develop an adaptive immunity that was capable of producing Ag-specific hIgG at a significant level via class switching. This unprecedented TB34N platform in humanized mice would be useful in dissecting human immunity, for generating human Abs and clinical applications.


Assuntos
Imunidade Adaptativa/imunologia , Anticorpos/imunologia , Antígenos CD34/metabolismo , Transplante Ósseo , Transplante de Tecido Fetal , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Baço/imunologia , Timo/transplante , Animais , Formação de Anticorpos , Hematopoese , Xenoenxertos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Baço/patologia
6.
Sci Rep ; 14(1): 15472, 2024 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-38969796

RESUMO

This study evaluated the determinants of mortality and the T cell immune response in patients with persistent Staphylococcus aureus bacteremia (SAB). This was a prospective cohort study and patients with confirmed SAB were enrolled from 2008 to 2020. We compared clinical, microbiological, and genotypic features between surviving and deceased patients with persistent SAB. The concentrations of cytokines and the proportions of IFN-γ secreting CD4+ T cells were measured serially during the bacteremia period. Of the 1760 patients, 242 had persistent bacteremia (PB), and 49 PB patients died within 30 days. In the multivariate analysis, the APACHE II score and female sex were independently associated with 30 days mortality. The level of IL-10 was significantly increased in the plasma of patients with a high Pitt bacteremia score and those who died within 12 weeks from the index day. The proportion of IFN-γ-secreting CD4+ T cells were the highest just before the positive-to-negative conversion of blood cultures in patients with a low Pitt bacteremia score and those who survived for 12 weeks. The level of IL-10 is correlated with clinical outcomes in PB patients. IFN-γ secreting CD4+ T cells might play a pivotal role in SAB PB.


Assuntos
Bacteriemia , Linfócitos T CD4-Positivos , Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Masculino , Feminino , Bacteriemia/mortalidade , Bacteriemia/microbiologia , Bacteriemia/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções Estafilocócicas/mortalidade , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Pessoa de Meia-Idade , Fatores de Risco , Idoso , Estudos Prospectivos , Interferon gama/sangue , Interferon gama/metabolismo , Interleucina-10/sangue , Adulto , Citocinas/sangue , Citocinas/metabolismo
7.
Blood ; 114(24): 4968-78, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19752395

RESUMO

Evidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from a genome-wide comparison of CpG methylation in human CD34(+) and CD34(-) cells, we identified a characteristic undermethylation dip around the transcription start site of promoters and an overmethylation of flanking regions in undifferentiated CD34(+) cells. This "bivalent-like" CpG methylation pattern around the transcription start site was more prominent in genes not associated with CpG islands (CGI(-)) than CGI(+) genes. Undifferentiated hematopoietic cells also exhibited dynamic chromatin associated with active transcription and a higher turnover of histone acetylation than terminally differentiated cells. Interestingly, inhibition of chromatin condensation by chemical treatment (5-azacytidine, trichostatin A) enhanced the self-renewal of "stimulated" HSCs in reconstituting bone marrows but not "steady-state" HSCs in stationary phase bone marrows. In contrast, similar treatments on more mature cells caused partial phenotypic dedifferentiation and apoptosis at levels correlated with their hematopoietic differentiation. Taken together, our study reveals that the undifferentiated state of hematopoietic cells is characterized by a unique epigenetic signature, which includes dynamic chromatin structures and an epigenetic plasticity that correlates to level of undifferentiation.


Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Células-Tronco Hematopoéticas/citologia , Acetilação , Animais , Antígenos CD34/biossíntese , Western Blotting , Ilhas de CpG , Epigênese Genética/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Sítio de Iniciação de Transcrição
8.
Immune Netw ; 20(1): e5, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32158593

RESUMO

The γδ T cells are unconventional lymphocytes that function in both innate and adaptive immune responses against various intracellular and infectious stresses. The γδ T cells can be exploited as cancer-killing effector cells since γδ TCRs recognize MHC-like molecules and growth factor receptors that are upregulated in cancer cells, and γδ T cells can differentiate into cytotoxic effector cells. However, γδ T cells may also promote tumor progression by secreting IL-17 or other cytokines. Therefore, it is essential to understand how the differentiation and homeostasis of γδ T cells are regulated and whether distinct γδ T cell subsets have different functions. Human γδ T cells are classified into Vδ2 and non-Vδ2 γδ T cells. The majority of Vδ2 γδ T cells are Vγ9δ2 T cells that recognize pyrophosphorylated isoprenoids generated by the dysregulated mevalonate pathway. In contrast, Vδ1 T cells expand from initially diverse TCR repertoire in patients with infectious diseases and cancers. The ligands of Vδ1 T cells are diverse and include the growth factor receptors such as endothelial protein C receptor. Both Vδ1 and Vδ2 γδ T cells are implicated to have immunotherapeutic potentials for cancers, but the detailed elucidation of the distinct characteristics of 2 populations will be required to enhance the immunotherapeutic potential of γδ T cells. Here, we summarize recent progress regarding cancer immunology of human γδ T cells, including their development, heterogeneity, and plasticity, the putative mechanisms underlying ligand recognition and activation, and their dual effects on tumor progression in the tumor microenvironment.

9.
Pathol Int ; 59(5): 279-87, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19432668

RESUMO

The aim of the present study was to determine the expression profile of the hedgehog (Hh) signaling molecules in normal, hyperplastic, and carcinomatous uterine endometrium. For this purpose, 271 endometrial tissue samples, (62 of normal endometrium, 127 of endometrial hyperplasias, and 82 endometrial adenocarcinomas) were studied using antibodies recognizing Hh-related signaling proteins, such as, sonic hedgehog (Shh), Patched (PTCH), Smoothened (Smo), Suppressor of fused [Su(Fu)], Gli-1, Gli-2, and Gli-3 by immunohistochemistry. The mRNA expression of these molecules was also assessed on reverse transcription-polymerase chain reaction. In the normal endometrium, the expression of Hh signaling molecules was generally downregulated except for Su(Fu), Gli-2, and Shh. In particular, the expression of both PTCH and Smo was very low or almost absent. Overall expression of Hh signaling molecules increased in hyperplastic endometrium; in particular, PTCH and Smo were significantly highly expressed in complex and atypical hyperplasia. In carcinoma samples extensive alterations were observed in the expression pattern of the signaling molecules. Nuclear Gli-2, cytoplasmic Gli-3, and Su(Fu) were overexpressed, whereas Shh, PTCH, and Smo expression were significantly reduced compared with the hyperplastic endometrium. The results suggest that the alteration of Hh signaling may be implicated in tumorigenesis of the endometrium.


Assuntos
Biomarcadores Tumorais/análise , Hiperplasia Endometrial/metabolismo , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Proteínas Hedgehog/biossíntese , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Hiperplasia Endometrial/genética , Neoplasias do Endométrio/genética , Feminino , Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Imuno-Histoquímica , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Microdissecção , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Receptores Patched , Receptor Patched-1 , RNA Mensageiro/análise , Receptores de Superfície Celular/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Smoothened , Análise Serial de Tecidos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de Zinco
10.
Oncoimmunology ; 7(12): e1500674, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30524895

RESUMO

Correct temporal and spatial control of actin dynamics is essential for the cytotoxic T cell effector function against tumor cells. However, little is known whether actin engineering in tumor-targeted T cells can enhance their antitumor responses, thereby potentiating the adoptive T cell therapy. Here, we report that TAGLN2, a 22-KDa actin-stabilizing protein which is physically associated with lymphocyte function-associated antigen-1 (LFA-1), potentiates the OTI TCR CD8+ T cells to kill the intercellular adhesion molecule-1 (ICAM-1)-positive/OVA-presenting E0771 cells, but not ICAM-1-negative OVA-B16F10 cells, suggesting an 'inside-out' activation of LFA-1, which causes more efficient immunological synapse formation between T cells and tumor cells. Notably, recombinant TAGLN2 fused with the protein transduction domain (TG2P) overcame the disadvantages of viral gene delivery, leading to a significant reduction in tumor growth in mice. TG2P also potentiated the CD19-targeted, chimeric antigen receptor (CAR)-modified T cells to kill Raji B-lymphoma cells. Our findings indicate that activating the TAGLN2-actin-LFA-1 axis is an effective strategy to potentiate the adoptive T-cell immunotherapy.

11.
Methods Mol Med ; 105: 359-68, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15492407

RESUMO

Embryonic stem (ES) cells differentiate efficiently in vitro and give rise to many different somatic cell types. Hematopoietic progenitors present within differentiated ES cells (embryoid bodies, EBs) can be identified by replating EB cells into semisolid media with hematopoietic growth factors. The developmental kinetics of various hematopoietic lineage precursors within EBs and molecular and cellular studies of these cells have suggested that the sequence of events leading to the onset of hematopoiesis within EBs is similar to that found within the mouse embryo. Thus, the in vitro differentiation model of ES cells provides a unique opportunity to study onset mechanisms involved in hematopoietic development.


Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Embrião de Mamíferos/fisiologia , Células Endoteliais/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Totipotentes/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Embrião de Mamíferos/citologia , Camundongos
12.
Transplantation ; 94(11): 1095-102, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23222735

RESUMO

BACKGROUND: In many humanized mouse models, there are few T cells in the engrafted human cell, whereas the number of B cells is high. We attempted to overcome this limitation and investigate whether the entire process of human T cell development arose similarly to the process in humans, as previously reported. METHODS: To produce an advanced humanized mice model, we transplanted human fetal liver/thymus tissue subrenally and injected human CD34(+) stem cells intravenously into NOD/SCID/IL2Rgamma null (NSG) mice. RESULTS: Humanized mice transplanted with fetal thymus/liver tissues and fetal liver-derived CD34(+) stem cells (FLT+FLCD34) showed higher levels of human cells and T cells than mice transplanted with fetal liver-derived CD34(+) stem cells only (FLCD34). In the transplanted thymus tissue of FLT+FLCD34 mice, thymus seeding progenitors (TSPs), early thymic progenitors (ETPs), pre-T cells, and all the other human T cell populations were identified. In the periphery, FLT+FLCD34 mice have high levels of CD45RA(+) T cells; conversely, FLCD34 mice have higher levels of CD45RO(+) T cells. The CD45RO(+) T cells of FLCD34 mice proliferated rapidly after stimulation and exhibited innate T cells properties, expressing PLZF (promyelocytic leukemia zinc finger protein). CONCLUSION: Human T cells educated by mouse MHC II in mice without a human thymus differ from normal human T cells. On the basis of these findings, numerous T cell-tropic human diseases could be explored in our humanized mice and molecular aspects of human T cell development could be also studied extensively.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Transplante de Fígado , Fígado , Linfócitos T/transplante , Timo/transplante , Animais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Proliferação de Células , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunidade Inata , Memória Imunológica , Imunofenotipagem/métodos , Interferon gama/metabolismo , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Fígado/embriologia , Fígado/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células Precursoras de Linfócitos T/imunologia , Células Precursoras de Linfócitos T/transplante , Proteína com Dedos de Zinco da Leucemia Promielocítica , Linfócitos T/imunologia , Timócitos/imunologia , Timócitos/transplante , Timo/embriologia , Timo/imunologia , Fatores de Tempo
13.
Br J Ophthalmol ; 94(5): 639-42, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20447966

RESUMO

BACKGROUND/AIM To determine if overexpression of the glaucoma gene MYOC is involved in the development of open-angle glaucoma (OAG) and if its promoter variants are associated with glaucoma in the Korean population. METHODS Human trabecular meshwork cells were cultured in the presence of ophthalmic steroids such as fluorometholone, fluorometholone acetate, dexamethasone, prednisolone acetate and rimexolone. The cells were cultured at a hydrostatic pressure of 32 mm Hg above atmospheric pressure and induction of MYOC was evaluated by northern blot analysis. Genomic DNA was extracted from blood samples obtained from 74 normal controls and 168 unrelated Korean patients with OAG, including primary OAG, normal tension glaucoma and steroid-induced glaucoma. A 461 base pair (bp) DNA fragment of the MYOC promoter region was amplified using PCR and its genotype was analysed by directly sequencing the product. RESULTS The potencies of steroid eye drops in MYOC induction in vitro was the same regardless of their potential for elevating intraocular pressure in vivo. Hydrostatic pressure had no effect on MYOC induction. A dinucleotide repeat polymorphism and three single nucleotide polymorphisms were identified, but no obvious differences in the genotype distribution and allele frequency of the variants between the control group and any type of OAG were observed. CONCLUSION Our data suggest that MYOC overexpression is not a cause or an effect of intraocular pressure elevation and that MYOC itself is not associated with OAG.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Frequência do Gene , Predisposição Genética para Doença , Glucocorticoides/farmacologia , Glicoproteínas/metabolismo , Humanos , Pressão Hidrostática , Polimorfismo de Nucleotídeo Único , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo
15.
Hum Gene Ther ; 19(7): 744-52, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18557700

RESUMO

Low in vivo transduction efficiency and safety concerns have been hurdles for effective hematopoietic stem cell (HSC) gene therapy. Here, we investigate whether the safety and efficiency of retroviral gene transfer into HSCs can be improved by using human allogeneic umbilical cord blood (UCB)-derived supplements instead of fetal bovine serum (FBS). When CD34(+) cells were cultured ex vivo in UCB-derived serum (CBS) or plasma (CBP), comparable or higher maintenance of HSCs was observed than in FBS and serum-free substitution medium (SFM) as assessed by the frequency of positive engraftment and the level of engraftment in NOD/SCID mice after transplantation of cultured cells. CBS and CBP also exhibited higher level stabilization of retroviral particles than SFM during in vitro culture of retrovirus pseudotyped with gibbon ape leukemia virus or vesicular stomatitis virus glycoprotein. Retroviral gene transfer into CD34(+) cells performed with CBS or CBP resulted in increased gene transfer into CD34(+) cells and increased transduction of reconstituted bone marrow cells compared to transfers with SFM or FBS. The increased transduction of bone marrow cells was associated with a larger number of transduced progenitors in the recipient mice. Significant oligoclonality in the transduced progenitors, as determined by ligation-mediated polymerase chain reaction, suggested efficient retroviral targeting of multiple HSCs in the CBS- or CBP-supplemented media. Combined, our results show that allogeneic UCB-derived serum or plasma is a safe and easily accessible serum supplement that can support efficient retroviral gene transfer into HSCs for the clinical-grade manipulation of HSCs.


Assuntos
Sangue Fetal , Técnicas de Transferência de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Retroviridae/genética , Transdução Genética , Transplante Homólogo , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea , Bovinos , Células Cultivadas , Meios de Cultura/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Hematopoéticas/virologia , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Vírus da Leucemia do Macaco Gibão/metabolismo , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Plasma , Soro , Proteínas do Envelope Viral
16.
Cell Stem Cell ; 2(5): 497-507, 2008 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-18462699

RESUMO

FLK1-expressing (FLK1(+)) mesoderm generates blood and vessels. Here, we show that combined BMP, Notch, and Wnt signaling is necessary for efficient FLK1(+) mesoderm formation from embryonic stem cells (ESCs). Inhibition of BMP, Notch, and Wnt signaling pathways greatly decreased the generation of FLK1(+) mesoderm and expression of the Ets transcription factor Er71. Enforced expression of ER71 in ESCs resulted in a robust induction of FLK1(+) mesoderm; rescued the generation of FLK1(+) mesoderm when blocked by BMP, Notch, and Wnt inhibition; and enhanced hematopoietic and endothelial cell generation. Er71-deficient mice had greatly reduced FLK1 expression, died early in gestation, and displayed severe blood and vessel defects that are highly reminiscent of the Flk1 null mouse phenotype. Collectively, we provide compelling evidence that ER71 functions downstream of BMP, Notch, and Wnt signals and regulates FLK1(+) mesoderm, blood, and vessel development.


Assuntos
Diferenciação Celular , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Animais , Vasos Sanguíneos/citologia , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula , Dipeptídeos , Endotélio Vascular/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Receptores Notch/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Alcaloides de Veratrum/metabolismo , Alcaloides de Veratrum/farmacologia , Proteínas Wnt/metabolismo
17.
Development ; 134(2): 393-405, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17166922

RESUMO

Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood. To define the underlying mechanisms, we compared gene expression profiles between embryonic stem (ES) cell-derived hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further characterized by utilizing the in vitro model of ES cell differentiation. Our studies revealed that Gata2 was a direct target of BMP4 and that enforced GATA2 expression upregulated Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2 additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, we provide compelling evidence that GATA2 plays specific, contextual roles in the generation of Flk-1+ mesoderm, the Flk-1+Scl+ hemangioblast, primitive erythroid and endothelial cells.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fator de Transcrição GATA2/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , Eritroblastos/citologia , Eritroblastos/metabolismo , Fator de Transcrição GATA2/deficiência , Fator de Transcrição GATA2/genética , Perfilação da Expressão Gênica , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Development ; 129(23): 5511-20, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12403720

RESUMO

Accumulating studies support the idea that a common progenitor, termed the hemangioblast, generates both hematopoietic and endothelial cell lineages. To better define the relationship between these cell lineages, we have generated knock-in embryonic stem (ES) cells carrying a non-functional human CD4 at the Scl locus. By using in vitro differentiated Scl(+/CD4) ES cells, we demonstrate that FLK1 and SCL are molecular determinants of the hemangioblast. Furthermore, our studies demonstrate that hematopoietic and endothelial cells develop via distinct, sequential generation of FLK1 and SCL-expressing cells. FLK1(+)CD4(-) cells first arise in developing embryoid bodies. The Scl gene is turned on within FLK1(+)CD4(-) cells to give rise to FLK1(+)CD4(+) cells. Alternatively, a subpopulation of the initial FLK1(+)CD4(-) cells remains as SCL negative. Within the FLK1(+)CD4(+) cells, FLK1 is down regulated to generate FILK1(-)CD4(+) cells. Replating studies demonstrate that hematopoietic progenitors are enriched within FLK1(+)CD4(+) and FLK1(-)CD4(+) cells, while endothelial cells develop from FLK1(+)CD4(+) and FLK1(+)CD4(-) cell populations.


Assuntos
Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Endotélio/crescimento & desenvolvimento , Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Antígenos CD4/genética , Separação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Endotélio/fisiologia , Citometria de Fluxo , Marcação de Genes , Humanos , Hibridização In Situ , Camundongos , Proteínas Proto-Oncogênicas/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
19.
Development ; 131(11): 2749-62, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15148304

RESUMO

The receptor tyrosine kinase FLK1 and the transcription factor SCL play crucial roles in the establishment of hematopoietic and endothelial cell lineages in mice. We have previously used an in vitro differentiation model of embryonic stem (ES) cells and demonstrated that hematopoietic and endothelial cells develop via sequentially generated FLK1(+) and SCL(+) cells. To gain a better understanding of cellular and molecular events leading to hematopoietic specification, we examined factors necessary for FLK1(+) and SCL(+) cell induction in serum-free conditions. We demonstrate that bone morphogenetic protein (BMP) 4 was required for the generation of FLK1(+) and SCL(+) cells, and that vascular endothelial growth factor (VEGF) was necessary for the expansion and differentiation of SCL-expressing hematopoietic progenitors. Consistently, Flk1-deficient ES cells responded to BMP4 and generated TER119(+) and CD31(+) cells, but they failed to expand in response to VEGF. The Smad1/5 and map kinase pathways were activated by BMP4 and VEGF, respectively. The overexpression of SMAD6 in ES cells resulted in a reduction of FLK1(+) cells. In addition, a MAP kinase kinase 1 specific inhibitor blocked the expansion of SCL(+) cells in response to VEGF. Finally, VEGF mediated expansion of hematopoietic and endothelial cell progenitors was inhibited by TGFbeta1, but was augmented by activin A. Our studies suggest that hematopoietic and endothelial commitment from the mesoderm occurs via BMP4-mediated signals and that expansion and/or differentiation of such progenitors is achieved by an interplay of VEGF, TGFbeta1 and activin A signaling.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Butadienos/farmacologia , Antígenos CD4/genética , Antígenos CD4/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , MAP Quinase Quinase 1 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Cadeias Pesadas de Miosina , Nitrilas/farmacologia , Miosina não Muscular Tipo IIB , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Smad , Proteína Smad1 , Proteína Smad5 , Proteína Smad6 , Células-Tronco/efeitos dos fármacos , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
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