RESUMO
This study aimed at establishing a protocol to increase the number of regenerated shoots and to limit the recovery of "escapes" during the regeneration of transgenic flax plants (cv Barbara). Here, we describe how light, adapted media and selection scheme could stimulate the transformation process, the organogenic potentiality of calli (by a factor of 3.2) and accelerate the transgenic shoot regeneration (by a factor of about 2). On comparison of the transformation rate observed while using low light (LL) and high light (HL) a considerable enhancement from 0.12 to 5.7% was evident. The promotive effect of light might also had a direct beneficial effect on transgenic plant production time leading to a reduction of more than 4 months in the time need to obtain transgenic seeds. All data indicate that HL plays a role on growth and on protein, rubisco and pigment contents by stimulating the gene implicated in photosynthetic and Calvin cycle processes.
Assuntos
Linho/fisiologia , Linho/efeitos da radiação , Luz , Regeneração , Meios de Cultura , Linho/genética , Regulação da Expressão Gênica de Plantas , Fotossíntese/genética , Brotos de Planta/genética , Brotos de Planta/fisiologia , Brotos de Planta/efeitos da radiação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Plantas Geneticamente Modificadas/efeitos da radiação , RNA de Plantas/metabolismo , Transformação GenéticaRESUMO
gamma9delta2 T lymphocytes are non-conventional lymphocytes presenting a direct cytotoxic effect against a broad range of tumour targets. These cells also secrete inflammatory cytokines that can boost the other components of the immune system. In contrast to conventional CD8(+) T cells, the cytotoxic effect of gamma9delta2 T lymphocytes does not depend on the expression of major histocompatibility complex molecules by target tumour cells. INNACELL gammadeltatrade mark is a cell therapy product obtained by ex vivo amplification of mononuclear cells. The stimulation is achieved by a specific synthetic agonist of gamma9delta2 T lymphocytes, bromohydrin pyrophosphate (BrHPP). After a single stimulation with BrHPP, gamma9delta2 T lymphocytes are expanded for 2 weeks in a closed system in culture medium with interleukin-2 (IL-2). On day 15, cells are washed and harvested in 4% human serum albumin. In this manufacturing process, the total cell population is expanded by approximately 10-fold and gamma9delta2 T lymphocytes undergo a specific 1000-fold expansion, corresponding to a gamma9delta2 T lymphocyte enrichment of more than 70% at the end of the culture. This manufacturing process is much simpler than most current cellular therapy approaches using conventional CD8(+) T-cell lines or clones: there is no final or initial separation, no purification step and no use of feeder cells; the specific T-cell receptor-mediated signal provided by BrHPP is sufficient to trigger the IL-2-dependent expansion of the gamma9delta2 subset, which then becomes predominant in the cell culture in large amounts.
Assuntos
Proliferação de Células , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T gama-delta/uso terapêutico , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/secundário , Carcinoma de Células Renais/terapia , Células Cultivadas , Humanos , Leucaférese , Contagem de Linfócitos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/transplanteRESUMO
Successful microarray experimentation can generate enormous amounts of data, potentially very rich but also very unwieldy. Bold outlooks and new methods for data analysis and presentation should yield additional insight into the complexities of the immune system.
Assuntos
Perfilação da Expressão Gênica , Genômica/métodos , Imunidade/genética , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Linfócitos B/metabolismo , Apresentação de Dados , Regulação da Expressão Gênica/genética , Humanos , Subpopulações de Linfócitos/metabolismo , Metanálise como Assunto , Camundongos , Processamento Pós-Transcricional do RNA , Splicing de RNA , Linfócitos T/metabolismo , Terminologia como Assunto , Transcrição GênicaRESUMO
Renal cell carcinoma represents 3% of solid malignancies in adults and nephrectomy remains the main treatment. Failure of conventional approaches for patients presenting with advanced disease has prompted the exploration of new strategies. This review describes the potential use of peripheral gammadelta (Vgamma9Vdelta2) T-cells in metastatic renal cell carcinoma. This peripheral lymphocyte population from the innate immune system has demonstrated an in vitro antitumor cytotoxicity against primary or established renal cell lines. Moreover, these Vgamma9Vdelta2 lymphocytes undergo a rapid and extensive expansion in vitro as well as in vivo upon stimulation with a synthetic potent agonist, the bromohydrin pyrophosphate molecule. Preclinical results obtained on specific in vitro amplification of Vgamma9Vdelta2 T-cells by bromohydrin pyrophosphate in renal cell carcinoma patients are presented in this review, while Phase I clinical trials are currently running. As there is growing evidence for the low efficiency of monotherapy in cancer patients, innovative approaches combining immunomodulatory gammadelta agonists with classic chemotherapies or administration of antiangiogenic agents are discussed.
Assuntos
Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/terapia , Neoplasias Renais/imunologia , Neoplasias Renais/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Proliferação de Células , Ensaios Clínicos como Assunto , Citocinas/imunologia , Humanos , Imunidade Celular , Imunidade Inata , Imunoterapia , Receptores de Antígenos de Linfócitos T gama-delta/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Osteoclasts are specialized macrophages that resorb bone. Mice lacking the AP-1 component c-Fos are osteopetrotic because of a lack of osteoclast differentiation and show an increased number of macrophages. The nature of the critical function of c-Fos in osteoclast differentiation is not known. Microarray analysis revealed that Nfatc1, another key regulator of osteoclastogenesis, was down-regulated in Fos(-/-) osteoclast precursors. Chromatin immunoprecipitation assay showed that c-Fos bound to the Nfatc1 and Acp5 promoters in osteoclasts. In vitro promoter analyses identified nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific Acp5 and Calcr promoters. Moreover, in Fos(-/-) precursors gene transfer of an active form of NFAT restored transcription of osteoclast-specific genes in the presence of receptor activator of the NF-kappaB ligand (RANKL), rescuing bone resorption. In the absence of RANKL, however, Fos(-/-) precursors were insensitive to NFAT-induced osteoclastogenesis unlike wild-type precursors. These data indicate that lack of Nfatc1 expression is the cause of the differentiation block in Fos(-/-) osteoclast precursors and that transcriptional induction of Nfatc1 is a major function of c-Fos in osteoclast differentiation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/fisiologia , Animais , Western Blotting , Proteínas de Transporte/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Técnicas de Cocultura , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Transferência de Genes , Humanos , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Microscopia de Fluorescência , Fatores de Transcrição NFATC , Análise de Sequência com Séries de Oligonucleotídeos , Osteoclastos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic beta cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/- mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/- mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/- and Amylin +/- mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.
Assuntos
Amiloide/metabolismo , Reabsorção Óssea , Osteogênese/fisiologia , Receptores da Calcitonina/metabolismo , Amiloide/genética , Animais , Densidade Óssea , Osso e Ossos/anormalidades , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoclastos/citologia , Osteoclastos/fisiologia , FenótipoRESUMO
The orphan nuclear estrogen receptor-related receptor alpha (ERRalpha) is expressed by osteoblastic cells and plays a functional role in osteoprogenitor proliferation and differentiation. To dissect further the role of ERRalpha in bone, we investigated the effects of estrogen (E2) on ERRalpha both in vitro and in vivo. Chronic treatment of fetal rat calvaria cells with E2-stimulated bone nodule formation and up-regulated ERRalpha mRNA expression at early (10 h and d 8) but not later times in culture, suggesting a link between ERRalpha and E2 during osteoprogenitor proliferation. ERRalpha mRNA levels were significantly lower in ovariectomized adult rat bones vs. those of sham-operated rats early (1 d and 1 wk) post surgery, but levels returned to control levels thereafter. ERRalpha is also expressed in osteoclasts (tartrate-resistant acid phosphatase + multinucleated cells) in vivo and in vitro (RAW 264.7 cells) and ovariectomization lowered the OPG/receptor activator of nuclear factor kappaB ligand expression ratio. Down-regulation of ERRalpha expression via antisense treatment of rat calvaria cells not only inhibited osteogenesis but also increased adipocyte colony formation and changed the OPG/receptor activator of nuclear factor kappaB ligand ratio. These data suggest that ERRalpha is regulated by estrogen in bone in which it may play a functional role at several levels (osteoblasts, adipocytes, and osteoclasts) in E2 deficiency diseases such as osteoporosis.