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1.
Clin Exp Immunol ; 177(1): 149-60, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24628444

RESUMO

Type 1 diabetes (T1DM) is a T cell-mediated autoimmune disease that selectively destroys pancreatic ß cells. The only possible cure for T1DM is to control autoimmunity against ß cell-specific antigens. We explored whether the natural compound curcumin, with anti-oxidant and anti-inflammatory activities, might down-regulate the T cell response that destroys pancreatic ß cells to improve disease outcome in autoimmune diabetes. We employed two accelerated autoimmune diabetes models: (i) cyclophosphamide (CYP) administration to non-obese diabetic (NOD) mice and (ii) adoptive transfer of diabetogenic splenocytes into NODscid mice. Curcumin treatment led to significant delay of disease onset, and in some instances prevented autoimmune diabetes by inhibiting pancreatic leucocyte infiltration and preserving insulin-expressing cells. To investigate the mechanisms of protection we studied the effect of curcumin on key immune cell populations involved in the pathogenesis of the disease. Curcumin modulates the T lymphocyte response impairing proliferation and interferon (IFN)-γ production through modulation of T-box expressed in T cells (T-bet), a key transcription factor for proinflammatory T helper type 1 (Th1) lymphocyte differentiation, both at the transcriptional and translational levels. Also, curcumin reduces nuclear factor (NF)-κB activation in T cell receptor (TCR)-stimulated NOD lymphocytes. In addition, curcumin impairs the T cell stimulatory function of dendritic cells with reduced secretion of proinflammatory cytokines and nitric oxide (NO) and low surface expression of co-stimulatory molecules, leading to an overall diminished antigen-presenting cell activity. These in-vitro effects correlated with ex-vivo analysis of cells obtained from curcumin-treated mice during the course of autoimmune diabetes. These findings reveal an effective therapeutic effect of curcumin in autoimmune diabetes by its actions on key immune cells responsible for ß cell death.


Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Antioxidantes/administração & dosagem , Curcumina/administração & dosagem , Células Dendríticas/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Células Th1/efeitos dos fármacos , Animais , Apresentação de Antígeno/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , NF-kappa B/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Células Th1/imunologia , Ativação Transcricional/efeitos dos fármacos
2.
Clin Exp Immunol ; 171(2): 135-46, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23286940

RESUMO

Type 1 diabetes mellitus (T1DM) results from death of insulin-secreting ß cells mediated by self-immune cells, and the consequent inability of the body to maintain insulin levels for appropriate glucose homeostasis. Probably initiated by environmental factors, this disease takes place in genetically predisposed individuals. Given the autoimmune nature of T1DM, therapeutics targeting immune cells involved in disease progress have been explored over the last decade. Several high-cost trials have been attempted to prevent and/or reverse T1DM. Although a definitive solution to cure T1DM is not yet available, a large amount of information about its nature and development has contributed greatly to both the improvement of patient's health care and design of new treatments. In this study, we discuss the role of different types of immune cells involved in T1DM pathogenesis and their therapeutic potential as targets and/or modified tools to treat patients. Recently, encouraging results and new approaches to sustain remnant ß cell mass and to increase ß cell proliferation by different cell-based means have emerged. Results coming from ongoing clinical trials employing cell therapy designed to arrest T1DM will probably proliferate in the next few years. Strategies under consideration include infusion of several types of stem cells, dendritic cells and regulatory T cells, either manipulated genetically ex vivo or non-manipulated. Their use in combination approaches is another therapeutic alternative. Cell-based interventions, without undesirable side effects, directed to block the uncontrollable autoimmune response may become a clinical reality in the next few years for the treatment of patients with T1DM.


Assuntos
Autoimunidade/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/imunologia , Transplante de Células-Tronco/métodos , Linfócitos T Reguladores/imunologia , Animais , Ensaios Clínicos como Assunto , Células Dendríticas/transplante , Modelos Animais de Doenças , Humanos , Insulina/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Transplante de Células-Tronco/tendências , Linfócitos T Reguladores/transplante
3.
Cancer Gene Ther ; 19(5): 352-7, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22402626

RESUMO

Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-ß receptor II (TßRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TßRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.


Assuntos
Adenocarcinoma/terapia , Terapia Genética/métodos , Glioma/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Neoplasias Pulmonares/terapia , Transgenes , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Glioma/genética , Glioma/cirurgia , Proteínas de Choque Térmico HSP70/genética , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética
4.
Gene Ther ; 7(8): 679-85, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10800091

RESUMO

Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-DeltaTK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic than its truncated version HSV1-DeltaTK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-DeltaTK, after administration of ganciclovir. The effectiveness of HSV1-DeltaTK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy.


Assuntos
Adenoviridae/efeitos dos fármacos , Sistema Nervoso Central/virologia , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Herpesvirus Humano 1/enzimologia , Timidina Quinase/genética , Animais , Antivirais/uso terapêutico , Neoplasias Encefálicas/terapia , Células Cultivadas , Ganciclovir/uso terapêutico , Glioma/terapia , Humanos , Neuroglia , Neurônios , Ratos
5.
J Clin Endocrinol Metab ; 85(3): 1296-305, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10720079

RESUMO

We tested the hypothesis that gene transfer using recombinant adenovirus vectors (RAds) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) might offer an alternative therapeutic approach for the treatment of pituitary prolactinomas that do not respond to classical treatment strategies. HSV1-TK converts the prodrug ganciclovir (GCV) to GCV monophosphate, which is in turn further phosphorylated by cellular kinases to GCV triphosphate, which is toxic to proliferating cells. One attractive feature of this system is the bystander effect, whereby untransduced cells are also killed. Our results show that RAd/HSV1-TK in the presence of GCV is nontoxic for the normal anterior pituitary (AP) gland in vitro, but causes cell death in the pituitary tumor cell lines GH3, a PRL/GH-secreting cell line, and AtT20, a corticotrophic cell line. We have used sulpiride- and oestrogen-induced lactotroph hyperplasia within the rat AP gland as an in vivo animal model. Intrapituitary infection of rats bearing oestrogen-induced lactotroph hyperplasia, with RAd/ HSV1-TK and subsequent treatment with GCV, decreases plasma PRL levels and reduces the mass of the pituitary gland. More so, there were no deleterious effects on circulating levels of other AP hormones, suggesting that the treatment was nontoxic to the AP gland in situ. In summary, our results show that suicide gene therapy using the HSV1-TK transgene could be further developed as a useful treatment to complement current therapies for prolactinomas.


Assuntos
Adenoviridae/genética , Estrogênios/farmacologia , Terapia Genética , Herpesvirus Humano 1/genética , Neoplasias Hipofisárias/terapia , Prolactinoma/terapia , Timidina Quinase/genética , Animais , Apoptose/genética , Linhagem Celular , Imunofluorescência , Herpesvirus Humano 1/enzimologia , Imuno-Histoquímica , Masculino , Adeno-Hipófise/virologia , Ratos , Células Tumorais Cultivadas
6.
Nat Med ; 5(11): 1256-63, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10545991

RESUMO

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression.


Assuntos
Neoplasias Encefálicas/terapia , Encefalite/etiologia , Terapia Genética/efeitos adversos , Glioma/terapia , Adenoviridae/genética , Animais , Astrócitos/imunologia , Sequência de Bases , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Ensaios Clínicos como Assunto , Primers do DNA , Encefalite/imunologia , Ganciclovir/efeitos adversos , Ganciclovir/uso terapêutico , Vetores Genéticos , Glioma/imunologia , Glioma/patologia , Herpesvirus Humano 1/enzimologia , Humanos , Linfócitos/imunologia , Ativação de Macrófagos , Microglia/imunologia , Bainha de Mielina/metabolismo , Ratos , Timidina Quinase/genética , Transgenes , Células Tumorais Cultivadas
7.
J Gen Virol ; 80 ( Pt 3): 571-583, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10091995

RESUMO

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.


Assuntos
Adenoviridae/genética , Glicoproteínas de Membrana/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , Adenoviridae/crescimento & desenvolvimento , Animais , Apoptose , Células Cultivadas , Citomegalovirus/genética , Proteína Ligante Fas , Feminino , Citometria de Fluxo , Genes Precoces/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteína Glial Fibrilar Ácida/genética , Hemorragia/etiologia , Hemorragia/patologia , Hemorragia/prevenção & controle , Humanos , Fígado/patologia , Hepatopatias/etiologia , Hepatopatias/patologia , Hepatopatias/prevenção & controle , Glicoproteínas de Membrana/efeitos adversos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Neuroglia/citologia , Neuroglia/virologia , Neurônios/citologia , Neurônios/virologia , Especificidade de Órgãos , Fosfopiruvato Hidratase/genética , Ratos , Transgenes/genética , Células Tumorais Cultivadas
8.
Gene Ther ; 5(4): 563-8, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9614583

RESUMO

Human embryonic kidney 293 cells contain the E1 region of adenovirus type 5, and thus sustain, through transcomplementation, the production of recombinant E1-deleted adenovirus vectors. During attempts to produce recombinant adenovirus expressing the apoptosis-inducing molecule Fas ligand (FasL) under the control of a very strong truncated major immediate-early human cytomegalovirus (MIEhCMV) promoter, we discovered that 293 cells were not surviving the initial cotransfection with a shuttle plasmid encoding the mouse FasL; and pJM17, a plasmid containing the genome of adenovirus type 5 with deletions in the E1-E3 regions, in an unpackagable form. Investigation of the reason for massive cell death after cotransfection led us to determine that 293 cells express the FasL receptor. Fas-Apo1 (CD95), and respond with apoptosis to the cross-linking of Fas-Apo1 with either IgM monoclonal antibodies or FasL. Therefore, we decided to generate adenoviral vectors expressing FasL, under the control of tissue-specific and/or-inducible promoter elements. Our findings can explain difficulties several groups have had in generating recombinant adenoviral vectors expressing FasL using 293 cells, as well as the lower titres reported.


Assuntos
Adenoviridae , Apoptose/fisiologia , Vetores Genéticos , Rim/embriologia , Glicoproteínas de Membrana/farmacologia , Receptor fas/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Proteína Ligante Fas , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Rim/metabolismo , Glicoproteínas de Membrana/metabolismo , Engenharia de Proteínas , Transfecção
9.
Mol Phylogenet Evol ; 8(1): 11-32, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9242593

RESUMO

The genus Tospovirus was thought to be composed only of the tomato spotted wilt virus (TSWV), but now at least four Tospovirus species have been proposed based on serological and molecular data. A classification of tospoviruses has been proposed taking into account global similarities of the N gene and N protein sequences of 7 isolates of Tospovirus. Because phylogenetic analyses based on global similarities can lead to classifications which do not mirror the genealogy of the group, we have employed a cladistic analysis using parsimony of this genus with RNA sequences of 450 nucleotides of the N gene from 14 new Argentinean isolates and 4 previously described isolates. Representatives of the Bunyaviridae family, Rift Valley Fever Virus (Phlebovirus) and Bunyamwera (Bunyavirus), were used as the outgroup in separate analyses.


Assuntos
Nucleocapsídeo/genética , Filogenia , Tospovirus/genética , Argentina , Sequência de Bases , Bunyaviridae/genética , Variação Genética , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , RNA Viral/genética , Vírus da Febre do Vale do Rift/genética , Seleção Genética , Alinhamento de Sequência
10.
J Virol Methods ; 56(1): 19-26, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8690762

RESUMO

Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.


Assuntos
Enzimas de Restrição do DNA , Reação em Cadeia da Polimerase/métodos , Tospovirus/isolamento & purificação , Sequência de Bases , Primers do DNA , Desoxirribonucleases de Sítio Específico do Tipo II , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Tospovirus/genética , Transcrição Gênica
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