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1.
J Vet Diagn Invest ; 29(2): 186-192, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28166712

RESUMO

Bovine viral diarrhea virus (BVDV) is a pathogen in cattle and alpacas ( Vicugna pacos), causing acute and persistent BVDV infections. We characterized the effect of acute BVDV infection on the immune system of alpacas by determining lymphocyte subpopulations in peripheral blood and gut-associated lymphoid tissues (GALT) as well as serum interferon levels. Alpacas were experimentally infected with BVDV-1b (strain CO-06). Peripheral blood leukocytes were isolated at 0, 3, 6, and 9 d postinfection (dpi), and leukocytes of GALT at 9 dpi, and evaluated using flow cytometry. Serum interferon levels were determined daily. Flow cytometric analyses of peripheral blood leukocytes showed a significant decrease in CD4+, CD8+, and αß T-lymphocytes at 3 dpi. CD8+ lymphocytes were significantly increased, and activated lymphocytes were significantly decreased in the C3-stomach region in BVDV-infected alpacas. Serum interferon concentrations significantly increased in BVDV-infected alpacas at 3-6 dpi, peaking at 3 dpi. Our study confirms that BVDV can be a primary acute pathogen in alpacas and that it induces an interferon response and alters leukocyte subset populations. The changes in the proportion of T-lymphocytes during the early stages of BVDV infection may result in transient immunosuppression that may contribute to secondary bacterial and viral infections, similar to cattle.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Camelídeos Americanos , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Animais , Bovinos , Citocinas/sangue , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/genética , Citometria de Fluxo/veterinária , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Leucócitos/classificação , Leucócitos/citologia
2.
J Immunol Methods ; 426: 86-94, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26268454

RESUMO

Antigen-specific, T cell hybridomas are useful to study the cellular, molecular and functional events, but their generation is a lengthy process. Thus, there is a need to develop robust methods to generate the hybridoma clones rapidly in a short period of time. To this end, we have demonstrated a novel approach using major histocompatibility complex (MHC) class II dextramers to generate T cell hybridomas for an autoantigen, proteolipid protein (PLP) 139-151. Using MHC class II dextramers assembled with PLP 139-151 as screening and sorting tools, we successfully obtained mono antigen-specific clones within seven to eight weeks. In conjunction with other T cell markers, dextramers permitted phenotypic characterization of hybridoma clones for their antigen specificity in a single step by flow cytometry. Importantly, we achieved successful fusions using dextramer(+) cells sorted by flow cytometry as a starting population, resulting in direct identification of multiple antigen-specific clones. Characterization of selected clones led us to identify chemokine receptor, CCR4(+) to be expressed consistently, but their cytokine-producing ability was variable. Our work provides a proof-of principle that the antigen-specific, CD4 T cell hybridoma clones can be generated directly using MHC class II dextramers. The availability of hybridoma clones that bind dextramers may serve as useful tools for various in vitro and in vivo applications.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridomas/imunologia , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Proliferação de Células , Células Cultivadas , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/química , Camundongos , Complexos Multiproteicos/imunologia , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores CCR4/biossíntese
3.
J Virol ; 88(21): 12541-50, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25142578

RESUMO

UNLABELLED: Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Chlorovirus PBCV-1 encodes a 187-amino-acid protein that resembles a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc (named cvSOD). cvSOD has an internal Met that results in a 165-amino-acid protein (named tcvSOD). Both cvSOD and tcvSOD recombinant proteins inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A k(cat)/K(m) value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 10(8) M(-1) s(-1), suggesting that cvSOD-catalyzed O2 (-) dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms. IMPORTANCE: Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.


Assuntos
Phycodnaviridae/enzimologia , Phycodnaviridae/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Cinética , Dados de Sequência Molecular , Nitroazul de Tetrazólio/metabolismo , Oxirredução , Filogenia , Riboflavina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vírion/enzimologia
4.
Helicobacter ; 18(6): 433-43, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23895367

RESUMO

BACKGROUND: Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated. MATERIALS AND METHODS: The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72 hours post-treatment by flow cytometry. RESULTS: Similar time course of HhepCDT-induced G2 /M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72 hours. The presence of all three HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2 /M arrest and apoptotic death. CONCLUSION: All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Epiteliais/citologia , Infecções por Helicobacter/fisiopatologia , Helicobacter hepaticus/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Infecções por Helicobacter/microbiologia , Helicobacter hepaticus/química , Humanos , Dados de Sequência Molecular
5.
Free Radic Biol Med ; 57: 92-104, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23291592

RESUMO

Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multiple physiological functions in cells and its bioavailability is negatively controlled through active-site binding to a specific thioredoxin-binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx and the effect of overexpression of TBP-2 in human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a sevenfold increase in TBP-2 and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day 7. Cell cycle analysis showed that TBP-2 OE cells arrested at the G2/M stage and that they displayed low expression of the cell cycle elements P-cdc2(Y15), cdc2, cdc25A, and cdc25C. Furthermore, TBP-2 OE cells were more sensitive to oxidation. Under H2O2 (200µM, 24h) treatment, these cells lost 80% viability and became highly apoptotic. Brief oxidative stress (200µM, 30min) to TBP-2 OE cells disrupted the Trx antiapoptotic function by dissociating the cytosolic and mitochondrial Trx-ASK binding complexes. The same H2O2-treated cells also showed activated ASK (P-ASK), increased Bax, lowered Bcl-2, cytochrome c release, and elevated caspase 3/7 activity. We conclude from these studies that high cellular levels of TBP-2 can potentially suppress Trx bioavailability and increase oxidation sensitivity. Overexpression of TBP-2 also causes slow growth by mitotic arrest and apoptosis by activating the ASK death pathway.


Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalino/metabolismo , Tiorredoxinas/metabolismo , Proteínas de Transporte/genética , Caspases/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Citocromos c/metabolismo , Ativação Enzimática , Células Epiteliais/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Pontos de Checagem da Fase M do Ciclo Celular , Mitocôndrias/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Proteína X Associada a bcl-2/metabolismo
6.
BMC Immunol ; 12: 40, 2011 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-21767394

RESUMO

BACKGROUND: Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent. RESULTS: The utility of MHC dextramers was evaluated in three autoimmune disease models: 1) proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s) mice; 2) myelin oligodendrocyte glycoprotein (MOG) 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b) mice; and 3) cardiac myosin heavy chain (Myhc)-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a) mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo. CONCLUSIONS: The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.


Assuntos
Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/química , Peptídeos/imunologia , Animais , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/síntese química , Peptídeos/metabolismo , Sensibilidade e Especificidade , Coloração e Rotulagem
7.
Virology ; 406(2): 270-9, 2010 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-20701940

RESUMO

The objective of this study was to identify porcine reproductive and respiratory syndrome virus (PRRSV)-encoded proteins that are responsible for the inhibition of TNF-α expression and the mechanism(s) involved in this phenomenon. Using a TNF-α promoter reporter system, the non-structural protein 1 (Nsp1) was found to strongly suppress the TNF-α promoter activity. Such inhibition takes place especially at the promoter's proximal region. Both Nsp1α and Nsp1ß, the two proteolytic fragments of Nsp1, were shown to be involved in TNF-α promoter suppression. Furthermore, using reporter plasmids specific for transcription factors (TFs) that bind to TNF-α promoter, Nsp1α and Nsp1ß were demonstrated to inhibit the activity of the TFs that bind CRE-κB(3) and Sp1 elements respectively. Subsequent analyses showed that Nsp1α moderately inhibits NF-κB activation and that Nsp1ß completely abrogates the Sp1 transactivation. These findings reveal one of the important mechanisms underlying the innate immune evasion by PRRSV during infection.


Assuntos
Regulação para Baixo , NF-kappa B/metabolismo , Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fator de Necrose Tumoral alfa/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Camundongos , Dados de Sequência Molecular , NF-kappa B/genética , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Ligação Proteica , Fator de Transcrição Sp1/genética , Suínos , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Proteínas não Estruturais Virais/genética
8.
Helicobacter ; 15(2): 98-107, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20402812

RESUMO

BACKGROUND: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram-negative bacteria is associated with double-stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells. MATERIALS AND METHODS: INT407 cells were incubated with or without recombinant HhCDT for 0-72 hours. H2AX phosphorylation and apoptotic parameters were analyzed. RESULTS: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro-apoptotic Bax protein was upregulated after 24 hours, while Bcl(2) expression decreased. Cytochrome c was released from mitochondria after 12-24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z-VAD-FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT-treated cells. CONCLUSION: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.


Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Células Epiteliais/microbiologia , Helicobacter hepaticus/patogenicidade , Mitocôndrias/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular , Citocromos c/análise , Citoplasma/química , Expressão Gênica , Histonas/metabolismo , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2/biossíntese
9.
Invest Ophthalmol Vis Sci ; 49(10): 4497-505, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18586881

RESUMO

PURPOSE: To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase(-/-)) lens epithelial cells (LECs) as a model. METHODS: Primary LEC cultures were obtained from wild-type (TTase(+/+)) and TTase(-/-) mice. Characterization and validation of the cells were determined by immunoblotting for TTase and alpha-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H(2)O(2) stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase(-/-) cells was achieved with reagent. RESULTS: Primary LEC cultures obtained from wild-type (TTase(+/+)) and TTase(-/-) mice were characterized and found to contain lens-specific alpha-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase(-/-) cells and its presence in the wild-type cells. TTase(-/-) LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase(+/+) cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H(2)O(2) after challenge with low levels of H(2)O(2). Reloading of purified TTase into the TTase(-/-) cells restored the antioxidant function in TTase(-/-) cells to a near normal state. CONCLUSIONS: These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.


Assuntos
Proliferação de Células , Glutarredoxinas/fisiologia , Cristalino/citologia , Estresse Oxidativo , Animais , Apoptose , Northern Blotting , Western Blotting , Sobrevivência Celular , Células Cultivadas , Citosol , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Citometria de Fluxo , Glutarredoxinas/farmacologia , Glutationa/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Peróxido de Hidrogênio/toxicidade , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/farmacologia , Cadeia A de alfa-Cristalina/metabolismo
10.
Transplantation ; 80(3): 362-9, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16082332

RESUMO

BACKGROUND: Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector. METHODS: To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice. RESULTS: After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days. CONCLUSION: Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.


Assuntos
Células da Medula Óssea/citologia , Transplante de Medula Óssea/métodos , Complexo Principal de Histocompatibilidade , Animais , Células da Medula Óssea/imunologia , Transplante de Células , DNA Complementar/metabolismo , Citometria de Fluxo , Rejeição de Enxerto , Sobrevivência de Enxerto , Antígeno HLA-A2/química , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Reação em Cadeia da Polimerase , Retroviridae/genética , Retroviridae/metabolismo , Transplante de Pele , Fatores de Tempo
11.
Cancer Biol Ther ; 4(5): 602-11, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15970678

RESUMO

BACKGROUND: Our previous investigations showed that retinoids, at specific concentrations, can inhibit cell proliferation. In this investigation, we hypothesize that high concentrations of retinoids can induce phenotypic changes (differentiation) and late apoptosis in pancreatic cancer cells in vitro. MATERIALS AND METHODS: To test our hypothesis, retinoid-induced differentiation was assessed: (1) phenotypically by light and electron microscopy and (2) biochemically by measuring carbonic anhydrase, aerobic metabolic and mucin producing activities. Modulation of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) autocrine pathways were utilized as mechanistic and differentiation markers. RESULTS: The extensive differentiation-indicative phenotypic changes correlated with several folds increase in the aerobic metabolism (MTT reduction and Mitochondrial mass), carbonic anhydrase activity and mucin production. There was a marked increase in TGF-beta (Bioassay and ELISA) and TGF-beta (RIA) secretion. EGF receptor density (Receptor binding assay) was reduced by 50% within six hours and was reflected on abolishment of EGFR ligand-induced proliferation. Cotreatment with the RAR-alpha antagonist, Ro41-5253 or pan-TGF-beta neutralizing antibody abolished the phenotypic and antiproliferative effects of all-trans retinoic acid. Apoptosis (TUNEL assay) was undetectable after three days of treatment with the maximum concentration used. However, apoptosis was extensively induced after six days of treatment. CONCLUSIONS: High concentrations of retinoids were able to induce phenotypic changes (differentiation) and late apoptosis in pancreatic cancer cells in vitro. The clinical ramifications of these observations await further investigations.


Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/fisiopatologia , Retinoides/farmacologia , Anidrases Carbônicas/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Neoplasias Pancreáticas/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptor alfa de Ácido Retinoico , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/farmacologia
12.
Cancer Biol Ther ; 4(4): 474-83, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15908778

RESUMO

BACKGROUND: The anticancer ability of natural retinoids on pancreatic adenocarcinoma, an aggressive tumor, is still controversial. This investigation tested the hypothesis that all-trans retinoic acid can inhibit proliferation and induce apoptosis in pancreatic cancer cell lines. MATERIALS AND METHODS: Using our previously optimized conditions, the effect of all-trans retinoic acid (atRA, 0.001-10 microM) was tested in ten human pancreatic adenocarcinoma cell lines with various degrees of differentiation. Proliferation was monitored by cell number, [3H]-thymidine incorporation and cell cycle arrest. Apoptosis was investigated morphologically by light and electron microscopy and biochemically by tissue transglutaminase activity (TGase), mitochondrial membrane potential, cell cycle analysis of sub-G1 cells and detection of fragmented DNA (fragmentation of prelabeled DNA, agarose electrophoresis and TUNEL assays). RESULTS: Retinoic acid caused potent concentration- and time-dependent inhibition of proliferation of all cell lines studied. Cell cycle was arrested at G1 or G2 with extensive reduction of number of cells at S-phase after 24 hours of treatment with apoptotic concentration of atRA. Complete inhibition of proliferation was followed by apoptosis as indicated by the progressive accumulation of sub-G1 apoptotic cells which was confirmed by the more specific DNA fragmentation assays. There were extensive apoptosis-indicative light and electron microscopic changes preceded by phenotypic redifferentiation. TGase was induced between 3-5-fold the control level and its inhibition partially reversed the antiproliferative effect of atRA. Cellular viability during the preapoptotic stage was confirmed by normal mitochondrial membrane potential in the first two days of treatment with the maximum atRA concentration used. However, the potential was progressively reduced with time as a preapoptotic change. Caspase 3-like activity was induced by the apoptotic concentrations of atRA at late time points. However, the redifferentiation indicative changes were not prevented by cotreatment with Ac-DEVE-CHO caspase 3 inhibitor. CONCLUSIONS: Together, our results demonstrated the efficient anticancer ability of natural retinoids on human pancreatic cancer cell lines tested, even those previously reported to be retinoid resistant.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Retinoides/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/fisiologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/ultraestrutura , Fatores de Tempo
13.
Transplantation ; 79(10): 1332-7, 2005 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-15912100

RESUMO

INTRODUCTION: Bone marrow cells expressing foreign MHC antigens survive poorly after transplantation. Stable mixed hematopoietic chimerism requires reconstitution with a relatively large number of foreign bone marrow cells and intensive depletion of host cells. In addition, when foreign MHC-transduced autologous bone marrow cells are transplanted, prolonged hematopoietic transgene expression requires extensive host conditioning. The competitive disadvantage associated with engraftment of donor cells expressing foreign MHC antigens is thought to result from a defect in engraftment secondary to donor-host incompatibility or immunologic resistance by the host. METHODS: We used a limiting-dilution competitive repopulation assay with cells from HLA-A2.1 transgenic mice to determine whether and to what extent foreign MHC antigen expression impairs engraftment in C57BL/6 hosts. Transplants were performed with Hoechst 33342 fluorescence-sorted side population (SP) cells, a subset of bone marrow enriched for stem cells. RESULTS.: Transplantation with 250 stem cell-enriched HLA-A2.1-transgenic side population cells successfully competed with nearly 5000 host C57BL/6 side population cells to produce stable long-term mixed chimerism. There was a direct relationship between the number of transplanted donor HLA-A2-expressing cells and the percentage of HLA-A2-expressing cells in the peripheral blood of reconstituted C57BL/6 mice (r2=0.1799, P=0.031). This correlation was maintained in secondary transplant recipients. CONCLUSIONS: HLA-A2-expressing hematopoietic cells do not have an engraftment defect when transplanted into C57BL/6 hosts and immunologic resistance did not limit chimerism following lethal irradiation. These results may have relevance to understanding long-term gene expression after hematopoietic stem cell based gene therapy.


Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Antígeno HLA-A2/metabolismo , Transplante de Células-Tronco Hematopoéticas , Doadores de Tecidos , Animais , Células Sanguíneas/metabolismo , Antígeno HLA-A2/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Período Pós-Operatório , Quimeras de Transplante , Imunologia de Transplantes , Tolerância ao Transplante
14.
Transplantation ; 79(8): 882-8, 2005 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-15849539

RESUMO

BACKGROUND: Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance. METHODS: Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions. RESULTS: After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice. CONCLUSIONS: Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.


Assuntos
Células da Medula Óssea/metabolismo , Expressão Gênica/genética , Sobrevivência de Enxerto , Lentivirus/genética , Transplante de Pele , Transdução Genética , Transgenes/genética , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Chlorocebus aethiops , Citocinas/metabolismo , Citocinas/farmacologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Masculino , Camundongos , Fatores de Tempo
15.
Front Biosci ; 9: 3145-55, 2004 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15353344

RESUMO

While most of the investigations into the causative events in the development of alcoholic liver disease (ALD) have been focused on multiple factors, increasing interest has centered around the possible role of immune mechanisms in the pathogenesis and perpetuation of ALD. This is because many of the clinical features of ALD suggest that immune effector mechanisms may be contributing to liver tissue damage, as evidenced by the detection of circulating autoantibodies, and the presence of CD4+ and CD8+ lymphoid cells in the livers of patients with ALD. One mechanism that has been associated with the development of autoimmune responses is the modification (haptenation or adduction) of liver proteins with aldehydes or other products of oxidative stress. This is because it has been shown that these adducted proteins can induce specific immune responses, to the adduct, the adduct plus protein (conformational antigens), as well as the unmodified parts of the protein. More importantly, it is possible to demonstrate that adducted self-proteins can induce reactivity to the normal self-protein and thereby induce autoimmune responses. Therefore, it is the purpose of this manuscript to outline the mechanism(s) by which these modified self proteins can induce autoimmune reactivity, and thus play a role in the development and/or progression of ALD.


Assuntos
Aldeídos/metabolismo , Autoimunidade , Hepatopatias Alcoólicas/metabolismo , Receptores Depuradores/metabolismo , Acetaldeído/química , Aldeídos/química , Animais , Apoptose , Doenças Autoimunes/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Morte Celular , Progressão da Doença , Haptenos/química , Hepatite/patologia , Humanos , Sistema Imunitário/patologia , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Malondialdeído/química , Necrose , Estresse Oxidativo , Tolerância a Antígenos Próprios
16.
Transplantation ; 76(5): 877-81, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-14501873

RESUMO

Nonmyeloablative allogeneic stem-cell transplantation (alloNST) is the focus of investigations searching for less-toxic transplantation regimens. We report studies on the kinetics of lymphodepletion and safety of pentostatin (PT) conditioning in alloNST. Patients with hematologic malignancy received mobilized blood from human leukocyte antigen-matched related (n=4) or unrelated (n=8) donors. PT 4 mg/m2 was administered on days -21, -20, and -19 and 200 cGy of total-body irradiation was administered on day -1, followed by cyclosporine A and mycophenolate mofetil. Mononuclear cell adenosine deaminase after PT was inhibited 84%. The absolute CD3+ cells decreased significantly by day -7 (49%) and CD19+ cells declined 92% by day -1. CD4+ cells were depressed more than CD8+ cells. Neutrophils and monocytes were minimally affected by PT. Median posttransplant peripheral blood chimerism on day 70 showed 95% donor leukocytes and 82.5% donor CD3 lymphocytes. PT demonstrated lymphodepleting effects and promising safety, supporting alloNST as early as 7 days after initiation of PT.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Imunossupressores/administração & dosagem , Pentostatina/administração & dosagem , Transplante de Células-Tronco , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Projetos Piloto , Transplante Homólogo
17.
Invest Ophthalmol Vis Sci ; 44(6): 2764-73, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12766085

RESUMO

PURPOSE: The present study describes a method for isolating neural stem cells/progenitors directly from the freshly dissociated embryonic retina (prospective identification) and compares their characteristics with those enriched from mitogen-exposed embryonic retinal cell culture. METHODS: Cell dissociates from embryonic rat retina and mitogen-exposed embryonic retinal cultures were stained with Hoechst 33342 fluorescent dye. The emission patterns of cells were analyzed in both blue and red wavelength using flow cytometry to enrich cells that retained or excluded the dye. The phenotype characteristics and differentiation potential of enriched cells were analyzed by immunocytochemical, RT-PCR, and electrophysiological analyses. RESULTS: The Hoechst dye efflux assay identified a minor population of cells, called side population (SP) cells, in fresh retinal dissociates. These cells that preferentially excluded the Hoechst 33342 fluorescent dye were proliferative and expressed both neural progenitor and retinal progenitor markers. The retinal SP cells generated functional neurons and glia and possessed the ability to differentiate along lineages of different late-born retinal cell types. Cells of similar phenotypes and potential were observed in the SP obtained from mitogen-exposed retinal culture. CONCLUSIONS: The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.


Assuntos
Benzimidazóis , Corantes Fluorescentes , Retina/citologia , Células-Tronco/citologia , Animais , Biomarcadores/análise , Técnicas de Cultura de Células , Separação Celular , Técnicas de Cocultura , Eletrofisiologia , Embrião de Mamíferos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Microscopia de Fluorescência , Fenótipo , Gravidez , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Verapamil/farmacologia
18.
Ann Surg ; 237(2): 265-72, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12560785

RESUMO

OBJECTIVE: To assess the use of donor pigs with cellular chimerism for prevention of acute rejection with modest immune suppression. The clinical use of pig organ xenografts is currently precluded by severe acute rejection, which resists standard immune suppression. SUMMARY BACKGROUND DATA: For long-term survival of pig organ xenografts, immune suppression significantly greater than used with allografts would currently be necessary, leaving the recipient immune deficient and at increased risk for infections. Induction of immune tolerance and tissue accommodation could enhance xenograft survival but would lead to complications and frequent graft failure. Induction of cellular chimerism within the donor pigs, however, could accomplish these goals before transplantation, significantly reducing the risk. METHODS: Marrow cells from sheep were infused into fetal pigs. Heart xenografts from chimeric or nonchimeric pigs were transplanted heterotopically into recipient sheep, simultaneous with infusion of splenocytes. Posttransplant suppression consisted of cyclosporine and tapered corticosteroids, comparable with allotransplants. RESULTS: All of the control grafts (n = 12) were rejected by acute vascular rejection in 4 to 8 days. In contrast, only one episode of vascular rejection was observed in the experimental group (n = 13). Four experimental recipients had an episode of moderate diffuse cellular rejection (grade 3) and one had moderate focal cellular rejection (grade 2). Each episode responded to pulse steroids. Seven grafts showed no significant rejection. There was little evidence of immune deficiency, infection, or toxicity. CONCLUSIONS: Acute vascular rejection was prevented in a large animal model without the need for severe immune suppression.


Assuntos
Transplante de Tecido Fetal/imunologia , Transplante de Coração/imunologia , Quimeras de Transplante/genética , Quimeras de Transplante/imunologia , Tolerância ao Transplante/genética , Tolerância ao Transplante/imunologia , Transplante Heterólogo/imunologia , Doença Aguda , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/uso terapêutico , Transplante de Medula Óssea , Ciclosporina/uso terapêutico , Transplante de Tecido Fetal/métodos , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/prevenção & controle , Coração/embriologia , Transplante de Coração/patologia , Síndromes de Imunodeficiência/etiologia , Síndromes de Imunodeficiência/prevenção & controle , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Hemissuccinato de Metilprednisolona/uso terapêutico , Modelos Animais , Ovinos , Suínos , Transplante Heterotópico
19.
In Vivo ; 16(6): 541-50, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12494899

RESUMO

BACKGROUND: Cord blood mononuclear cells (MNC) are a rich source of precursor cytotoxic effector cells. Earlier we have shown that interleukin-2 (IL-2)-activated MNC from cord blood have significant cytotoxic activity against human leukemia and breast cancer cells in vitro and in vivo, compared to MNC from peripheral blood. MATERIALS AND METHODS: In order to further improve the antitumor cytotoxic ability of cord blood MNC, IL-2 was combined with IL-15 and colony stimulating factors GMCSF, G-CSF and M-CSF for the activation. The activated cells were examined for their cytotoxic effects in vitro against human breast cancer cell lines MDA-231, MDA453 and SKB43 and in vivo against MDA-231 grown in SCID mice. Phenotypes of these activated cells were determined using flow cytometry. The expression of immune response related genes in activated cells was measured using RT-PCR techniques. RESULTS: There was a significant increase in cytotoxicity of the effector cells activated with IL-2, IL-15 and some colony stimulating factors compared to cells activated with each of these cytokines alone or other combinations. Our results demonstrated the increase in cytotoxicity appears to be due to: 1) increase in CD56-positive cytotoxic cells; 2) cytokine/cytotoxic factors produced by the effector cells, such as Interferon-7 and Perforin; 3) stimulation by accessory cells, such as dendritic cells. In vivo administration of in vitro-activated cord blood cells into SCID mice bearing MDA-231 tumors reduced the number of metastases and increased survival compared to untreated tumor bearing controls. CONCLUSION: The combination of IL-2 with IL-15 and CSF is better for the activation of cord blood effector cells than to IL-2 alone.


Assuntos
Neoplasias da Mama/imunologia , Fatores Estimuladores de Colônias/farmacologia , Sangue Fetal/imunologia , Interleucina-15/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Animais , Neoplasias da Mama/terapia , Citotoxicidade Imunológica , Combinação de Medicamentos , Proteína Ligante Fas , Feminino , Sangue Fetal/citologia , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Fígado/efeitos dos fármacos , Fígado/patologia , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Transplante de Neoplasias , Perforina , Proteínas Citotóxicas Formadoras de Poros , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
20.
Mol Reprod Dev ; 63(3): 309-17, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12237946

RESUMO

Embryonal carcinoma (EC) cells are recognized as an excellent model system for studying the early stages of mammalian development. Many studies performed with EC cells involve transient transfection with promoter/reporter gene constructs and/or mammalian expression vectors. One of the limitations of working with EC cells is their inability to be transfected at high efficiency. In most cases, EC cells are transfected using the calcium phosphate method. The objective of this study was to identify protocols and culture conditions that significantly increase the transfection efficiency of EC cells. F9 EC cells were used for this purpose, because they are the EC cell line studied most commonly. We show that the transfection efficiency of F9 EC cells using the calcium phosphate method is less than 5%; whereas, their transfection efficiency can be improved approximately 15-fold using optimized culture conditions and liposome-based transfection reagents. Specifically, we demonstrate that more than 50% of F9 EC cells can be transfected using LipofectAMINE 2000. In addition to higher levels of transfection, there is much less plate-to-plate variation with liposome-based reagents as compared to transfection with calcium phosphate. Interestingly, transfection efficiency using these reagents was found to be inversely related to cell density. This contrasts sharply with the recommendation that transfection with LipofectAMINE 2000 or LipofectAMINE in conjunction with the PLUS reagent be performed at high cell densities. Given the improvements in transfection efficiency reported here, it will now be possible to perform studies with F9 EC cells that require transfection at significantly higher levels than that achieved using the calcium phosphate method. Overall, the highest transfection efficiencies were consistently obtained using LipofectAMINE 2000.


Assuntos
Portadores de Fármacos , Vetores Genéticos , Lipossomos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco de Carcinoma Embrionário , Genes Reporter , Transfecção
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