Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Janus Quinase 2/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Transtornos Mieloproliferativos/diagnóstico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Trombocitose/induzido quimicamente , Idoso de 80 Anos ou mais , Substituição de Aminoácidos/fisiologia , Antineoplásicos/uso terapêutico , Benzamidas , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/complicações , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/genética , Fenilalanina/genética , Trombocitose/genética , Valina/genéticaRESUMO
We report one case of unilateral ovarian Sertoli-Leydig tumor with intermediate grade and heterologous element, in a young woman with clinical and biological typical presentation. We discuss 2D and 3D ultrasonographic and doppler features.
Assuntos
Neoplasias Ovarianas/diagnóstico por imagem , Tumor de Células de Sertoli-Leydig/diagnóstico por imagem , Ultrassonografia Doppler/métodos , Adulto , Feminino , Humanos , Imageamento Tridimensional , Neoplasias Ovarianas/patologia , Tumor de Células de Sertoli-Leydig/patologiaRESUMO
BACKGROUND: The solitary fibrous tumor (SFT) is a spindle-cell tumor that very rarely involves the orbit. We report a new case that we compare to reports in the literature. CASE: A 72-year-old woman presented a conjunctival inflammation of the right eye developing over 5 months with progressive proptosis. Magnetic resonance imaging revealed an extraconal and homogeneous mass, which showed hypointensity on T1-weighted images and hyperintensity on T2-weighted images, without specificity. Histological examination of the lesion removed by anterior orbitotomy confirmed the diagnosis of the SFT of the orbit. The patient was doing well without recurrence after 9 months. DISCUSSION: The diagnosis of SFT is histological. It is a mesenchymal tumor. Immunohistochemically, the tumor cells are strongly positive for CD34 and vimentin. CONCLUSION: The SFT of the orbit is a very rare and generally benign tumor. It must be immunohistochemically differentiated from other spindle-cell tumors of the orbit. The treatment is a complete surgical excision, and long-term follow-up is necessary because recurrence may appear long after treatment.
Assuntos
Exoftalmia/etiologia , Neoplasias Orbitárias/complicações , Idoso , Feminino , Humanos , Neoplasias Orbitárias/patologiaRESUMO
Inhibition of human immunodeficiency virus type 1 reverse transcriptase (RT) by both nucleoside and nonnucleoside RT inhibitors profoundly inhibits virus replication. Nucleoside RT inhibitors are known to be toxic, but there is little information regarding the toxicities of nonnucleoside RT inhibitors (NNRTI). We demonstrate that efavirenz (an NNRTI) induces caspase- and mitochondrion-dependent apoptosis of Jurkat T cells and human peripheral blood mononuclear cells. The clinical relevance of these observations is not yet clear.
Assuntos
Apoptose/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Oxazinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Alcinos , Anexina A5 , Benzoxazinas , Caspases/fisiologia , Ciclopropanos , Inibidores Enzimáticos , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Células Jurkat , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologiaRESUMO
By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.
Assuntos
Antígenos CD8/análise , Células Dendríticas/fisiologia , Interferon gama/biossíntese , Interleucina-12/fisiologia , Linfócitos T Citotóxicos/fisiologia , Animais , Linfócitos T CD4-Positivos/fisiologia , Ligante de CD40/fisiologia , Feminino , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaRESUMO
Because the persistence of human immunodeficiency virus (HIV) in cellular reservoirs presents an obstacle to viral eradication, we evaluated whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induces apoptosis in such reservoirs. Lymphocytes and monocyte-derived macrophages (MDM) from uninfected donors do not die following treatment with either leucine zipper human TRAIL (LZhuTRAIL) or agonistic anti-TRAIL receptor antibodies. By contrast, such treatment induces apoptosis of in vitro HIV-infected MDM as well as peripheral blood lymphocytes from HIV-infected patients, including CD4(+) CD45RO(+) HLA-DR(-) lymphocytes. In addition, LZhuTRAIL-treated cells produce less viral RNA and p24 antigen than untreated controls. Whereas untreated cultures produce large amounts of HIV RNA and p24 antigen, of seven treated CD4(+) CD45RO(+) HLA-DR(-) cell cultures, viral RNA production was undetectable in all, p24 antigen was undetectable in six, and proviral DNA was undetectable in four. These data demonstrate that TRAIL induces death of cells from HIV-infected patients, including cell types which harbor latent HIV reservoirs.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Infecções por HIV/imunologia , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Terapia Antirretroviral de Alta Atividade , Proteínas Reguladoras de Apoptose , Linfócitos T CD4-Positivos/fisiologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Memória Imunológica , Células Jurkat , Macrófagos/fisiologia , RNA Viral/análise , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/análise , Receptores do Fator de Necrose Tumoral/fisiologia , Ligante Indutor de Apoptose Relacionado a TNFRESUMO
Although treatment with combination antiretroviral therapy leads to a reduction in the level of plasma viremia and an improvement in CD4 T cell count for most patients, for a minority of patients, an improvement in CD4 T cell count occurs despite the failure of treatment to suppress viral replication. Recent reports suggest that these discordant improvements in CD4 T cell count may last for months to years and are associated with improved clinical outcomes. In a retrospective observational study, we evaluated the effect of therapy cessation on 8 patients with discordant immunologic responses to therapy and found that improved CD4 T cell responses are dependent upon ongoing drug pressure. If antiretroviral agents that are likely to resuppress the virus are not available, we suggest that patients continue the therapy associated with immunologic improvement to maximize the clinical benefit of the discordant response.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Esquema de Medicação , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Carga ViralRESUMO
Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.
Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Anilidas/farmacologia , Inibidores Enzimáticos/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Neoplasias do Córtex Suprarrenal , Glândulas Suprarrenais/enzimologia , Sítios de Ligação , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de Lipoproteínas/metabolismo , Esteroides/metabolismo , Esterol O-Aciltransferase/metabolismo , Células Tumorais CultivadasRESUMO
Infection with the human immunodeficiency virus (HIV) is associated with a progressive decrease in CD4 T-cell number and a consequent impairment in host immune defenses. Analysis of T cells from patients infected with HIV, or of T cells infected in vitro with HIV, demonstrates a significant fraction of both infected and uninfected cells dying by apoptosis. The many mechanisms that contribute to HIV-associated lymphocyte apoptosis include chronic immunologic activation; gp120/160 ligation of the CD4 receptor; enhanced production of cytotoxic ligands or viral proteins by monocytes, macrophages, B cells, and CD8 T cells from HIV-infected patients that kill uninfected CD4 T cells; and direct infection of target cells by HIV, resulting in apoptosis. Although HIV infection results in T-cell apoptosis, under some circumstances HIV infection of resting T cells or macrophages does not result in apoptosis; this may be a critical step in the development of viral reservoirs. Recent therapies for HIV effectively reduce lymphoid and peripheral T-cell apoptosis, reduce viral replication, and enhance cellular immune competence; however, they do not alter viral reservoirs. Further understanding the regulation of apoptosis in HIV disease is required to develop novel immune-based therapies aimed at modifying HIV-induced apoptosis to the benefit of patients infected with HIV.
Assuntos
Apoptose , Infecções por HIV/imunologia , HIV/imunologia , Linfócitos/fisiologia , Linfócitos/virologia , Linfócitos B/patologia , Linfócitos B/fisiologia , Linfócitos B/virologia , Infecções por HIV/patologia , Humanos , Linfócitos/patologia , Macrófagos/patologia , Macrófagos/fisiologia , Macrófagos/virologia , Linfócitos T/patologia , Linfócitos T/fisiologia , Linfócitos T/virologiaRESUMO
Metabolites of AZT can inhibit HIV-1 integrase in vitro (Mazumder A, et al., Proc Natl Acad Sci USA 1994;91:5771-5775). To determine if long-term dideoxynucleoside therapy can lead to the emergence of HIV-1 AZT-resistant variants containing mutations in the integrase, we have sequenced the proviral DNA encoding the HIV-1 integrase of nine HIV-1-infected patients at different time points during treatment. Four of the nine patients developed mutations during the course of treatment. Although most mutations occurred at nonconserved amino acids, one patient developed a mutation at codon (R166T), a residue that is conserved among all integrases from known HIV-1 isolates. This mutation was introduced in the recombinant HIV-1 integrase protein to determine if it could confer resistance to AZT in vitro. We show that the R166T integrase mutant is still proficient at carrying 3'-processing and 3' end-joining but that the enzyme is not resistant to AZT-TP. Our results suggest that it is unlikely that integrase inhibition contributes to the antiviral activity of AZT.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , Integrase de HIV/genética , HIV-1/efeitos dos fármacos , Mutação , Inibidores da Transcriptase Reversa/farmacologia , Zidovudina/farmacologia , Sequência de Aminoácidos , Fármacos Anti-HIV/uso terapêutico , DNA Viral/análise , Resistência Microbiana a Medicamentos , Infecções por HIV/tratamento farmacológico , Integrase de HIV/química , HIV-1/enzimologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Provírus , Inibidores da Transcriptase Reversa/uso terapêutico , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Tempo , Zidovudina/uso terapêuticoRESUMO
Genetic immunization through ex vivo transduction of dendritic cells has been suggested as an effective approach to enhance antitumor immunity by activating both CD4+ and CD8+ T cells. Immunizing mice with dendritic cells transduced with an adenovirus expressing the human melanoma antigen glycoprotein 100 (DCAdhgp100) as a cancer vaccine, we demonstrated complete protective immunity and a potent CTL response against melanomas expressing murine glycoprotein 100 in a CD4+ cell-dependent manner. Surprisingly, however, effective tumor rejection was not the result of cooperation between CD4+ and CD8+ T cells. Protective immunity was completely lost when CD4+ cells were depleted immediately before tumor challenge, whereas it was unaffected by removal of CD8+ cells, establishing a principal role for CD4+ cells in the effector phase of tumor rejection. Neither protective immunity nor CTL generation in this model required interleukin 12, in spite of high levels of IFN-gamma secretion by tumor-reactive T cells. Most notably, the DCAdhgp100 vaccine could elicit protective antitumor CD4+ cells in the absence of CD40 ligand, although it does not bypass the need for CD40-mediated signals to generate melanoma-reactive CTLs. Thus, in contrast to the current thinking that the optimal cancer vaccine should include determinants for both CD4+ and CD8+ cells, the potency of the DCAdhgp100 vaccine appears to be a result of its ability to directly prime autoreactive CD4+ cells through a process that does not require interleukin 12 and CD40 signals.
Assuntos
Autoimunidade , Vacinas Anticâncer , Células Dendríticas/imunologia , Interleucina-12/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Adenoviridae/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40 , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos , Humanos , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Baço/imunologia , Linfócitos T Citotóxicos/imunologia , Fatores de TempoRESUMO
Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.
Assuntos
Dano ao DNA , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Ribossômico/metabolismo , Camptotecina/farmacologia , DNA/biossíntese , DNA de Cadeia Simples , Inibidores Enzimáticos/farmacologia , Células HT29 , Humanos , Cinética , Família Multigênica , Fosforilação , Inibidores da Topoisomerase IRESUMO
Apolipoproteins of high density lipoprotein (HDL) and especially apolipoprotein (apo)AI and apoAII have been demonstrated as binding directly to the class B type I scavenger receptor (SR-BI), the HDL receptor that mediates selective cholesteryl ester uptake. However, the functional relevance of the binding capacity of each apolipoprotein is still unknown. The human adrenal cell line, NCI-H295R, spontaneously expresses a high level of SR-BI, the major apoAI binding protein in these cells. As previously described for murine SR-BI, free apoAI, palmitoyl-oleoyl-phosphatidylcholine (POPC)-AI, and HDL are good ligands for human SR-BI. In vitro displacement of apoAI by apoAII in HDLs or in Lp AI purified from HDL by immunoaffinity enhances their ability to compete with POPC-AI to bind to SR-BI and also enhances their direct binding capacity. The next step was to determine whether the higher affinity of apoAII for SR-BI correlated with the specific uptake of cholesteryl esters from these HDLs. Free apoAII and, to a lesser extent, free apoAI that were added to the cell medium during uptake experiments inhibited the specific uptake of [(3)H]cholesteryl esters from HDL, indicating that binding sites on cells were the same as cholesteryl ester uptake sites. In direct experiments, the uptake of [(3)H]cholesteryl esters from apoAII-enriched HDL was highly reduced compared with the uptake from native HDL. These results demonstrate that in the human adrenal cell line expressing SR-BI as the major HDL binding protein, efficient apoAII binding has an inhibitory effect on the delivery of cholesteryl esters to cells.
Assuntos
Apolipoproteína A-II/metabolismo , Ésteres do Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos/metabolismo , Receptores de Lipoproteínas , Neoplasias do Córtex Suprarrenal/metabolismo , Animais , Apolipoproteína A-I/farmacologia , Apolipoproteína A-II/farmacologia , Ligação Competitiva , Antígenos CD36 , Humanos , Lipoproteínas/química , Lipoproteínas/metabolismo , Lipoproteínas HDL/química , Camundongos , Fosfatidilcolinas/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Trítio , Células Tumorais CultivadasRESUMO
CC10/uteroglobin is a remarkable protein whose physiological roles have only recently been explored in vivo. Both transgenic mice that have been rendered deficient and humans that have been characterized as deficient in this protein exhibit tendencies toward inflammatory, fibrotic, and oncologic disease, demonstrating the potential of the protein as a therapeutic agent. The protein itself is an excellent candidate for clinical development because of its inherent physical properties. It is relatively small, resistant to proteases, stable to extremes of heat and pH, and can be produced by recombinant methods. The physiological roles of this multifunctional protein continue to be uncovered as research progresses in vitro, in animals, and eventually in humans. The pathways through which CC10 mediates its effects, its receptors, and other family members will be a rich source of exciting research, as well as potential diagnostic and therapeutic agents. This paper is an introductory, noncomprehensive review of some of the scientific and medical rationale in support of CC10-based therapies in selected clinical applications.
Assuntos
Pneumonia/tratamento farmacológico , Proteínas/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Uteroglobina , Doença Aguda/terapia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Asma/fisiopatologia , Displasia Broncopulmonar/tratamento farmacológico , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/fisiopatologia , Doença Crônica/terapia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Humanos , Recém-Nascido , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Nefropatias/fisiopatologia , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/fisiopatologia , Diálise Renal/efeitos adversos , Síndrome do Desconforto Respiratório do Recém-Nascido/tratamento farmacológico , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/fisiopatologiaAssuntos
Adesão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Fibronectinas/efeitos dos fármacos , Fibronectinas/metabolismo , Proteínas/metabolismo , Proteínas/farmacologia , Uteroglobina , Animais , Ligação Competitiva/fisiologia , Adesão Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Camundongos , Pneumonia/metabolismo , Pneumonia/fisiopatologiaRESUMO
In rodents, cholesterol for adrenal steroidogenesis is derived mainly from high-density lipoproteins (HDL) via the HDL receptor, scavenger receptor-BI (SR-BI). In humans cholesterol for steroidogenesis is considered to be derived from the low-density lipoprotein (LDL) receptor pathway, and the contribution of SR-BI to that is unknown. In the present study SR-BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortical cells and compared with LDL receptor expression. In addition, the functional contribution of both receptors for cholesteryl ester delivery to human adrenocortical cells was compared. Northern blot and reverse transcription-PCR amplification and sequence analysis demonstrated the presence of SR-BI mRNA in foetal and adult human adrenal cortex. Furthermore, SR-BI mRNA was expressed to similar levels in human primary adrenocortical and adrenocortical carcinoma NCI-H295 cells, indicating its presence in the steroid-producing cells. Treatment of NCI-H295 cells with 8Br-cAMP, a stimulator of glucocorticoid synthesis via the protein kinase A second messenger signal transduction pathway, resulted in an increase of both SR-BI and LDL receptor mRNA levels in a time- and dose-dependent manner. The induction of SR-BI and LDL receptor by cAMP was independent of ongoing protein synthesis and occurred at the transcriptional level. Ligand blot experiments indicated that a protein of similar size to SR-BI is the major HDL-binding protein in NCI-H295 cells. Western blot analysis demonstrated that cAMP treatment increased the levels of LDL receptor and, to a lesser extent, SR-BI protein in NCI-H295 cells. Binding and uptake of cholesterol was quantitatively smaller from HDL than from LDL, both in basal as well as in cAMP-stimulated cells. Scatchard analysis under basal conditions indicated that NCI-H295 cells express twice as many specific binding sites for LDL than for HDL. Dissociation constant values (Kd; in nm) were approximately five times higher for HDL than for LDL, indicating a lower affinity of HDL compared with LDL. The combined effects of these two parameters and the low cholesteryl ester content of HDL subfraction 3 (HDL3) contributes to a lower cholesteryl ester uptake from HDL than from LDL by the NCI-H295 cells. In conclusion, both the SR-BI and LDL receptor genes are expressed in the human adrenal cortex and coordinately regulated by activators of glucocorticoid synthesis. In contrast to rodents, in human adrenocortical cells the HDL pathway of cholesterol delivery appears to be of lesser importance than the LDL pathway. Nevertheless, the SR-BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway.
Assuntos
Antígenos CD36/genética , Proteínas de Membrana , Receptores Imunológicos , Receptores de LDL/genética , Receptores de Lipoproteínas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical , Sítios de Ligação , Colesterol/farmacologia , Ésteres do Colesterol/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Cinética , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Ligação Proteica/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Células Tumorais CultivadasRESUMO
Glycoprotein 100 (gp100) is one of a series of well-characterized human melanoma-associated antigens expressed by most melanoma cells. Immunization of C57BL/6 mice with an adenovirus (Ad) vector encoding human gp100 (Adhgp100) has been shown to induce limited protective immunity against challenge with murine melanoma B16 cells. In the current study we determined whether gp100-specific immunity can be enhanced using bone-marrow-derived dendritic cells (DCs) transduced with Adhgp100 ex vivo. Subcutaneous injection of Adhgp100-infected DCs resulted in potent T-cell-mediated protective immunity and a greater than 80% reduction of established tumors when administered to B16 tumor-bearing hosts. Compared to direct injection of Adhgp100 vector alone, immunization with Adhgp100-infected DCs induced markedly greater antitumor activity. In vitro CTL analysis demonstrated that DC-Adhgp100 immunization activated both CD4(+) and CD8(+) CTLs, while no lytic activity was generated by vaccination with Adhgp100 alone. In vivo depletion of CD4(+) T cells, but not CD8(+) T cells, completely abrogated CTL activity, suggesting that Adhgp100-transduced DCs result in activation of both CD4(+) and CD8(+) CTLs via a CD4(+)-dependent mechanism. We speculate that this improved efficacy of Adhgp100-transduced DCs compared to direct immunization with Adhgp100 may be the result of direct DC-mediated CD4(+) T cell activation. These results emphasize the importance of CD4(+) T cells in the development of therapeutic antigen-specific cancer vaccines.
Assuntos
Adenoviridae/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Melanoma/imunologia , Animais , Feminino , Técnicas de Transferência de Genes , Imunização , Interferon gama/biossíntese , Interleucina-4/biossíntese , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma/terapia , Camundongos , Linfócitos T Citotóxicos/imunologiaRESUMO
This study addresses the relationship of epitope-specific Ab responses and alternative autoantibody responses in a model system in which an antigenized self protein serves as the carrier for a defined heterologous B cell epitope. Ubiquitin, a nonimmunogenic self protein, was engineered to present heterologous B and T cell epitopes in the recombinant molecule. Fusion to the C terminus introduced a universal T cell epitope from a Mycobacterium tuberculosis Ag. The B cell epitope was created by inserting a 12-residue loop sequence of HIV-1 gp120 at a surface-exposed position of ubiquitin. These modifications preserved the ubiquitin fold, allowing a new conformational epitope to be presented among native self epitopes. Mice immunized with the hybrid protein bearing only the mycobacterial T cell epitope elicited a strong autoantibody response to native ubiquitin. In contrast, antisera elicited against hybrid ubiquitin presenting the HIV B cell epitope reacted specifically with the foreign epitope but not with native ubiquitin. Absence of autoantibody in the response was attributed to poor competition of autoreactive B cells for limiting T cell help. Both types of responses were associated with Th responses to defined epitopes of the ubiquitin hybrid protein. These results may have implications for a tolerance mechanism dependent on B-T cell cooperation.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Autoanticorpos/biossíntese , Epitopos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteínas Recombinantes de Fusão/imunologia , Ubiquitinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Autoanticorpos/imunologia , Feminino , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-1/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Especificidade da Espécie , Ubiquitinas/genéticaRESUMO
HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3'-azido-3'-deoxythymidine (AZT) analog 3',5-diazido-2', 3'-dideoxyuridine 5'-monophosphate (5N3-AZTMP) and proteolytic mapping identified the amino acid 153-167 region of integrase as the site of photocrosslinking. Docking of 5N3-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.
Assuntos
Integrase de HIV/química , Integrase de HIV/metabolismo , HIV-1/fisiologia , Estrutura Secundária de Proteína , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Marcadores de Afinidade , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Sequência Conservada , Primers do DNA , Didesoxinucleotídeos , HIV-1/enzimologia , Ligação de Hidrogênio , Lisina , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Nucleotídeos de Timina/química , Replicação Viral , Zidovudina/química , Zidovudina/farmacologiaRESUMO
We used purified mammalian topoisomerases I (top1) and oligonucleotides containing a unique top1 cleavage site to study top1-mediated cleavage and recombination in the presence of nicks and short gaps mimicking DNA damage. In general, top1 cleavage was not induced opposite to the nicks, and nicks upstream from the top1 cleavage site suppressed top1 activity. Irreversible top1 cleavage complexes ("suicide products" or "aborted complexes") were produced in DNA containing nicks or short gaps just opposite to the normal top1 cleavage site. Camptothecin enhanced the formation of the aborted top1 complexes only for nicks downstream from the cleavage site. These aborted (suicide) complexes can mediate DNA recombination and promote illegitimate recombination by catalyzing the ligation of nonhomologous DNA fragments (acceptors). We report for the first time that top1-mediated recombination is greatly enhanced by the presence of a phosphate at the 5' terminus of the top1 aborted complex (donor DNA). By contrast, phosphorylation of the 3' terminus of the gap did not affect recombination. At concentrations that strongly enhanced inhibition of intramolecular religation, resulting in an increase of top1 cleavable complexes, camptothecin did not reduce recombination (intermolecular religation). Nicks or gaps with 5'-phosphate termini would be expected to be produced directly by ionizing radiations or by processing of abasic sites and DNA lesions induced by carcinogens or drugs used in cancer chemotherapy. Thus, these results further demonstrate that DNA damage can efficiently trap top1-cleavable complexes and enhance top1-mediated DNA recombination.