RESUMO
PURPOSE: Helical Tomotherapy (HT) appears as a valuable technique for total body irradiation (TBI) to create highly homogeneous and conformal dose distributions with more precise repositioning than conventional TBI techniques. The aim of this work is to describe the technique implementation, including treatment preparation, planning and dosimetric monitoring of TBI delivered in our institution from October 2016 to March 2019. MATERIAL AND METHOD: Prior to patient care, irradiation protocol was set up using physical phantoms. Gafchromic films were used to assess dose distribution homogeneity and evaluate imprecise patient positioning impact. Sixteen patients' irradiations with a prescribed dose of 12Gy were delivered in 6 fractions of 2Gy over 3 days. Pre-treatment quality assurance (QA) was performed for the verification of dose distributions at selected positions. In addition, in-vivo dosimetry was carried out using optically stimulated luminescence dosimeters (OSLD). RESULTS: Planning evaluation, as well as results of pre-treatment verifications, are presented. In-vivo dosimetry showed the strong consistency of OSLD measured doses. OSLD mean relative dose differences between measurement and calculation were respectively +0,96% and -2% for armpit and hands locations, suggesting better reliability for armpit OSLD positioning. Repercussion of both longitudinal and transversal positioning inaccuracies on phantoms is depicted up to 2cm shifts. CONCLUSION: The full methodology to set up TBI protocol, as well as dosimetric evaluation and pre-treatment QA, were presented. Our investigations reveal strong correspondence between planned and delivered doses shedding light on the dose reliability of OSLD for HT based TBI in-vivo dosimetry.
Assuntos
Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia de Intensidade Modulada/métodos , Irradiação Corporal Total/métodos , Fracionamento da Dose de Radiação , Transplante de Células-Tronco Hematopoéticas , Humanos , Linfoma Anaplásico de Células Grandes/terapia , Linfoma de Células T/terapia , Posicionamento do Paciente/métodos , Imagens de Fantasmas , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Radiometria/métodos , Reprodutibilidade dos Testes , Condicionamento Pré-Transplante/métodosRESUMO
Using molecular simulation, we determine the critical properties of Si as well as the loci for several remarkable thermodynamic contours spanning the supercritical region of the phase diagram. We consider a classical three-body potential as well as a quantum (tight-binding) many-body model, and determine the loci for the ideality contours, including the Zeno line and the H line of ideal enthalpy. The two strategies (classical or quantum) lead to strongly asymmetric binodals and to critical properties in good agreement with each other. The Zeno and H lines are found to remain linear over a wide temperature interval, despite the changes in electronic structure undergone by the fluid along these contours. We also show that the classical and quantum model yield markedly different results for the parameters defining the H line, the exponents for the power-laws underlying the line of minima for the isothermal enthalpy and for the density required to achieve ideal behavior, most notably for the enthalpy.
RESUMO
The human HIV transactivator protein Tat is essential for efficient viral transcription that occurs by a complex mechanism involving interaction of Tat with the TAR RNA element. This interaction appears to require the mediation of a cellular protein, cyclin T1. However, the possibility that Tat and TAR associate in a binary Tat-TAR complex has been little investigated. Using a chemically synthesized active Tat protein, the kinetic and equilibrium parameters of its interaction with TAR were determined by surface plasmon resonance technology. Independently of partner and method of immobilization onto the sensor chip, the association (k(a) = 5-9 x 10(5) M(-1) s(-1)) and dissociation rate constants (k(d) = 1.7-4.3 x 10(-3) s(-1)) yielded similar equilibrium dissociation constants (K(d) = 2-8 nM). A truncated peptide encompassing residues 30-86 of Tat did not bind to TAR at all. We conclude that Tat can form a high-affinity complex with TAR in the absence of cyclin T1 and that the N-terminal domain of Tat is essential for this interaction, suggesting a conformational link between this domain and the basic domain of Tat. These results are important in our quest for developing therapeutic compounds that impair viral replication.
Assuntos
Produtos do Gene tat/metabolismo , RNA Viral/metabolismo , Produtos do Gene tat/química , Humanos , Imobilização , Cinética , Análise Serial de Proteínas , Ligação Proteica/fisiologia , RNA Viral/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estreptavidina/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
The Tat regulatory protein of human immunodeficiency virus type 1 (HIV-1) is secreted by infected cells and plays a key role in viral pathogenesis and replication. Tat protein has been proposed as a target antigen for vaccine design since anti-Tat antibodies may interfere with virus spread and disease progression. The aim of this study was to analyse the serum antibody response of mice, rabbits, macaques and humans immunized with recombinant Tat, synthetic Tat, Tat toxoid or Tat peptides and to examine the biological properties of these antibodies in terms of Tat-induced transactivation and HIV-1 replication. Only sera with antibody specificity to both N-terminal and basic functional domains were able to inhibit extracellular Tat-dependent transactivation significantly in vitro. Antibodies from a human subject immunized with Tat also reduced HIV-1 replication in acutely infected T cells and blocked reactivation of virus replicating low levels in chronically infected cells by exogenous Tat. These results demonstrate that immunization with Tat protein or a combination of synthetic Tat peptides elicits the production of Tat-neutralizing serum antibodies and suggest that Tat vaccination could be used to block in vivo extracellular Tat autocrine/paracrine transactivation of HIV-1 replication.
Assuntos
Vacinas contra a AIDS/imunologia , Epitopos de Linfócito B , Produtos do Gene tat/imunologia , Anticorpos Anti-HIV/sangue , HIV-1/imunologia , Animais , Repetição Terminal Longa de HIV , HIV-1/fisiologia , Humanos , Imunização , Macaca , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Ativação Transcricional , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
More than 25 years after their discovery, monoclonal antibodies are now the most rapid expanding pharmaceutical viable drugs in clinical trials. The emergence of these antibodies was made possible by the development of genetic recombinant techniques. It is now possible to obtain engineered antibodies: chimearic or humanized or fully human monoclonal antibodies via the use of phage display technology or of transgenic mice. These antibodies are tolerable to the human immune system and eleven have been approved for therapeutic by the US Food and Drug Administration (FDA), the majority of them in the past four years. At least an additional 400 monoclonal antibodies are in clinical trials to treat cancer, transplant rejection or to combat autoimmune or infectious diseases. Important advances have been made in the design of highly specific fragment antibodies, fused or not with drugs or radioisotopes, and in the large industrial scale production with different expression systems (bacteria, yeasts, mammalian cells and transgenic plants and animals). In the next future new molecular promising strategies will enhance affinity, stability and expression levels and reduce the price of these engineering monoclonal to permit their use to treat a large number of diseases.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Rejeição de Enxerto/tratamento farmacológico , Humanos , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Estados Unidos , United States Food and Drug AdministrationRESUMO
Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Sialoglicoproteínas/genética , Fator de Transcrição AP-1/metabolismo , Uridina Trifosfato/farmacologia , Angiotensina II/farmacologia , Animais , Aorta Torácica/citologia , Sítios de Ligação/genética , Northern Blotting , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Osteopontina , Fator de Crescimento Derivado de Plaquetas/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.
Assuntos
Movimento Celular/fisiologia , Espaço Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Nucleotídeos/farmacologia , Sialoglicoproteínas/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Aorta , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Cultura em Câmaras de Difusão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Osteopontina , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Difosfato de Uridina/farmacologia , Uridina Trifosfato/farmacologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.
Assuntos
Proteínas Fúngicas/genética , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Escherichia coli , Proteínas Fúngicas/química , Guanosina/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Purina-Núcleosídeo Fosforilase/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The aims of the present study were to determine the prevalence of human herpesvirus type 8 (HHV-8) in HIV-positive Brazilian patients with (HIV+/KS+) and without Kaposi's sarcoma (HIV+/KS-) using PCR and immunofluorescence assays, to assess its association with KS disease, to evaluate the performance of these tests in detecting HHV-8 infection, and to investigate the association between anti-HHV-8 antibody titers, CD4 counts and staging of KS disease. Blood samples from 66 patients, 39 HIV+/KS+ and 27 HIV+/KS-, were analyzed for HHV-8 viremia in peripheral blood mononuclear cells by PCR and HHV-8 antigenemia for latent and lytic infection by immunofluorescence assay. Positive samples for latent nuclear HHV-8 antigen (LNA) antibodies were titrated out from 1/100 to (1/4)09,600 dilution. Clinical information was collected from medical records and risk behavior was assessed through an interview. HHV-8 DNA sequences were detected by PCR in 74.3% of KS+ patients and in 3.7% of KS- patients. Serological assays were similar in detecting anti-LNA antibodies and anti-lytic antigens in sera from KS+ patients (79.5%) and KS- patients (18.5%). HHV-8 was associated with KS whatever the method used, i.e., PCR (odds ratio (OR) = 7.4, 95% confidence interval (CI) = 2.16-25.61) or anti-LNA and anti-lytic antibodies (OR = 17.0, 95%CI = 4.91-59.14). Among KS+ patients, HHV-8 titration levels correlated positively with CD4 counts (rho 0.48, P = 0.02), but not with KS staging. HHV-8 is involved in the development of KS in different geographic areas worldwide, as it is in Brazil, where HHV-8 is more frequent among HIV+ patients. KS severity was associated with immunodeficiency, but no correlation was found between HHV-8 antibody titers and KS staging.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/virologia , Soropositividade para HIV/virologia , Herpesvirus Humano 8/isolamento & purificação , Sarcoma de Kaposi/virologia , Adulto , Anticorpos Antivirais/sangue , Brasil , Intervalos de Confiança , Estudos Transversais , Feminino , Imunofluorescência , Humanos , Masculino , Razão de Chances , Reação em Cadeia da Polimerase , Estatísticas não ParamétricasRESUMO
The aims of the present study were to determine the prevalence of human herpesvirus type 8 (HHV-8) in HIV-positive Brazilian patients with (HIV+/KS+) and without Kaposi's sarcoma (HIV+/KS-) using PCR and immunofluorescence assays, to assess its association with KS disease, to evaluate the performance of these tests in detecting HHV-8 infection, and to investigate the association between anti-HHV-8 antibody titers, CD4 counts and staging of KS disease. Blood samples from 66 patients, 39 HIV+/KS+ and 27 HIV+/KS-, were analyzed for HHV-8 viremia in peripheral blood mononuclear cells by PCR and HHV-8 antigenemia for latent and lytic infection by immunofluorescence assay. Positive samples for latent nuclear HHV-8 antigen (LNA) antibodies were titrated out from 1/100 to 1/409,600 dilution. Clinical information was collected from medical records and risk behavior was assessed through an interview. HHV-8 DNA sequences were detected by PCR in 74.3 percent of KS+ patients and in 3.7 percent of KS- patients. Serological assays were similar in detecting anti-LNA antibodies and anti-lytic antigens in sera from KS+ patients (79.5 percent) and KS- patients (18.5 percent). HHV-8 was associated with KS whatever the method used, i.e., PCR (odds ratio (OR) = 7.4, 95 percent confidence interval (CI) = 2.16-25.61) or anti-LNA and anti-lytic antibodies (OR = 17.0, 95 percentCI = 4.91-59.14). Among KS+ patients, HHV-8 titration levels correlated positively with CD4 counts (rho 0.48, P = 0.02), but not with KS staging. HHV-8 is involved in the development of KS in different geographic areas worldwide, as it is in Brazil, where HHV-8 is more frequent among HIV+ patients. KS severity was associated with immunodeficiency, but no correlation was found between HHV-8 antibody titers and KS staging
Assuntos
Humanos , Masculino , Feminino , Infecções Oportunistas Relacionadas com a AIDS/virologia , Herpesvirus Humano 8/isolamento & purificação , Sarcoma de Kaposi/virologia , Anticorpos Antivirais/sangue , Brasil , Intervalos de Confiança , Estudos Transversais , Imunofluorescência , Razão de Chances , Reação em Cadeia da Polimerase , Estatísticas não ParamétricasRESUMO
A 32-basepair deletion polymorphism in the CCR5 chemokine receptor gene (CCR5Delta32) has been identified and shown to have functional significance in determining susceptibility to infection by human immunodeficiency virus type 1 (HIV-1) and possibly in influencing disease progression in HIV-1 positive individuals. These findings led to an interest in studies of DeltaCCR5 allele geographical distribution in human population, for complete understanding of the role of CCR5 in HIV-1 epidemiology. Inter-population variation in CCR5Delta32 frequency may be a significant factor in the prediction of AIDS endemicity. In this report we assessed the frequency of DeltaCCR5 in a Chilean population (63 HIV-1 infected and 62 non-infected individuals). No homozygous CCR5Delta32 individual was identified, and no significant difference was observed between HIV-1 infected (3/63) and non-infected (3/62) individuals for the heterozygote CCR5Delta32 state. This is the first evidence of the contribution of DeltaCCR5 allele to the genetic background of the Chilean population, which is characterized by intense ethnic admixture and by gene flow from the European Spanish gene pool.
Assuntos
Infecções por HIV/genética , HIV-1 , Receptores CCR5/genética , Deleção de Sequência , Chile , HumanosRESUMO
Sequencing of the Saccharomyces cerevisiae genome revealed an open reading frame (YJR105w) encoding a putative protein highly similar to adenosine kinases from other species. Disruption of this gene (renamed ADO1) affected utilization of S-adenosyl methionine (AdoMet) as a purine source and resulted in a severe reduction of adenosine kinase activity in crude extracts. Furthermore, knock-out of ADO1 led to adenosine excretion in the medium and resistance to the toxic adenosine analogue cordycepin. From these data we conclude that ADO1 encodes yeast adenosine kinase. We also show that ADO1 does not play a major role in adenine utilization in yeast and we propose that the physiological role of adenosine kinase in S. cerevisiae could primarily be to recycle adenosine produced by the methyl cycle.
Assuntos
Adenosina Quinase/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Adenosina/metabolismo , Sequência de Aminoácidos , Desoxiadenosinas/farmacologia , Resistência a Medicamentos , Dados de Sequência Molecular , Mutação , Fenótipo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Terminologia como AssuntoRESUMO
Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.
Assuntos
Arteriosclerose/metabolismo , Calcinose/metabolismo , Osteocalcina/metabolismo , Osteonectina/metabolismo , Sialoglicoproteínas/metabolismo , Túnica Média/metabolismo , Angioplastia com Balão/efeitos adversos , Animais , Aorta/metabolismo , Doenças da Aorta/etiologia , Doenças da Aorta/metabolismo , Calcinose/etiologia , Imuno-Histoquímica , Masculino , Microscopia Confocal , Osteopontina , CoelhosRESUMO
In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca(2+) sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y(1), P2Y(2), P2Y(4), or P2Y(6) receptor subtypes. Our data demonstrate that P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.
Assuntos
Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Purinérgicos P2/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células 3T3 , Actinas/fisiologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Citoesqueleto/fisiologia , Ativação Enzimática/fisiologia , Fibroblastos/metabolismo , Membranas Intracelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Nucleotídeos/farmacologia , Proteínas Serina-Treonina Quinases/fisiologia , Agonistas do Receptor Purinérgico P2 , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Vasoconstrição/fisiologia , Quinases Associadas a rhoRESUMO
A human lymphoid cell line (F172-D8) excreting a human immunodeficiency virus type 1 (HIV-1) anti-gp41 monoclonal antibody was used to construct a plasmid containing the cDNA of the single-chain variable fragment (scFvD8) corresponding to this antibody. A stable human osteosarcoma cell line was obtained which expressed the scFvD8 protein in the cytoplasm. Whereas a cell line transfected with a control construct (pCI-neo) was readily and productively infected with laboratory (Ba-L) or primary HIV-1 isolates, the scFvD8 cell line did not support productive infection. Binding of the virus, internalization, and reverse transcription were not altered by scFvD8 expression, but gp160 expression was dramatically reduced. These data suggest that cytoplasmic expression of this artificial single-chain antibody can interfere with gp160 expression, thereby reducing the production of mature viral envelope proteins.
Assuntos
Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Engenharia de Proteínas , Linhagem Celular , Sequência Conservada , Citoplasma/química , Epitopos/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Testes de Neutralização , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Células Tumorais Cultivadas , Replicação ViralRESUMO
Infection with human immunodeficiency virus type 1 (HIV-1) has been shown to elicit a serum antibody response with neutralizing activity against T cell line-adapted HIV strains and primary HIV-1 isolates. Mucosal surfaces are the primary route of HIV-1 infection. Evidence is presented here for the presence of HIV-neutralizing antibodies in secretions. Infection of mucosal cells with HIV stimulates systemic and mucosal immune responses and results in the generation of neutralizing antibodies. Serum IgG and IgA neutralize HIV-1MN infection of susceptible T cell lines; serum IgG inhibits more effectively. Mucosal IgA purified from parotid saliva of HIV-1-seropositive individuals could neutralize both a T cell line-adapted strain and a primary isolate. The neutralizing activity of IgA was not directed against the anti-third-variable-loop or the anti-ELDKWA epitope. Thus, the specificity of mucosal IgA for HIV-1 neutralization epitopes remains to be determined and may provide insight into development of a mucosal vaccine.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Anticorpos Anti-HIV/análise , Infecções por HIV/imunologia , Soropositividade para HIV/imunologia , HIV-1/imunologia , Imunoglobulina A Secretora/análise , Glândula Parótida/imunologia , Síndrome da Imunodeficiência Adquirida/sangue , Adulto , Feminino , Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , Soropositividade para HIV/sangue , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Testes de NeutralizaçãoRESUMO
We have compared two types of plasmids for DNA immunization against HTLV-I envelope glycoproteins. One type of plasmid contains the coding DNA of the complete envelope gene of HTLV-I under the control of the CMV promoter with (CMVenvLTR) or without (CMVenv) the tax/rex genes. The second type contains the coding DNA of the complete env gene of HTLV-I under the control of the human desmin muscle specific promoter (DesEnv). These plasmids were inoculated into mice and the humoral response was studied by flow cytometry, ELISA and neutralization assays. Inoculation of the DesEnv construct elicited a higher humoral response with better neutralization properties than the injection of CMVenvLTR or CMVenv plasmids. The choice of vectors will be important for the design of genetic HTLV-I vaccines.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/imunologia , Músculos/metabolismo , Vacinas de DNA/imunologia , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Feminino , HIV-1/imunologia , Células HeLa , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Proteínas Recombinantes/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genéticaRESUMO
Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A(1)/A(2)-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P(1) purinoceptor subtypes, only alloxazine, an antagonist of A(1)- and A(2)-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate, which are A(1)-, A(2a)-, and A(3)-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A(2b)-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A(2b)-adenosine receptor and involves a cAMP-dependent pathway.
Assuntos
Adenosina/fisiologia , Apoptose/fisiologia , Artérias/fisiologia , Espaço Extracelular/metabolismo , Músculo Liso Vascular/fisiologia , Receptores Purinérgicos P1/fisiologia , Artérias/citologia , Artérias/metabolismo , Células Cultivadas , AMP Cíclico/fisiologia , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Receptor A2B de Adenosina , Receptores Purinérgicos P1/metabolismoRESUMO
Many adult T-cell leukaemia/lymphoma (ATLL) patients who respond to induction treatment, then relapse. Knowing the clonality pattern of residual tumourous clones during treatment could help understand disease evolution and aid therapeutic decisions. We developed a sensitive and semi-quantitative molecular analysis of these clones in ATLL patients. DNA samples from PBMCs derived from eight ATLL patients were studied over time by quadruplicate linker mediated PCR (LMPCR) amplification of HTLV-1 integration sites. Patients were treated with combination chemotherapy, zidovudine-interferon-alpha and/or by peripheral stem cell transplantation or allogeneic bone marrow transplantation. Persistence of tumourous clones at a high frequency (>1/300 PBMCs) was frequently observed, even in complete responders, and was invariably correlated with relapse and/or poor outcome. Fluctuation in the frequency of some tumourous clones was observed with evidence for clonal change under treatment in one patient, indicating that treatment of ATLL can result in the selection of resistant clones. Finally, allogeneic bone marrow transplantation (BMT) using an HTLV-1 infected sibling as donor was found to be associated with long-lasting disappearance of tumourous clones and a possible cure of the disease. Long-term persistent clonal expansion of circulating HTLV-1 bearing T cells which derived from the donor bone marrow was evidenced in this patient. In conclusion, variable success in treatment of ATLL is probably due to the clonal heterogeneity which results in the selection of resistant clones. Semi-quantitative assessment of residual disease (RD) through LMPCR may predict treatment failure. Accordingly, additional therapy may be tailored to the clonality pattern observed after first-line therapy.