RESUMO
BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano , Vacinas de DNA , Camundongos , Humanos , Animais , Linfócitos T CD8-Positivos , Granzimas/genética , Fator de Necrose Tumoral alfa , Vacinas de DNA/genética , Proteínas Virais/metabolismo , Vaccinia virus/genética , DNA , Fatores de Transcrição de Zíper de Leucina Básica , Proteínas dos Retroviridae/genéticaRESUMO
The cervical spine is part of the axial skeleton and is responsible for protecting vital structures, such as the spinal cord and the vertebral arteries and veins. Traumatic injury to the cervical spine occurs in approximately 3% of blunt trauma injuries, and approximately 80% are below the level of C2. The AO Spine society divides the spine into four segments: the upper cervical spine (C0-C2), subaxial spine (C3-C7), thoracolumbar spine, and sacral spine. Various classifications have been proposed for the subaxial segment since that of Allen and Ferguson in 1982; however, none is universally accepted, and treatment remains controversial. The complex anatomy and biomechanics of the subaxial spine and the lack of a widely accepted classification system make these injuries difficult to evaluate on imaging. The Subaxial Injury Classification System (SLIC) uses fracture morphology, the integrity of discoligamentous complex, and neurological status to score the patient and determine between operative and non-operative management; however, other factors may influence management, such as time for immobilisation, osteoporosis, surgeon's experience, and hospital circumstances. SLIC classifies fracture morphology in a crescent order of severity based on Allen and Ferguson's classification. Compression fractures are the simpler ones, while both distraction injuries and translation/rotation are severe injuries, which are always associated with some degree of discoligamentous complex (DLC) injury. This article will review the indications for imaging, the basis of the SLIC classification, the different types of fracture morphology, evaluation of the DLC, and other features important in decision making in subaxial spine trauma.
Assuntos
Vértebras Cervicais/lesões , Traumatismos da Coluna Vertebral/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Traumatismos da Coluna Vertebral/classificação , Traumatismos da Coluna Vertebral/etiologia , Tomografia Computadorizada por Raios XRESUMO
The liver plays an important role in the clearance, by receptor-mediated endocytosis, of circulating glycoproteins. It has been demonstrated that tissue kallikreins, which are acid glycoproteins, circulate in plasma, where they are poorly inhibited by plasma proteins. We have shown that the liver is the main organ that clears tissue kallikreins from the circulation. We now report the identification of receptors involved in this clearance. Using a perfused rat-liver system, and as models, pig pancreatic (PPK) and horse urinary (HoUK) kallikreins, we have found that: (a) the binding of PPK to the perfused liver was inhibited by 50 mM methyl alpha-D-mannoside and 20 microM mannan, was partially inhibited by 50 mM mannose and was unaffected by 1.5 microM asialofetuin; (b) binding of HoUK to the perfused liver was inhibited by 1.5 microM asialofetuin, 50 mM galactose and 50 mM lactose and was unaffected by 50 mM mannose; (c) the clearance rate of both kallikreins followed the equation y = a.xb; (d) their binding was Ca2+-dependent and their clearance was inhibited by 3 mM chloroquine and 10 mM methylamine. Using isolated liver cells and tritiated HoUK, we calculated that 500,000 receptors/cell were present and the Scatchard plot showed that there were two apparent affinity constants: 0.24.10(9) l/M) (high-affinity) and 0.3.10(8) l/M (low-affinity). These results show that PPK is recognized by a liver mannose receptor and HoUK by the galactose receptor. The liver uptake of native and circulating tissue kallikreins thus emerges as a mechanism by which their levels in plasma are regulated.
Assuntos
Endocitose , Calicreínas/sangue , Fígado/metabolismo , Receptores Imunológicos/análise , Animais , Receptor de Asialoglicoproteína , Separação Celular , Cavalos , Calicreínas/fisiologia , Cinética , Fígado/fisiologia , Especificidade de Órgãos , Perfusão , Ratos , Receptores Imunológicos/fisiologia , SuínosRESUMO
A four-step procedure was used to purify rat plasma kallikrein (RPK) with a relative molecular mass (Mr) of 87 kD (obtained both by gel filtration and SDS-PAGE), which indicates the purification of an alpha (intact) kallikrein, in contrast to previously described RPK preparations which had lower Mr (beta or degraded form). RPK is a neutral (pI 6.7) serine proteinase glycoprotein (15% carbohydrates) and contains (residues/mol): galactose (27), N-acetylglucosamine (24), mannose (13), glucose (13) and fucose (7). This purified alpha form of RPK has properties very similar to those of pure human and bovine kallikreins.
Assuntos
Calicreínas/isolamento & purificação , Animais , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Calicreínas/sangue , Conformação Molecular , RatosRESUMO
Previous studies have shown that perfused rat liver in situ is able to clear recirculating rat plasma kallikrein (RPK) in two phases: an initial clearance lasting a few minutes, followed by a slow exponential phase. Using purified RPK preparations we now show that: RPK is a glycoprotein; clearance was inhibited by human serum against blood group B and 0.1 M melibiose but was not affected by human serum against blood group A, 0.1 M lactose, 0.1 M mannose, 0.05 M N-acetyl galactosamine, 0.05 M galactose or 15 microM asialofetuin. Prolonged incubation of RPK with alpha-galactosidase reduced RPK clearance. Oligosaccharide structures in RPK may have terminal galactose units since treatment of RPK with neuraminidase did not affect the clearance rate; RPK clearance occurs in the absence of added Ca2+, with either EDTA or EGTA in the perfusion fluid; the exponential phase is reversibly inhibited by the addition of NH4Cl or chloroquine to the perfusion fluid. This observation, along with experiments using liver homogenates, suggests that RPK catabolism is carried out by lysosomal enzymes, probably cathepsin B of possible hepatocyte origin.
Assuntos
Calicreínas/sangue , Fígado/metabolismo , Animais , Soros Imunes/farmacologia , Taxa de Depuração Metabólica/efeitos dos fármacos , Perfusão , Ratos , alfa-Galactosidase/farmacologia , beta-Galactosidase/farmacologiaRESUMO
An isolated rat liver perfusion model was used to study the effects of acute exposure of the organ to either ethanol or a molasses distillate (cachaça). When ethanol (72 mM) or a molasses distillate (68 mM ethanol) was added to the perfusion fluid, lysosomal injury was indicated by the increased release of tartrate-inhibited acid phosphatase activity at the end of a 3 h period of perfusion. Other cellular compartments were not significantly damaged in these acute experiments, as judged by the release of aspartate and alanine aminotransferases, lactate dehydrogenase and alkaline phosphatase. The behavior of both ethanol itself and the alcoholic beverage was similar as far as enzyme release is concerned but only the molasses distillate caused significant acidosis (a decrease in perfusate pH) at the end of a 3 h period of perfusion. These data may be of importance for a better understanding of the hepatic damage caused by alcohol abuse and useful for laboratory investigation of alcohol intoxication.