Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

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.

Subaxial spine trauma: radiological approach and practical implications.

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.

Decoupled ecomorphological evolution and diversification in Neogene-Quaternary horses.

Evolutionary theory has long proposed a connection between trait evolution and diversification rates. In this work, we used phylogenetic methods to evaluate the relationship of lineage-specific speciation rates and the mode of evolution of body size and tooth morphology in the Neogene and Quaternary radiation of horses (7 living and 131 extinct species). We show that diversification pulses are a recurrent feature of equid evolution but that these pulses are not correlated with rapid bursts in phenotypic evolution. Instead, rapid cladogenesis seems repeatedly associated with extrinsic factors that relaxed diversity bounds, such as increasing productivity and geographic dispersals into the Old World. This evidence suggests that diversity dynamics in Equinae were controlled mainly by ecological limits under diversity dependence rather than rapid ecomorphological differentiation.

Cross resistance to accase herbicide in Lolium rigidum.

Lolium rigidum is a cross-pollinating grass weed present in Europe and occurring in winter wheat and orchard crops. Several graminicides such as chlorotoluron and/or isoproturon and diclofop-methyl in mixtures or alone have been used successfully to control this weed in Spain during the past decade. However, several L rigidum populations have developed resistance to these herbicides following selection due to their continuous use. Four resistance mechanisms have been found in this grass weed, an enhanced metabolic detoxiflcation of the herbicides and an insensitive isoform of ACCase being the most important ones. The extent of cross-resistance depends on the type of mechanism. The biotype with an enhanced metabolic detoxification showed cross-resistance to ACCase-, ALS-, PSII- and tubuline-inhibiting herbicides, while the biotype resistant due to a mutation of the target site (ACCase) presented cross-resistance to ACCase-inhibiting herbicides only.

Identification of receptors in the liver that mediate endocytosis of circulating tissue kallikreins.

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.

Purification and characterization of the alpha form of rat plasma kallikrein.

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.

The liver is the main organ to clear plasma and tissue kallikreins from rat plasma, in vivo.

We report observations regarding the in vivo distribution of labelled kallikreins in plasma, liver and some other organs, twenty minutes following their intravenous injection in the rat. The kallikreins used were: tritiated homogeneous human plasma (HuPK) and horse urinary (HoUK) as well as highly purified iodinated rat plasma kallikrein (RPK). The main findings were: the liver cleared 15% of HuPK, 38% of RPK and 69% HoUK; with both types (plasma and tissue) of native kallikreins the liver was the main clearing organ.

Receptor-mediated clearance of tissue kallikreins by rat liver.

The exsanguinated, isolated and perfused rat liver clears from the perfusate, at comparable rates, some native tissue kallikreins: human and horse urinary as well as hog pancreatic; the clearance rates were dependent on the initial enzyme concentration in the perfusing fluid. Contrary-wise, rat urinary kallikrein was cleared at negligible rates. Neuraminidase pretreatment of these four kallikreins did not alter their clearance rates. Horse urinary kallikrein binding to isolated prefixed hepatocytes was calcium-dependent and inhibited by asialofetuin (but not by fetuin) and some sugars; these characteristics are compatible with the interpretation that this native tissue kallikrein is recognized by the hepatocyte asialoglycoprotein-receptor. It was calculated that there are about 300,000 receptor sites per cell either using perfusion experiments at 4 degrees C or isolated hepatocytes.

Rat plasma kallikrein clearance by perfused rat liver.

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.

Kinin-inactivating endopeptidase from rat liver.

A kinin-inactivating serine-endopeptidase from rat liver was purified to an activity of 912 mU/mg of protein, when measured on bradykinin. The endopeptidase molecular weight, estimated by gel filtration, was 68,000. Its isoelectric point was close to pH 4.9. Vm for the hydrolysis of bradykinin, was 1.25 mumol/min/mg protein; Km was 28 microM. The two hydrolysis products from bradykinin were the pentapeptide Arg1-Phe5 and the tetrapeptide Ser6-Arg9.

Lysosome injury by perfusion of the isolated rat liver with ethanol or a molasses distillate (cachaça).

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.

Acute turpentine inflammation and kinin release in rat-paw thermic oedema.

Livers from rats at 2-3 days after s.c. injection of turpentine, when perfused, synthesized prekallikrein nearly 3 times faster than did livers from normal rats. On the other hand paw oedema, produced by heating to 46 degrees, in rats injured in this way was less marked. Likewise in such rats the amount of bradykinin release by 50 min. of coaxial perfusion of the paw was only 13.6 +/- 4.6 compared with 63.1 +/- 13.4 ng in normal rats. A possible explanation for the observed reduction in production of bradykinin may be inhibition of kallikrein due to an increased concentration of alpha 2-macroglobulin.