[Magnetic resonance spectroscopy data in the prognosis of consciousness recovery in patients with vegetative state]. / Prognozirovanie vosstanovleniia soznaniia u patsientov v vegetativnom sostoianii s primeneniem metoda vodorodnoi magnitno-rezonansnoi spektroskopii.

AIM: To study the prognostic value of magnetic resonance spectroscopy (MRS) in patients with vegetative state/unresponsive wakefulness syndrome (VS/UWS). MATERIAL AND METHODS: Thirty-four patients with VS/UWS underwent multi-voxel MRS (thalamus, globus pallidus, putamen, internal capsules, fornix, brainstem, temporal and frontal cortex). Subjects were grouped according to etiology: 22 patients with traumatic brain injury (TBI) (group 1) and 12 patients with a hypoxia (group 2). The groups were matched by age and duration of UWS (mean 2, 3 months). The CRS-R was used to identify the first signs of consciousness during hospitalization and 6-12 months later. Outcomes of the patients with TBI were as follows: chronic VS/UWS (n=6), minimally conscious state (MCS) plus (n=9), emergence from MCS (EMCS) (n=7). Outcomes of the patients with hypoxia were: chronic vegetative state (n=10), minimally conscious state (MCS) (n=2). RESULTS: The decrease in the NAA/Cr ratio in thalamus, capsula interna, temporal cortex are correlated with poor outcome in both groups. Higher rates of NAA/Cr in these structures are correlated with further recovery of consciousness. The decrease in the ratio of NAA Cr and NAA/NAA+Cho+Cr in the midbrain is correlated with poor outcome only in UWS with hypoxia. CONCLUSION: The results suggest that the MRS allows to more accurately predicting the outcome in VS/UWS patients with hypoxic brain damage, as well as in UWS patients with TBI, who have recovered consciousness to the level of EMCS.

Prenatal diagnosis of 22q11.2 deletion when ultrasound examination reveals a heart defect.

PURPOSE: The incidence of 22q11.2 deletion syndrome is approximately 1 in 5,000 births, and accounts for 5-30% of all heart defects, making it one of the more common genetic conditions in the population. METHODS: We employed fluorescence in situ hybridization (FISH) to study the incidence of 22q11.2 deletions in fetuses with cardiac anomalies detected on ultrasound examination. RESULTS: Of 64 cases, 18 had visible chromosome anomalies. FISH testing for 22q11.2 deletion was performed on the remaining 46 cases, and five exhibited a 22q11.2 deletion. Three of the five had de novo deletions, one was maternally inherited, and one family declined testing. CONCLUSION: FISH analysis for 22q11.2 deletion should be performed on all fetuses with cardiac defects (excluding hypoplastic left heart and echogenic focus) and a normal G-banded karyotype.

Melanosomal and lysosomal alterations in murine melanocytes following transfection with the v-rasHa oncogene.

Melanomas exhibiting mutated ras genes are frequently invasive and amelanotic. Transfecting melanocytes with ras oncogenes causes transformation and a loss of visible pigmentation. We analyzed murine melanocytes rendered amelanotic by transfection with the v-rasHa oncogene. Consistent with previous reports, tyrosinase and tyrosinase-related protein-1 (TRP-1) were not expressed by transformed cells. In addition, lack of expression of TRP-2 and the product of the silver locus was documented. Levels of melanosomal matrix antigens, the pink-eyed dilution locus protein and lysosome-associated membrane protein-1 were markedly reduced. Residual matrix antigens were localized by immunofluorescence to large vacuoles distributed peri-nuclearly in transfected cells. Electron microscopy demonstrated the absence of typical melanosomes and the presence of large vacuolar structures, also in a peri-nuclear distribution. Although levels of lysosomal hydrolases, such as beta-glucuronidase and cathepsin D, were diminished, marked elevations were observed in the expression of cathepsins B and L, 2 thiol proteases implicated in the acquisition of invasiveness. Our data demonstrate that transfection of melanocytes with v-rasHa is sufficient to disrupt the biogenesis of melanosomes and to up-regulate thiol protease synthesis, providing insights into the amelanotic and invasive nature of melanomas exhibiting mutations in ras genes.

Processing of a viral glycoprotein in the endoplasmic reticulum for class II presentation.

Endogenous processing of viral glycoproteins for presentation to CD4+T cells is a poorly investigated aspect of antigen processing and presentation. This pathway may involve not only pathogens, but also self proteins, and may thus be involved in self-tolerance. We have characterized the processing of the endoplasmic reticulum-restricted glycoprotein (G) of vesicular stomatitis virus, termed poison tail (Gpt), biochemically and enzymatically, and by T cell recognition assays. Expressed with a vaccinia vector, Gpt remains endoglycosidase H-sensitive and does not mature to endoglycosidase D sensitivity. The protein is degraded in the ER with a T1/2 of 4 h. Gpt peptides are not secreted since Gpt-infected cells are unable to sensitize uninfected antigen-presenting cells in an innocent bystander assay. Using flow cytometry, Gpt is undetectable on the plasma membrane; in contrast, wild-type G is readily found on the surface or secreted into the milieu as soluble G following infection of A20 cells with a vaccinia recombinant expressing G. The degradation of Gpt is sensitive to the thiol reagent diamide and occurs optimally at physiological pH. A series of proteolytic inhibitors were tested: 3,4-dichloroisocoumarin and 1-chloro-3-tosylamido-7-amino-2-heptanone inhibited degradation, which suggests the involvement of a serine protease. The degradation does not require transport to the Golgi complex, and is not sensitive to a variety of lysosomotropic agents. We show that the degradation products include the immunogenic epitopes recognized by a panel of T cell clones and hybridomas.

Destructive proteolysis by cysteine proteases in antigen presentation of ovalbumin.

Most native antigens require digestion by acidic proteases in order to be recognized in the context of major histocompatibility complex class II by T helper cells (Th). We have studied the roles of three different acidic proteases, cathepsin D, cathepsin B and cathepsin L, in the processing of ovalbumin (OVA) for presentation in the context of I-Ad. We report that digestion of OVA in vitro with the aspartyl protease cathepsin D generates the epitope OVA322-336, which is recognized by I-Ad-restricted OVA-specific Th in the presence of paraformaldehyde-fixed antigen-presenting cells (APC). In contrast, digestion of OVA with the cysteine proteases cathepsin B and L not only failed to generate an epitope, but also destroyed OVA322-336. In the presence of fixed APC expressing I-Ad. OVA322-336 was protected from destructive proteolysis by cathepsin L. These results illustrate the dependence of epitope selection on the intracellular proteolytic environment in APC, and suggest that mechanisms must exist for protection of epitopes from destructive proteolysis in the processing compartments.

Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells.

Melanosomes, the subcellular site of melanin synthesis and deposition, may be related to the endolysosomal lineage of organelles. To determine if melanosomes contain lysosomal hydrolases, we examined the subcellular distribution of five of these enzymes in melanocytes cultured from C57BL/6J mice. Analyses of Percoll gradient density centrifugations demonstrated that beta-hexosaminidase, beta-galactosidase, beta-glucuronidase, and cathepsins B and L all co-sedimented with tyrosinase-rich densely sedimenting melanosomes. The melanosomal distribution of these enzymes was confirmed in studies of melanocytes cultured from albino mice and of melanocytes rendered amelanotic by transfection with the v-rasHa oncogene (which lack dense, melanized melanosomes). In these cells, only a less dense peak of activity for each hydrolase was present. The level of each hydrolase was elevated in black cells when compared with albino cells. Metabolic labeling studies confirmed that the increase in beta-glucuronidase in black versus albino cells resulted mainly from increased synthesis of this enzyme. The data suggest that melanosomes represent specialized lysosomes present within melanocytes, that they contain a broad array of lysosomal hydrolases, and that the levels of these hydrolases are elevated in cells actively engaged in pigment production.

Selective processing of exogenous antigens by antigen-presenting cells with deleted MHC genes.

Recently, APCs expressing genes for the alpha- and beta-chains of MHC class II, but not other proteins encoded in the MHC class II locus, have been found to be defective in processing proteins by the MHC class II Ag presentation pathway. Ag presentation of hen egg OVA has been examined in one of these cell lines, T2.Ak. OVA was processed normally by T2.Ak when compared with OVA processed by T1.Ak, a cell line that expresses the MHC genes missing in T2.Ak. By contrast, native hen egg lysozyme (HEL) was not presented as Ag by T2.Ak, which is in agreement with earlier results. Digestion with thiol proteases is important for Ag processing of HEL. We therefore analyzed the expression of these enzymes in T1.Ak and T2.Ak by using a reagent that specifically radiolabels thiol proteases. In these experiments, the repertoire of proteases expressed in the microsomes of T1.Ak was found to be distinct from the repertoire expressed by T2.Ak. Finally, in in vitro digestion experiments, the group of thiol proteases active in T1.Ak microsomes digested HEL differently from the group identified in T2.Ak. These results provide evidence that the defect in Ag presentation that is encoded by MHC genes and manifests itself in defective processing of HEL is not absolute. Further, the mutation in T2.Ak coincides with altered activities of thiol proteases, a class of enzymes involved in processing exogenous Ags.

Role of cathepsin D in antigen presentation of ovalbumin.

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.

Immunolocalization of endosomal cathepsin D in rabbit alveolar macrophages.

Intravacuolar proteolysis appears to be an important component of antigen presentation, the activation of peptide hormones, and the conversion of biologically important mediators from inactive precursors. Cathepsin D has been identified in the endosomes of rabbit alveolar macrophages by biochemical analyses [Diment and Stahl, J. Biol. Chem. 260,15311, 1985; Diment et al., J. Biol. Chem. 263,6901, 1988]. Using affinity-purified goat antirabbit cathepsin D IgG, we have localized cathepsin D to the endosomes of rabbit alveolar macrophages. Immunofluorescent staining of frozen sections showed labeling in lysosomes and small vesicles in the periphery of the cell. Label was not seen on the plasma membrane. With immunoperoxidase labeling at the electron microscopic level on cells containing endocytosed mannose-BSA gold, we saw labeling in endosomes and classical lysosomes. When the results were quantitated using immunogold labeling of thin cryosections, we found that the majority of cathepsin D (62.2%) was present in lysosomes, 4.0% in large clear vacuoles, a surprisingly high percentage (29.3%) in small vesicles, 4.9% in endosomes, and none on the plasma membrane. We conclude from this study that, in addition to being present in lysosomes, cathepsin D is present in endosomes and in small peripheral vesicles.

Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin.

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.

Cleavage of parathyroid hormone in macrophage endosomes illustrates a novel pathway for intracellular processing of proteins.

Most ligands which are taken up by macrophages are transported to lysosomes where they are degraded to their constituents by a concert of acid hydrolases. This process requires a number of intracellular events which result in the transport of ligands from light density endosomes to the more dense lysosomes. In contrast, our studies have shown that macrophages may process some incoming ligands in endosomes (Diment, S., and Stahl, P. D. (1985) J. Biol. Chem. 260, 15311-15317) and that cathepsin D, an aspartyl protease, is localized in these organelles (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). Using rabbit alveolar macrophages, which can be subjected to subcellular fractionation, we have traced the intracellular transport and processing of bovine parathyroid hormone (PTH-(1-84]. We present evidence that macrophages internalize PTH-(1-84). Once in endosomes the hormone is cleaved to fragments which include a bioactive peptide, PTH-(1-34), and then the fragments are returned to the extracellular medium, without delivery to lysosomes. The entire cycle from initial binding to release of PTH-(1-34) is achieved within 10-15 min, a time period consistent with findings in vivo. Our data provide evidence for a novel route for processing of an endocytosed ligand.

Cathepsin D is membrane-associated in macrophage endosomes.

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.

Generation of macrophage variants with 5-azacytidine: selection for mannose receptor expression.

Mannose receptors are expressed only in primary macrophages. Established macrophage-derived cell lines, although apparently possessing the potential to synthesize mannose receptors, do not express them on their plasma membranes. Using the drug 5-Azacytidine, mannose receptor expression was induced in the macrophage-derived mouse cell line J774. Receptor positive cells were sorted through a fluorescent activated cell sorter (FACS) prior to cloning. Clones were isolated which continuously express mannose receptors in culture. These macrophages were able to endocytose beta-glucuronidase and phagocytose yeast particles via mannose receptors. Secretion of the lysosomal enzyme beta-hexosaminidase was also reduced in proportion to the degree of mannose receptor expression.

Macrophage endosomes contain proteases which degrade endocytosed protein ligands.

Rabbit alveolar macrophages rapidly internalize and degrade mannosylated bovine serum albumin (125I-mannose-BSA). Trichloroacetic acid-soluble degradation products appear in the cells as early as 6 min after uptake at 37 degrees C, and in the extracellular medium after 10 min. Incubation of endocytic vesicles containing this ligand in isotonic buffers at pH 7.4 + ATP resulted in intravesicular proteolysis, which was inhibited by monensin, nigericin, or ammonium chloride. At pH 5.0, degradation proceeded rapidly and was abolished by lysis of the vesicles with 0.1% Triton X-100. Readdition of lysosomes to the incubation mixture did not increase the rate of prelysosomal degradation. Proteolysis of 125I-mannose-BSA was optimal at pH 4.5, and inhibited by low concentrations of the cathepsin D inhibitor pepstatin A. After subcellular fractionation of the macrophages on Percoll gradients, 125I-mannose-BSA sedimented with prelysosomal vesicles and was not transported to secondary lysosomes. Addition of pepstatin A to extracellular medium during internalization of prebound 125I-mannose-BSA partially inhibited degradation of ligand, and resulted in transfer of undegraded 125I-mannose-BSA to lysosomes after 20 min. Using 125I-bovine serum albumin as a substrate for the protease in the presence of 0.1% Triton X-100, we have shown that as much as 36% of the total pepstatin A-sensitive activity sediments with nonlysosomal membranes. After intraendosomal iodination using lactoperoxidase, a labeled protease was isolated by affinity chromatography on pepstatin-agarose. The labeled protease, which had a subunit size of 46 kDa, was detected in endocytic vesicles after 5 min of internalization. These results suggest that a cathepsin D-like protease is responsible for the degradation of 125I-mannose-BSA in macrophages, and that this ligand is degraded in a prelysosomal vesicle.

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

Mannose-specific oligosaccharide recognition by mononuclear phagocytes.

Glycoproteins terminating in mannose are recognized by receptors on macrophages. The mannose receptor is expressed by a variety of macrophages but expression is closely regulated. Activated macrophages, for example, express little mannose receptor activity. Kinetic and fractionation experiments suggest that cell surface mannose receptors recycle to and from an acidic, pre-lysosomal compartment. Preliminary evidence suggests that the mannose receptor is a large polypeptide and that it is structurally related to the mannose binding protein found in serum. The mannose receptor may, among other possibilities, regulate the extracellular levels of lysosomal hydrolases.

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages.

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.