Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity.

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpss1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37% when the disaccharide Galpss1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ss-Gal-globotriaosylceramide (Galpss1-3Galpa1-4Galpss1-4Glc pss1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2% formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpss1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ss-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Selective distribution of the heparin in mammals: conspicuous presence of heparin in lymphoid tissues.

The systematic study on the distribution of heparin in 12 tissues of eight mammalian species is reported. It is shown that heparin varies from 1 microgram/g of dry tissue in cat liver up to 1000 micrograms/g of dry tissue in bovine ileum. Heparin was present in relatively high amounts in lung, ileum and skin, of most of the species analysed. It was also observed that heparin was conspicuously present in high amounts in thymus and lymph nodes of all the species except for rabbits. Conversely, heparin was not detected in brains of all the species and also in none of the rabbit tissues. Based on this characteristic distribution the possible physiological role of heparin is discussed.

Absence of heparin or heparin-like compounds in mast-cell-free tissues and animals.

Three models were used for the analysis of heparin concentration and the presence of mast cells, namely different fetal and adult bovine tissues, mast-cell-deficient mice and athymic mice. It was observed that heparin and mast cells are present mainly in spleen and liver during fetal development and in skin, lung and ileum in adults. A good correlation between the concentration of heparin and the number of mast cells was observed in all tissues examined. No heparin was detected in animals that did not have mast cells, such as the WBB6Fl W/Wv mice, again suggesting a correlation between mast cells and heparin. No differences in the other sulfated glycosaminoglycans were observed between the mast cell-deficient mice and the normal littermates and breeders. Studies in 'nude' mice have shown that the heparin concentration in different tissues is similar to normal strains.

Selective appearance of heparin in mammalian tissues during development.

The distribution of heparin in tissues of bovine and porcine fetuses of different ages as well as in the adult animals is reported. Heparin is present almost exclusively in the hematopoietic and lymphoid tissues of fetuses (spleen, liver, thymus and lymph nodes), whereas ileum, skin and lung besides lymph nodes and bovine thymus are the heparin-rich tissues in adult animals. The small amount of heparin in fetal tissues, its decrease in porcine liver and spleen, its appearance in skin, ileum and lung, tissues directly exposed to the environment after birth, its preferential localization at the maternal side of the placenta together with other data led to the suggestion that heparin may be involved with defense mechanisms.

Sphingolipids of the mycopathogen Sporothrix schenckii: identification of a glycosylinositol phosphorylceramide with novel core GlcNH(2)alpha1-->2Ins motif.

Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss-Y1, -Y2, and -Y6, respectively) have been isolated. By a combination of 1- and 2-D 1H-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and gas chromatography/mass spectrometry (GC/MS), Ss-Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Manalpha1-->3Manalpha1-->6GlcNH(2)alpha1-->2Ins1-P-1Cer (where Ins=myo-inositol, P=phosphodiester). While the GlcNH(2)alpha1-->6Ins1-P- motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH(2)alpha1-->2Ins1-P- has not been previously observed in any glycolipid. Ss-Y1 and Ss-Y2 were both found to have the known glycan structure Manalpha1-->3Manalpha1-->2Ins1-P-1Cer. Together with the results of a prior study [Toledo et al. (2001) Biochem. Biophys. Res. Commun. 280, 19-24] which showed that the mycelium form expresses GIPCs with the structures Manalpha1-->6Ins1-P-1Cer and Manalpha1-->3Manalpha1-->6Ins1-P-1Cer, these results demonstrate that S. schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core linkages.

Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1).

We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1.

Inhibition of macrophage invasion by monoclonal antibodies specific to Leishmania (Viannia) braziliensis promastigotes and characterisation of their antigens.

Monoclonal antibodies that specifically recognise Leishmania (Viannia) braziliensis promastigotes were produced and termed SST-2, SST-3 and SST-4. SST-2 recognises a conformational epitope present in a 24-28 kDa doublet and in a 72 kDa component, as verified by Western blotting. Indirect immunofluorescence showed that the antigen recognised by SST-2 is distributed homogeneously on the parasite surface. SST-3 recognises a flagellar glycoprotein of approximately 180 kDa. The reactivity of this mAb was abolished by sodium m-periodate treatment, indicating that SST-3 reacts with a carbohydrate epitope of the 180 kDa antigen. SST-4 recognises a conformational epitope of a 98 kDa antigen. SST-2, SST-3 and SST-4 were specific to L. (V.) braziliensis promastigote forms. Indirect immunofluorescence did not show reactivity of SST-2 or SST-3 with amastigotes of L. (V.) braziliensis, or with promastigotes of Leishmania (Viannia) panamensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Viannia) lainsoni, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major, or Leishmania (Leishmania) chagasi. We also evaluated the involvement of SST-2, SST-3 and SST-4 antigens in parasite-macrophage interaction. Fab fragments of SST-3 and SST-4 significantly inhibited the infectivity of L. (V.) braziliensis promastigotes to mouse peritoneal macrophages.

Identification of GM3 as a marker of therapy-resistant periradicular lesions.

The purpose of this study was to analyze the profile of glycosphingolipids (GSLs) in periradicular lesions refractory to endodontic treatment. Sixteen periapical lesions were removed surgically from patients (experimental group) and compared with 10 samples of periodontal ligament removed from extracted intact third molars (control group). After the GSLs extraction and purification procedures were performed the neutral and acidic GSL fractions were analyzed by high-performance thin-layer chromatography and quantified by densitometry. Data reported herein show that: (i) tissues in the experimental group presented about twice as much GSLs as the control group; (ii) lesion tissues express lactoneotetraosylceramide, and lactofucopentaosyl (IV) ceramide, whereas these neutral GSLs are absent in normal tissues; and (iii) normal tissues express GT1b, whereas lesions cells do not express this ganglioside. In contrast lesion tissues express GM3, which is conspicuously absent in normal tissues.

Production and characterization of monoclonal antibodies to shark cartilage proteoglycan.

1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10(6) Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller than PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and further characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteoglycans react with MST1, indicating that the antibody does not recognize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does not recognize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 and PG2 aggregate with hyaluronic acid.

Immunochemical characterization of carbohydrate antigens from fungi, protozoa and mammals by monoclonal antibodies directed to glycan epitopes.

Cell surface carbohydrates constitute the major antigenic determinants of fungi and protozoa. Glycoconjugates also represent a large variety of antigens or markers present in mammals such as histo-blood groups ABO, differentiation and heterophile antigens, among others. This article focuses on the general properties of glycoconjugate antigens and production and characterization of the anti-carbohydrate monoclonal antibodies (MoAbs). It describes the specificity and some properties of monoclonal antibodies directed against carbohydrate epitopes present in tumor-associated glycoproteins, in glycosaminoglycans of higher eukaryotes and in glycolipid antigens of protozoa and fungi. The epitopes recognized by the anti-carbohydrate MoAbs range from one sugar unit up to ten sugar units. Although most anti-carbohydrate MoAbs are directed predominantly toward terminal sugar residues, a few MoAbs are also reactive with internal sugar residues. The fine structure of the carbohydrate epitopes has been chemically defined by [1H] NMR, GC/MS of alditol acetates of partially permethylated compounds, -FAB/MS, degradation with exoglycosidases and inhibition with different methyl-glycosides and oligosaccharides.

Isolation and possible composition of glucosylceramides from Paracoccidioides brasiliensis.

Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated from mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose. Analysis of 1H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcp beta 1-->Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides). The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry. No glycosphingolipid other than glucosylceramide was detected in P. brasiliensis.

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo.

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.

Structural properties of lipid reconstructs and lipid composition of normotensive and hypertensive rat vascular smooth muscle cell membranes.

Multiple cell membrane alterations have been reported to be the cause of various forms of hypertension. The present study focuses on the lipid portion of the membranes, characterizing the microviscosity of membranes reconstituted with lipids extracted from the aorta and mesenteric arteries of spontaneously hypertensive (SHR) and normotensive control rat strains (WKY and NWR). Membrane-incorporated phospholipid spin labels were used to monitor the bilayer structure at different depths. The packing of lipids extracted from both aorta and mesenteric arteries of normotensive and hypertensive rats was similar. Lipid extract analysis showed similar phospholipid composition for all membranes. However, cholesterol content was lower in SHR arteries than in normotensive animal arteries. These findings contrast with the fact that the SHR aorta is hyporeactive while the SHR mesenteric artery is hyperreactive to vasopressor agents when compared to the vessels of normotensive animal strains. Hence, factors other than microviscosity of bulk lipids contribute to the vascular smooth muscle reactivity and hypertension of SHR. The excess cholesterol in the arteries of normotensive animal strains apparently is not dissolved in bulk lipids and is not directly related to vascular reactivity since it is present in both the aorta and mesenteric arteries. The lower cholesterol concentrations in SHR arteries may in fact result from metabolic differences due to the hypertensive state or to genes that co-segregate with those that determine hypertension during the process of strain selection.

Isolation of Leishmania (Viannia) braziliensis glycolipid antigens and their reactivity with mAb SST-1, specific for parasites of Viannia subgenus.

Specific glycolipids (GLs) from Leishmania (Viannia) braziliensis promastigotes were isolated and purified. A monoclonal antibody directed to carbohydrate epitopes of these GLs was produced. mAb SST-1 recognizes a low molecular weight GL as established by solid-phase radioimmunoassay and HPTLC immunostaining, and does not cross-react with lipophosphoglycan isolated from L. (V.) braziliensis promastigotes. An indirect immunofluorescence study indicated that the antigenic GLs are present at the L. (V.) braziliensis promastigote surface. SST-1 reacted with promastigotes of L. (V.) naiffi and L. (V.) guyanensis, but not with species in the L. Leishmania subgenus i.e. L. (L.) amazonensis, L. (L.) chagasi, or L. (L.) major. All L. (V.) braziliensis serodemes tested were reactive with SST-1. These results indicate that SST-1 recognizes specific GLs expressed by species of the Viannia subgenus, and will be particularly useful for identification of L. (V.) braziliensis.

Structural differences of heparan sulfates according to the tissue and species of origin.

Some structural features of thirteen heparan sulfates isolated from different mammalian tissues and species are reported. Two N-acetylated disaccharides, one of then O-sulfated and two N-sulfated disaccharides, one of then 6-sulfated are formed from these compounds by the combined action of heparitinases I and II from Flavobacterium heparinum. The relative proportions of the four disaccharide units vary quite significantly among the thirteen heparan sulfates indicating that the structure of these polymers are tissue and species specific. Based on the frequency of appearance of each one of the disaccharides it was calculated that 10(36) types of heparan sulfates might theoretically be found. The possible role of these polyanions in cell-cell recognition is discussed in view of the present findings.

A monoclonal antibody (ST-1) directed to the native heparin chain.

A mouse monoclonal antibody, ST-1, was raised against heparin complexed to Salmonella minnesota. Characterization of this antibody showed that it recognizes an epitope in the intact molecule of heparin that is present regardless of its source or anticoagulant activity. ST-1 is the first monoclonal antibody specific for the intact unmodified molecule of heparin to be described. 3H-labeled heparin in solution was immunoprecipitated by ST-1, and the formation of the 3H-labeled immunocomplex was selectively inhibited by unlabeled heparin. No cross-reactivity of ST-1 was observed with other glycosaminoglycans such as heparan sulfate, chondroitin sulfate, hyaluronic acid, dermatan sulfate, and keratan sulfate, or with polyanionic polymers such as dextran sulfate. Selective removal of the N-sulfate groups or N,O-desulfation of heparin strongly reduced the binding of ST-1. Inhibition of binding was also observed after carbodiimide reduction of the carboxyl groups of the uronic acid units of heparin. Competitive assays of ST-1 binding to heparin immobilized on poly-L-lysine-coated plates using oligosaccharides of different sizes that arose from HNO2 cleavage of heparin showed that the minimum fragment required for reactivity of ST-1 is a decasaccharide.

Mechanism of fibronectin-mediated cell migration: dependence or independence of cell migration susceptibility on RGDS-directed receptor (integrin).

Cell migration on fibronectin (FN)-coated substrata was studied using 10 cell lines, of which only 2 showed clear enhancement and 1 showed marginal enhancement of cell migration. The migration of the other 7 cell lines was not affected on FN-coated substrata, although they all showed FN-dependent cell adhesion. The migration-enhancing activity of FN was found in the fragment including the cell-adhesion and Hep-2 domains, but not other domains (Hep-1/Fib-1, Gel, Fib-2). No difference in the migration-enhancing effect was seen among FNs from plasma, fibroblasts, or transformed cells. FN-dependent cell migration was inhibited by polyclonal antibodies directed to the C-terminal half region including the cell binding domain, but not by antibodies directed to five other domains. Since these results indicated that FN-mediated cell migration could be controlled by the cell-adhesion domain of FN and its receptor, studies were then focused on the effect of antibodies directed to receptors for FN and collagen, and on the effect of tetrapeptide sequences recognized by these receptors. It was found that (i) cell migration on FN-coated surfaces was specifically inhibited by anti-FN receptor antibody P1F8 but not by anticollagen receptor antibody P1H5; (ii) the migration was strongly inhibited by Arg-Gly-Asp-Ser but not by other oligopeptide sequences. However, the majority of those cell lines not susceptible to FN-dependent cell migration were characterized by having FN receptors and the ability to adhere on FN-coated matrix. Based on these findings, it was concluded that FN-dependent cell migration shares the same recognition mechanism as FN-dependent cell adhesion, but that the majority of cell lines not exhibiting FN-dependent migration still show FN-dependent cell adhesion and express the FN receptor (integrin); i.e., cell migration and adhesion involve the same receptor and the same FN loci, but migration is controlled by still-unidentified cellular factors which determine the susceptibility of the cell to the dynamic function of the FN receptor (integrin) unit.

A monoclonal antibody directed to terminal residue of beta-galactofuranose of a glycolipid antigen isolated from Paracoccidioides brasiliensis: cross-reactivity with Leishmania major and Trypanosoma cruzi.

A mouse monoclonal antibody, MEST-1, was produced against Band 1 glycolipid antigen of Paracoccidioides brasiliensis. The glycan structure of Band 1 antigen was recently elucidated and the monosaccharides sequence was defined as: Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins. The reactivity of MEST-1 MAb was determined by solid-phase radioimmunoassay and high performance thin layer chromatography immunostaining. Selective oxidation of galactofuranose residues and inhibition assays with different methyl-glycosides, revealed that MAb MEST-1 is directed against the terminal residue of beta-D-galactofuranose of Band 1, a phosphoglyceroglycolipid antigen of P. brasiliensis. By indirect immunofluorescence, it was observed that the epitope recognized by MEST-1 is accessible to the antibody in yeast forms of this fungus. Reactivity of MEST-1 with parasites known to express galactofuranose containing glycoconjugates was also analyzed by indirect immunofluorescence. A positive fluorescence was observed with promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. GIPL-1 was identified as the antigen recognized by MEST-1 in Leishmania major, indicating that the MAb MEST-1 recognizes terminal galactofuranose residue in either beta 1-->6 or beta 1-->3 linkage to the mannose.