Reduction of inflammatory slan (6-sulfo LacNAc) dendritic cells in psoriatic skin of patients treated with etanercept.

Dermal dendritic cells (DCs) play a central role in the immunopathology of psoriasis. We previously identified slanDCs as pro-inflammatory TNF-α, IL-23- and IL-12-producing DCs in human blood and as prominent inflammatory dermal TNF-α secreting and CD11c-positive DC subset in psoriasis. Here, we ask for the effects of TNF-α-inhibition on inflammatory slanDCs in skin and blood of 10 patients with psoriasis during 24 weeks of treatment with etanercept. Treatment with etanercept reduced the frequency of dermal slanDCs but did not induce apoptosis as determined by lack of increased active caspase-3-expression. In parallel, we found increased frequencies of slanDCs in blood which expressed lower levels of HLA-DR. Stimulating slanDCs isolated from the blood of healthy donors in vitro induced a strong production of IL-1ß, IL-6, IL-23 and IL-12p70. This capacity was efficiently reduced in the presence of etanercept, thereby indicating that TNF-α is an autocrine stimulus for maturation and pro-inflammatory cytokine production of slanDCs. In vivo, we noticed that treatment with etanercept did reduce the number of dermal slanDCs in parallel to the overall expression of TNF-α and IL-23p19. However, successful treatment did not down-regulated the percentage of dermal slanDCs that stained positive for TNF-α and IL-23p19 indicating that remaining slanDCs kept their pro-inflammatory capacity. This study provides novel insights into the immune regulatory properties of etanercept at the level of inflammatory slanDCs in vivo in skin and blood as well as in vitro.

Improved high-performance liquid chromatographic method to estimate aminosugars and its application to glycosaminoglycan determination in plasma and serum.

An improved isocratic high-performance liquid chromatography (HPLC) method for the analysis of L-(-)-fucose. D-(+)-galactosamine, D-(+)-glucosamine, D-(+)-galactose, obtained by hydrolysis of glycosaminoglycans (GAGs) and D-(+)-glucose and D-(+)-mannose is described. The presence in circulation of GAGs, acid polysaccharide sequences of alternate monosaccharide units, aminosugar and uronic acid (galactose in keratan sulfate), has been measured in terms of their sugar components. To evaluate concentration of these circulating sugars we considered blood samples obtained from healthy humans. Plasma or serum was filtered through weak anion-exchange Ecteola-cellulose either untreated or after mild alkaline treatment. GAGs adhering to resin were recovered by salt elution, and desalted on Bio-Gel P-2 resin. GAG fractionation by charge was carried out on a strong anion exchanger. GAG composition was evaluated in terms of galactose and aminosugars, measured in HPLC by the proposed procedure using anion-exchange resin and pulsed amperometric detection. The mobile phase consisted of 0.02 M NaOH and elution was carried out at flow-rate of 1.0 ml/min. The amperometric detector was set as follows: t1 (0.5 s), E1 (+0.1 V); t2 (0.09 s), E2 (+0.6 V); t3 (0.05 s), E3 (-0.6 V). The analysis required 14 min. Calibration standard curves for the six analytes were linear from 0.25 to 40 microM. RSD values for intra- and inter-day variabilities were < or = 5.3% at concentrations between 0.25 and 40 microM. Accuracy, expressed as percentage error, ranged from - 16 to 14%. The method was specific and sensitive with quantitation limits of 1 pmol for L-(-)-fucose, D-galactosamine and D-glucosamine, 3 pmol for D-(+)-galactose and D-(+)-glucose and 5 pmol for D-(+)-mannose. The results of the assay showed higher GAG concentrations in serum than in plasma.

Enhanced liver blood concentrations of adenine arabinoside accomplished by lactosaminated poly-L-lysine coupling: implications for regional chemotherapy of hepatic micrometastases.

Conjugates of antiviral and antiblastic nucleoside analogs (NAs) with galactosyl-terminating peptides selectively enter hepatocytes after binding of the carrier galactose residues to the asialoglycoprotein receptor. Since NAs, when set free from the carrier within hepatocytes, partly exit from these cells into the bloodstream, we considered the possibility that administration of galactosyl-terminating conjugates of NAs could result in plasma concentrations of these drugs that would be higher in liver sinusoids than in capillaries of other organs. In the present study we demonstrated the validity of this hypothesis. We injected rats with a conjugate of adenine arabinoside (ara-A) with lactosaminated poly-L-lysine and found that the plasma concentrations of ara-A were >2-fold higher in blood of liver than in systemic circulation. Liver blood was collected from the inferior vena cava after closing below and above the outflows of the hepatic veins. The present result suggests that conjugation with galactosyl-terminating peptides might be a way to selectively increase the concentrations of NAs not only in hepatocytes, which have the asialoglycoprotein receptor, but also in cells infiltrating the liver, such as neoplastic cells of micrometastases nourished by hepatic sinusoids.

Enzymatic midchain branching of polylactosamine backbones is restricted in a site-specific manner in alpha 1,3-fucosylated chains.

Branched polylactosamines on animal cell surfaces are believed to contribute to multivalent interactions in cell adhesion and cell signalling. Their biosynthesis proceeds via linear precursors that become branched by beta1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous work has identified the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (1) and the hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc (4) as acceptors for a rat serum enzyme activity (cIGnT6), which transfers GlcNAcbeta1-6 units to the midchain galactose residues. Thereby, 1 is converted to the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc and 4 to the doubly branched octasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3(GlcNAcb eta1-6)Galbeta1-4GlcNAc [Leppänen, A., Salminen, H., Zhu, Y., Maaheimo, H., Helin, J., Costello, C. E., & Renkonen, O. (1997) Biochemistry 36, 7026-7036]. Here we report that neither the alpha1, 3-fucose-containing derivatives of 1 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)G lcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Gl cNAc] nor a similar derivative of 4 [Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)+ ++GlcNAcbeta1-3Galbeta1- 4GlcNAc] were acceptors for the rat serum cIGnT6 activity. Hence, the enzyme's branch-forming action was completely prevented at sites in the immediate neighborhood of the fucosylated loci of the polylactosamines. In Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, the inhibition of the branch-forming reaction was restricted to the fucose-carrying LacNAc unit; at the middle LacNAc, the branching proceeded normally. However, in the isomeric Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, the fucose residue prevented branching completely at the middle LacNAc and almost completely at the reducing end LacNAc. In summary, alpha1,3-fucose residues in polylactosamine chains inhibited the cIGnT6 reaction in a site-specific manner, at the fucosylated LacNAc unit itself and also at sites one and two LacNAc units upstream, but not at the LacNAc units downstream from the fucosylated locus. These data imply that site-directed branching in polylactosamines is possible in vitro with the aid of specifically positioned alpha1,3-fucosyl units, that can be removed afterward without harming the branched backbones.

Enzymatic analysis of cell surface lactosaminyl glycans by flow cytometry.

Cell surface expressed lactosaminyl glycans were determined on live cells by flow cytometry using a sialyltransferase mediated labeling procedure. Fluorescent CMP-sialic acid and Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase were applied to probe expression of acceptor glycans on untreated or sialidase pretreated erythrocytes. After enzymatic fluorescence labeling, erythrocytes were treated with endo-beta-galactosidase or trypsin to distinguish polylactosaminyl- and complex-type glycans. The expression of lactosaminyl sequences on cord- was 20% lower than on adult cells. After sialidase treatment fluorescence incorporation on both cell types increased twofold compared to untreated cells indicating a low sialylation extent. A recombinant alpha 2,3-sialyltransferase was preferentially labeling polylactosaminyl glycans. Taking advantage of the different fine specificity as determined here, alpha 2,6- and alpha 2,3-sialyltransferase can be applied to distinguish certain types of lactosaminyl glycans.

Polylactosamines are not obligate receptors for invasion of Plasmodium falciparum malaria as shown in HEMPAS variant II-gal- erythrocytes.

A HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum test) erythrocyte, atypical Variant II (referred to herein as Variant II-gal-), lacking long-chain polylactosamine on both glycoproteins (Band 3 and 4.5) and glycosphingolipids, was characterized by the carbohydrate profile of the erythrocyte membrane according to Fukuda et al. (Blood, 73, 1331-1339, 1989). Two laboratories previously reported that polylactosamine isolated from the erythrocyte protein Band 3 inhibited invasion of red blood cells by Plasmodium falciparum in malarial culture, suggesting a role for this carbohydrate in adhesion of the parasite. Therefore, HEMPAS erythrocyte Variant II-gal- presented a unique opportunity to further examine this premise. Freshly drawn blood samples (normal and HEMPAS Variant II-gal-) were separately incubated with P. falciparum from mannitol-synchronized cultures. The parasite was found to invade HEMPAS Variant II-gal- erythrocytes at a 30% lower rate through two life cycles, as shown by microscopic evaluation of invasion and by [3H]hypoxanthine incorporation into parasite. This observation, along with the published fact that glycophorin-deficient MkMk cells are also infectable, but at a lower rate, indicates that neither sialoglycoproteins nor polylactosamines are an obligate adhesive ligand for P. falciparum, although the possibility remains that either may still contribute to adhesive events during infection.

[Carbohydrate composition of native and desialylated low density lipoproteins in the plasma of patients with coronary atherosclerosis]. / Uglevodnyi sostav nativnykh i decialirovannykh lipoproteidov nizkoi plotnosti plazmy krovi bol'nykh koronarnym aterosklerozom.

The monosaccharide content was examined in apoprotein B and lipids of total low density lipoprotein (LDL) preparations isolated from the blood of healthy individuals and patients with coronary atherosclerosis, as well as in desialylated and sialylated LDL obtained by affinity chromatography on Ricinus Communis Agglutinin-agarose. The glycoconjugates of total apoprotein-B of healthy subjects' LDL were found to consist of N-acetylglycosamine, galactose, mannose and sialic acid in a molar ratio of 2:1:2.5:1. The content of protein-bound neutral sugars was the same in healthy subjects and patients, while there was a lower sialic acid amount in the patients. The level of protein-bound sialic acid in patients' desialylated LDL was 2- to 3-fold lower than that in sialylated LDL. In contrast to apoprotein-B glycoconjugated, the lipid-bound carbohydrate chains of total LDL preparations had N-acetylgalactosamine and glucose, but not mannose. In total LDL from patients, the content of all lipid-bound carbohydrates was 1.5- to 4-fold lower than that in LDL from healthy subjects. In healthy subjects and in patients alike, the neutral sugar content of a lipid subfraction of desialylated LDL was lower than in those of sialylated LDL. It is concluded that atherogenic desialylated LDL differ from non-atherogenic sialylated LDL in neutral sugar levels.

[The effect of endo-beta-galactosidase on lactosamine sialoantigens Gd, Fl, Vo, Li]. / Der Effekt von Endo-beta-Galaktosidase auf die Laktosamin-Sialoantigene Gd, Fl, Vo, Li.

Human red cells (RBC) were treated with endo-beta-galactosidase from Bacteroides fragilis removing type 2 polylactosamine chains [Galssl-4GlcNAc]n from the RBC surface by cleaving internal Galssl-4Glc(NAc) bonds of linear lactosamine sequences. I and i built up by unsubstituted branched and linear chains were inactivated. The sialo-antigen Vo resembling i antigen expression on adult, newborn and i adult RBC was also inactivated, demonstrating that the Vo determinant is created by sialylation of linear chains. Fl and Gd antigens known to be sialylated polylactosamine chains were not inactivated. Because Gd antigenicity is increased by increasing the length of sugar sequences and Vo determinants are obviously short chain sequences, the action of endo-beta-galactosidase could depend on the length of sialylated sequences on the RBC membrane.

Effect of acute and chronic ethanol administration on rat liver alpha 2,6-sialyltransferase activity responsible for sialylation of serum transferrin.

The effect of a single administration and a 6-week treatment with ethanol on rat liver sialyltransferase activity towards asialoglycoproteins and N-acetyllactosamine (Gal beta 1,4GlcNAc) was studied. Since only the alpha 2,6-sialyltransferase is involved in the in vivo sialylation of transferrin, Gal beta 1,4GlcNAc was chosen as an acceptor and alpha 2,6-sialyl-N-acetyllactosamine was separated from the corresponding alpha 2,3-sialyl isomer present in the sialyltransferase reaction mixture by high-performance liquid chromatography. After a single ethanol administration there was a low (about 20%) but significant (p less than 0.005) reduction of sialyltransferase activity towards asialotransferrin as well as a reduced alpha 2,6-sialyltransferase activity towards N-acetyllactosamine. An opposite result was found in the chronically ethanol-treated rats: in these animals either the total or alpha 2,6-sialyltransferase activity was slightly higher than in control animals. Blood ethanol concentration was significantly high (3.3 +/- 1.2 mg/ml) only in the acute-treated animals, suggesting that the accumulation in the body of ethanol and/or its metabolites induces a reduction of liver alpha 2,6-sialyltransferase activity responsible for the transferrin sialylation. Current results are consistent with the finding (Stibler H, Hultcrantz R: Alcohol Clin Exp Res 11:468-473, 1987) that an enhanced level of hyposialylated transferrin isoforms is a marker of present but not previous alcohol abuse.

Primary defect of congenital dyserythropoietic anemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II.

Congenital dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) is a genetic disease caused by membrane abnormality. Previously we have found that Band 3 and Band 4.5 are not glycosylated by lactosaminoglycans in HEMPAS erythrocytes, whereas normally these proteins have lactosaminoglycans (Fukuda, M. N., Papayannopoulou, T., Gordon-Smith, E. C., Rochant, H., and Testa, U. (1984) Br. J. Haematol. 56, 55-68). In order to find out where glycosylation of lactosaminoglycans stops, we have analyzed the carbohydrate structures of HEMPAS Band 3. By fast atom bombardment-mass spectrometry, methylation analysis, and hydrazinolysis followed by exoglycosidase treatments, the following structure was elucidated: (formula; see text) N-Linked glycopeptides synthesized in vitro by reticulocyte microsomes from HEMPAS were shown to be predominantly the above short oligosaccharide, whereas those from normal reticulocytes contain large molecular weight carbohydrates. The N-acetylglucosaminyltransferase II, which transfers N-acetylglucosamine to the C-2 position of the Man alpha 1----6Man beta 1----arm of the biantennary core structure, was therefore examined by using Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcol as an acceptor. N-Acetylglucosaminyltransferase II activity was demonstrated in the lymphocyte microsome fraction from normal individuals. However, this enzyme activity was found to be decreased in those from HEMPAS patients. These results suggest that the primary defect of HEMPAS lies in the lowered activity of N-acetylglucosaminyltransferase II.

Elevated serum alpha(1----3)-L-fucosyltransferase activity with synthetic low molecular weight acceptor in human ovarian cancer.

Serum alpha(1----3)-L-fucosyltransferase activity was measured in 29 ovarian cancer patients with active disease, 26 ovarian cancer patients with no clinical evidence of disease and 23 healthy females. N-Acetyl-2'-O-methyl-lactosamine was used as the acceptor for the enzyme. The level of the enzyme activity was significantly (P less than 0.05) higher in the serum of patients with known tumor when compared to healthy controls and patients with no clinical evidence of disease.

Structures of sialylated fucosyl polylactosaminoglycans isolated from chronic myelogenous leukemia cells.

Polylactosaminoglycans were isolated from human chronic myelogenous leukemia cells and their structures were elucidated. The lactosaminoglycan saccharides were isolated by hydrazinolysis and fractionated by QAE-Sephadex. The structures of fractionated oligosaccharides were analyzed by fast atom bombardment-mass spectrometry and methylation before and after treatment with specific exoglycosidases, such as alpha 2----3 specific neuraminidase. Based on these experiments, the structures of sialyl polylactosaminoglycans of chronic myelogenous leukemia cells were found to contain the following unique structure which is absent in normal mature granulocytes: (formula; see text) In addition to this, chronic myelogenous leukemia polylactosaminoglycans can be distinguished from normal granulocyte polylactosaminoglycans by the following characteristics. Leukemic polylactosaminoglycans are (a) shorter, (b) more highly sialylated and contain fully sialylated, tetrasialosyl polylactosaminoglycans, (c) are less fucosylated at C-3 of N-acetylglucosamine of polylactosaminyl side chains, and (d) contain a significant amount of sialyl Lex, NeuNAc alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3, structure. These results indicate that chronic myelogenous leukemia cells express unique polylactosaminoglycan structures which are distinct from normal mature granulocytes.

Effect of slate dust on the rat erythrocyte membrane composition: in vitro and in vivo studies.

The biochemical composition of rat erythrocyte ghost membrane obtained by hypotonic lysis and slate-dust-induced lysis were compared in vitro. The phospholipids and glycosamine contents decreased in slate-dust-exposed erythrocyte membrane, whereas there were no changes in protein content. The in vitro and in vivo association of silica, leaching from slate dust, with the components of rat erythrocyte ghost membrane, has also been demonstrated. The interaction of silica with membrane constituents is proposed as a mechanism of action of slate-dust toxicity.

[Turnover of the amino sugars of erythrocyte and thrombocyte glycoproteins in rats maintained on different carbohydrate diets]. / Obnovlenie aminosakharov glikoproteidov éritrotsitov i trombotsitov krys, soderzhavshikhsia na razlichnykh uglevodnykh ratsionakh.

The turnover of erythrocyte and platelet glycoprotein amino sugars in rats fed carbohydrates has been studied. Partial replacement of starch by sucrose results in an almost 3-fold increase in the half-life of erythrocyte glycoprotein amino sugars and in a 2-fold increase in that of platelets as early as 14 days after keeping the animals on carbohydrates. It is concluded that study of dynamic parameters of cell amino sugars can be used for evaluation of the role played by food in metabolic and adaptive reactions both in experimental animals and man.

Selective penetration and pharmacological activity of lactosaminated albumin conjugates of adenine arabinoside 5-monophosphate (ara-AMP) in mouse liver.

With the aim of improving the chemotherapeutic index of adenine arabinoside 5-monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes where it is digested in lysosomes. In mice, the L-[3H]SA-ara-AMP conjugates, intravenously injected, selectively penetrated the liver, only small quantities were taken up by cells of spleen, bone marrow, intestine, and brain. After administration of the conjugate to mice with Ectromelia virus hepatitis, ara-AMP was selectively concentrated in liver in a pharmacologically active form. If L-SA-ara-AMP conjugates behave in man as in mouse, their administration to patients with chronic hepatitis B should result in a selective concentration of ara-AMP in liver with a more efficient inhibition of virus replication accompanied by lower toxicity for other tissues.

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes.

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.

Structure of fetal lactosaminoglycan. The carbohydrate moiety of Band 3 isolated from human umbilical cord erythrocytes.

The structure of lactosaminoglycan prepared from Band 3 glycoprotein of umbilical cord blood erythrocytes was elucidated. The glycopeptides were digested by endo-beta-galactosidase and oligosaccharides, core glycopeptides, and intact glycopeptides were analyzed by permethylation, exoglycosidase digestion, and fast atom bombardment mass spectrometry. The structure of one of the major components was found to be: sequence in text This structure is unique in that 1) two linear polylactosaminyl chains are attached to the core portion, 2) the polylactosaminyl side chain is much longer on the Man alpha 1----6 side than on the Man alpha 1----3 side, 3) alpha 2----3-linked sialic acid is present at the terminal of the long polylactosaminyl side chain, whereas alpha 2----6-linked sialic acid is present in the other short side chain, and 4) fucose is linked only to the external position of the lactosaminyl side chain and fucose, alpha 1----6-linked to the innermost N-acetylglucosamine, is absent.

Isolation and characterization of polyfucosylated lactosaminoglycan from human granulocytes.

Lactosaminoglycan was isolated from human granulocytes and the structure of neutral lactosaminoglycan was elucidated. The lactosaminoglycan glycopeptides and lactosaminoglycan saccharides obtained by hydrazinolysis were analyzed by permethylation. In addition, the lactosaminoglycan was digested by endo-beta-galactosidase and the "core" portion and released oligosaccharides were analyzed by specific glycosidases, permethylation, and fast atom bombardment mass spectrometry. The structure of the major component in the neutral lactosaminoglycan was found to be: sequence in text where m + n + o + p greater than 6, and the mean value of fucose content = 4.5. This structure is unique in that 1) four linear polylactosaminyl chains are attached to the core portions, 2) N-acetylglucosamine residues in the polylactosaminyl side chains are substituted with fucose through an alpha 1----3 linkage and at least 1 mol of Gal beta 1----4(Fuc alpha 1----3)GlcNAc terminal structure is present, and 3) the tetraantennary core is a major component. Since this polyfucosylated lactosaminoglycan is abundantly present in human granulocytes, we propose that this lactosaminoglycan is a major carrier for the granulocyte-specific antigen, Gal beta 1----4(Fuc alpha 1----3)GlcNAc, which is recognized by the My-1 monoclonal antibody (Huang, L. C., Civin, C. I., Magnani, J. L., Shaper, J. H., and Ginsburg, V. (1983) Blood 61, 1020-1023).

Changes in the concentration of serum proteins and their carbohydrate components during immobilization stress.

Changes in different protein fractions during immobilization stress were demonstrated. The concentration of albumins increased and that of all globulin fractions decreased, especially the concentration of alpha 2, and gamma globulins. Increased concentration of the carbohydrate components of serum proteins was also observed. Since the latter change was associated with a fall in globulin concentration, and globulins, contrary to albumins, contain carbohydrate components, it may be supposed that the proteins investigated during stress have molecules rich in carbohydrates.