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Scarcely understood defects lead to asthenozoospermia, which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitrofertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm function. We have performed a differential proteomics analysis of human sperm and seminal plasma and identified and quantified 667 proteins in sperm and 429 proteins in seminal plasma data set, which were used for further analysis. Statistical and mathematical analysis combined with pathway analysis and self-organizing maps clustering and correlation was performed on the data set.

Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of an oviductal sperm reservoir, all of which appear to be important in the fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification of the glycoproteins, glycosylation sites, glycan compositions, and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. The majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated, or both. Many Lewis x/a and y/b epitopes bearing glycans were found, suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to the prefractionation and complexity of the sample.

Epithelial cells lining the urinary tract secrete urinary exosomes (40–100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk.

Allergies are a global health problem with rapidly increasing prevalence but still lacking pathogenetic knowledge or optimal treatment. The objective is to add to the conventional thinking that allergies are caused by overactive, mainly T-cell-mediated, immunological responses and thus to raise the putative role of altered epithelial functions.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLex). It is thought that multivalent 6-sulfo SLex expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLex in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin–binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLex epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLex. N-glycans containing potential 6-sulfo SLex epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLex epitopes on O-glycans that are important for its recognition by L-selectin.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLex). It is thought that multivalent 6-sulfo SLexexpression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLexin total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin–binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLexepitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLex. N-glycans containing potential 6-sulfo SLexepitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLexepitopes on O-glycans that are important for its recognition by L-selectin.

Background Chronic rhinosinusitis (CRS) is characterized by persistent inflammation of the nasal and paranasal mucosa with numerous emigrated leukocytes. L-Selectin on leukocytes and its endothelial glycosylated ligands initiate leukocyte infiltration into inflamed tissues. Endoscopic sinus surgery (ESS) is the major approach for restoring sinus physiology after failure of conservative therapy; however, the effect of enlarging the maxillary sinus ostium is still unknown. Here, we compared two histological markers of local inflammation, the number of mucosal eosinophils, and the expression of endothelial L-selectin ligands, with clinical outcomes after enlarging or saving the maxillary sinus ostium.Methods Twenty-three patients with CRS underwent uncinectomy on one side and additional middle meatal antrostomy on the other side. Maxillary sinus mucosa biopsy specimens from these patients and nine healthy subjects were taken for immunohistochemical evaluations of the number of mucosal eosinophils and endothelial L-selectin ligands. Also, symptoms and mucociliary clearance were measured.Results The postoperative reduction of the endothelial L-selectin ligands was independent of the operation technique. There was a correlation between postoperative number of mucosal eosinophils and symptom score, which was also independent of the surgical technique. The postoperative decrease of mucosal eosinophils, as well as the correlation of the intraoperative eosinophils with the postoperative symptom score, was found only on antrostomy side.Conclusion ESS decreases the expression of endothelial L-selectin ligands, which might lead to decreased eosinophil traffic into maxillary sinus mucosa, putatively more when enlarging the maxillary sinus ostium. Both intra- and postoperative low number of eosinophils seem to be indicators of good subjective recovery.

It has been suggested that excessive activation of the antiinflammatory pathways in sepsis may lead to poor outcome of patients with sepsis. The aim of this study was to test the value of histocompatibility leukocyte antigen HLADRexpression on blood monocytes and plasma levels of interleukin IL4 and 10 in prediction of hospital mortality in patients with sepsis. Sixtyone critically ill patients with sepsis were prospectively enrolled to this study in two university hospital intensive care units. Survivors n 41 and nonsurvivors n 20 differed significantly in HLADR expression at admission survivors' median 84 interquartile range 6498 versus nonsurvivors' median 62 interquartile range 4783, P0.025 by MannWhitney test. Similarly, the analysis revealed statistically significant differences between survivors and nonsurvivors in admission plasma IL10 levels and in admission Sequential Organ Failure Assessment SOFA and Acute Physiology and Chronic Health Evaluation APACHE II scores, but not in IL4 levels. The areas under receiver operating curves AUC showed that both monocyte HLADR expression and plasma IL4 level showed poor discriminative power in prediction of hospital mortality AUC < 0.70. Only IL10 levels on days 1 and 2 showed reasonable predictive power AUCs 0.706 and 0.725, respectively. The highest AUC values were those of APACHEII 0.786 and admission SOFA score 0.763. In conclusion, APACHE II and SOFA scores on admission showed better discriminatory power than HLADR expression and IL10 and IL4 levels in prediction of hospital mortality in critically ill patients with sepsis.

Hydrocortisone reduces the number of inflammatory leukocytes within tissues, but thus far the site of action on the multistep adhesion cascade leading to leukocyte extravasation has not been identified. We have recently developed a noninvasive in vivo reflected-light confocal microscopy technique to study this at sites of inflammation in human patients. In the present study, we evaluated the effect of preoperative intravenous hydrocortisone treatment on leukocyte trafficking after conjunctival inflammation induced by cataract surgery in human subjects in vivo. The surgery generated leukocyte rolling along the endothelial lining of conjunctival vessels. While preoperative hydrocortisone did not reduce the number of rolling cells, it significantly raised the velocity of individual rolling leukocytes and concomitantly reduced leukocyte emigration into conjunctival tissue. Immunohistology of conjunctival biopsies excised from the individuals studied provided circumstantial evidence that endothelial P-selectin might play a role in the surgery-induced up-regulation of the leukocyte rolling. Furthermore, hydrocortisone reduced surgery-induced P-selectin induction, suggesting a role for this selectin in the regulation of local leukocyte traffic into sites of inflammation in human conjunctiva. Taken together, these results suggest that control of the rolling velocity might be an effective way to adjust leukocyte traffic in vivo in human subjects.

Hydrocortisone reduces the number of inflammatory leukocytes within tissues, but thus far the site of action on the multistep adhesion cascade leading to leukocyte extravasation has not been identified. We have recently developed a noninvasive in vivo reflected-light confocal microscopy technique to study this at sites of inflammation in human patients. In the present study, we evaluated the effect of preoperative intravenous hydrocortisone treatment on leukocyte trafficking after conjunctival inflammation induced by cataract surgery in human subjects in vivo. The surgery generated leukocyte rolling along the endothelial lining of conjunctival vessels. While preoperative hydrocortisone did not reduce the number of rolling cells, it significantly raised the velocity of individual rolling leukocytes and concomitantly reduced leukocyte emigration into conjunctival tissue. Immunohistology of conjunctival biopsies excised from the individuals studied provided circumstantial evidence that endothelial P-selectin might play a role in the surgery-induced up-regulation of the leukocyte rolling. Furthermore, hydrocortisone reduced surgery-induced P-selectin induction, suggesting a role for this selectin in the regulation of local leukocyte traffic into sites of inflammation in human conjunctiva. Taken together, these results suggest that control of the rolling velocity might be an effective way to adjust leukocyte traffic in vivo in human subjects.

Inflammatory diseases are characterized by the leukocyte infiltration into tissues. L-selectin on lymphocytes and its endothelial glycosylated ligands are instrumental in the initiation of lymphocyte extravasation. Immunohistochemical stainings with monoclonal antibodies against functionally active glycan-decorated L-selectin ligands, ie, sialyl-Lewis x (sLex, 2F3, and HECA-452) or sulfated extended core 1 lactosamine (MECA-79), were performed on more than 400 specimen representatives for thyroiditis, myocarditis, psoriasis, vasculitis, ulcerative colitis, and their corresponding noninflamed tissues. The endothelial expression of sLex or sulfo sLex glycans in postcapillary venules was either absent or low in control tissues. The de novoinduction of endothelial expression of sLex or sulfo sLex glycans was detected in all inflamed tissues. Furthermore, each organ carried its own modification of sLex or sulfo sLex glycans, ie, zip code. Our results suggest that these zip code glycans may provide means for organ selective leukocyte traffic that could be used in selective leukocyte traffic inhibition.

Acute heart allograft rejection is characterized by leukocyte infiltration and myocyte damage, key elements in the histological grading of rejection. The induction of selectins and their ligands on the graft postcapillary venular endothelium increases leukocyte tethering to, rolling on, and extravasation through the endothelium into graft parenchyma. We have characterized peptide mimicking selectin ligands by screening phage peptide libraries using anti-Lewis A antibodies and E-selectin as target molecules. The effect of this selectin- binding peptide, IELLQAR, on the prevention of inflammation and tissue damage and on the prolongation of graft survival in inbred DA (RT1a) rat heart allografts transplanted to WF (RT1v) recipients was tested. Bovine serum albumin (0.1, solvent), VTSIAQA (control peptide), or IELLQAR were either continuously infused into the peritoneum via osmotic mini pumps or injected twice daily IV. Treatment with bovine serum albumin and VTSIAQA did not alter the number of graft infiltrating leukocytes or the histological grade of acute rejection, all scored as grade 4. On the contrary, the selectin binding peptide, IELLQAR, dose-dependently reduced inflammation and at the highest dose (6.0 mg/kg per day) eliminated the majority of graft infiltrating leukocytes, reduced the histological grade from 4 to 1B, but had no effect on graft survival. These data indicate that the intensity of inflammation related to the allograft rejection does not correlate to the graft survival.

α1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galβ1-4GlcNAc) to Galβ1-4(Fucα1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of α1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNβ1-2Man, LNβ1-6Manα1-OMe, LNβ1-2Manα1-3(LNβ1-2Manα1-6)Manβ1-4GlcNAc, and Galβ1-3(LNβ1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcβ1-3′LN (underscored site reacted) and GlcNAcβ1-3′LNβ1-3′LN (both LNs reacted), but very poorly with the reducing end LN of LNβ1-3′LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNβ1-3′LN and LNβ1-3′LNβ1-3′LNβ1-3′LN, confirming our previous data on LNβ1-3′LNβ1-OR. In contrast, the sialylated glycan Neu5Acα2-3′LNβ1-3′LNβ1-3′LNβ1-3′LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile α1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of α2,3-sialylated i-type polylactosamines.

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcbeta1-3'LN (underscored site reacted) and GlcNAcbeta1-3'LNbeta1-3'LN (both LNs reacted), but very poorly with the reducing end LN of LNbeta1-3'LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNbeta1-3'LN and LNbeta1-3'LNbeta1-3'LNbeta1-3'LN, confirming our previous data on LNbeta1-3'LNbeta1-OR. In contrast, the sialylated glycan Neu5Acalpha2-3'LNbeta1-3'LNbeta1-3'LNbeta1-3'LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile alpha1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of alpha2,3-sialylated i-type polylactosamines.

The GalNAcbeta1-4GlcNAc determinant (LdN) occurs in some human and bovine glycoconjugates and also in lower vertebrates and invertebrates. It has been found in unsubstituted as well as terminally substituted forms at the distal end of conjugated glycans, but it has not been reported previously at truly internal positions of polylactosamine chains. Here, we describe enzyme-assisted conversion of LdNbeta1-OR oligosaccharides into GlcNAcbeta1-3GalNAcbeta1-4GlcNAcbeta1-OR. The extension reactions, catalyzed by human serum, were modeled after analogous beta3-GlcNAc transfer processes that generate GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-OR. The newly synthesized GlcNAcbeta1-3GalNAc linkages were unambiguously identified by nuclear magnetic resonance data, including the appropriate long-range correlations in heteronuclear multiple bond correlation spectra. The novel GlcNAcbeta1-3'LdN determinant proved to be a functional acceptor for several mammalian glycosyltransferases, suggesting that human polylactosamines may contain internal LdN units in many distinct forms. The GlcNAcbeta1-3'LdN determinant was unusually resistant toward jackbean beta-N-acetylhexosaminidase; the slow degradation should lead to a convenient method for the search of putative internal LdN determinants in natural polylactosamine chains.

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogasterto search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novopathway from GDP-mannose through enzymatic reactions catalyzed by GDP-d-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-d-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). TheDrosophilagenome does not have orthologs for the salvage pathway enzymes, i.e.fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze α1,3- and α1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode α1,2-specific fucosyltransferases were found. We also identified two novel genes coding forO-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophilagenome. Finally, using the DrosophilaCG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.

We report here on in vitroacceptor and site specificity of recombinant α3-fucosyltransferase V (Fuc-TV) with 40 oligosaccharide acceptors. Galβ1–4GlcNAc (LN) and GalNAcβ1–4GlcNAc (LDN) reacted rapidly; Galβ1–3GlcNAc (LNB) reacted moderately, and GlcNAcβ1–4GlcNAc (N,N′-diacetyl-chitobiose) reacted slowly yet distinctly. In neutral and terminally α3-sialylated polylactosamines of i-type, the reducing end LN unit reacted rapidly and the distal (sialyl)LN group very slowly; the midchain LNs revealed intermediate reactivities. The data suggest that a distal LN neighbor enhances but a proximal LN neighbor reduces the reactivity of the midchain LNs. This implies that Fuc-TV may bind preferably the tetrasaccharide sequence Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcfor transfer at the underlined monosaccharide. Terminal α3-sialylation of i-type polylactosamines almost doubled the reactivities of the LN units at all positions of the chains. We conclude that, in comparison with human Fuc-TIV and Fuc-TIX, Fuc-TV reacted with a highly distinct site specificity with i-type polylactosamines. The Fuc-TV reactivity of free LNB resembled that of LNBβ1–3′R of a polylactosamine, contrasting strongly with the dissimilarity of the reactivities of the analogous pair of LN and LNβ1–3′R. This observation supports the notion that LN and LNB may be functionally bound at distinct sites on Fuc-TV surface. Our data show that Fuc-TV worked well with a very wide range of LN-glycans, showing weak reactivity only with distal (sialyl)LN units of i-type polylactosamines, biantennaryN-glycans, and I branches of polylactosamines.

Two rapid and simple procedures for the quantitative analysis of GDP-

Polylactosamines Neu5Acα2‐3′Lexβ1‐3′Lexβ1‐3′Lex and Neu5Acα2‐3′LNβ1‐3′Lexβ1‐3′Lex [Lex, Galβ1‐4(Fucα1‐3)GlcNAc; LN, Galβ1‐4GlcNAc] decorate selectin counterreceptors in human HL‐60 cells. Here, we show that HL‐60 cell lysates catalyze distal α3‐sialylation of LNβ1‐3′LNβ1‐3′LN and LNβ1‐3′Lexβ1‐3′Lex efficiently, outlining two potential sets of biosynthetic pathways leading to the selectin ligands. In one set, α3‐sialylation precedes internal fucosylation of the polylactosamine backbone, whereas in the other one, internal fucosylation is initiated before α3‐sialylation. In contrast to α3‐sialylation, LNβ1‐3′Lexβ1‐3′Lex was α6‐sialylated much less efficiently than LNβ1‐3′LNβ1‐3′LN by HL‐60 cell lysates. Hence, internal fucosylation may regulate the extent of α6‐sialylation of polylactosamines in these cells.

L‐selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6‐sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active α(1,3)fucosyltransferase Fuc‐TVII, the enzyme responsible for the final fucosylation of sialyl‐N‐acetyllactosamine during sLex synthesis, and express sLex on the cell surface. Furthermore, these cells degrade sLex, primarily by desialylating it to neutral Lex epitopes by α(2,3)sialidase(s).

L-selectin-dependent lymphocyte extravasation is a hallmark of acute heart allograft rejection in rats. On screening over 600 endomyocardial biopsies (EMBs), taken at different time points after heart transplantation in man, we identified 91 samples with histological signs of acute rejection. Rejection and nonrejection EMBs were analyzed for the presence of properly glycosylated, ie, sulfated sialyl Lewis-x (sLex) decorated L-selectin ligands. Two anti-sLex (2F3 and HECA-452) and one anti-6- or 6′-sulfated and/or 6,6′-bisulfation (MECA-79) monoclonal antibodies were used. Nonrejecting heart endothelium did not express, or expressed only weakly, sulfated and or sLex decorations of L-selectin ligands. On the contrary, these epitopes were readily detectable on endothelium of capillaries and venules at the onset and during acute rejection episodes. The more intense the sulfated sLex expression was, the more severe the rejection episode was in histological grading. The endothelial expression of L-selectin ligands decreased to background levels as the rejection resolved. Our data demonstrate a complete correlation between the level of expression of the sulfated sLex-decorated ligands on the one hand and the histological severity of acute heart allograft rejection on the other hand. These data suggest that functionally active endothelial L-selectin ligands are instrumental in lymphocyte extravasation into the human heart allografts at the onset and during acute rejection episodes.

Abstract: Purpose: E- and P-selectins, expressed on vascular endothelium, and their sialyl-Lewisx (sLex)- and/or sialyl-Lewisa (sLea)-containing ligands have a crucial role in extravasation and metastasis of circulating cells. We wanted to analyse the role of selectins and their ligands in head and neck tumours. Methods: A total of 40 consecutive biopsy specimens were collected from surgery performed at the Helsinki University Central Hospital between September 1995 and November 1996. The series of specimens contained both benign and malignant head and neck tumours of epithelial, lymphoid or mesenchymal origin. All these were analysed with immunohistochemistry for epithelial and endothelial expression of sLex and sLea glycans and E- and P-selectins. Results: Epithelial expression of sLex and sLea glycans was higher in benign than in malignant lesions in both epithelial and lymphoid tumours. On the other hand, endothelial expression of sLex, sLea, E- and P-selectin was lower in benign than in malignant lesions in both epithelial and lymphoid tumours. Conclusions: These data suggest that altered epithelial and endothelial expression of sLex and sLea glycans acting on selectin ligands is linked to the development of head and neck tumours.

Tumor cells can invade and generate metastasis via either lymphatics or blood vessels. When tumor cells are circulating in the blood, they must adhere to the vessel wall, which is lined by endothelium, before they can extravasate and form new metastases. Several families of adhesion molecules have been identified to play a role in the extravasation cascade. Selectins and their sialyl Lewis^x and/or sialyl Lewis^a (sLe^x and sLe^a, respectively) containing ligands play an initiating role in this cascade; we have now analyzed their role in the generation of metastatic breast carcinoma lesions. We examined expression of endothelial E- and P-selectin, expression of epithelial and endothelial sLe^x and sLe^a normal tissues compared with primary and metastatic breast in carcinoma lesions within individual patients. While normal breast epithelial cells do not express sLe^x or sLe^a, epithelial expression of these oligosaccharide epitopes was enhanced in primary breast carcinoma lesions. Furthermore, epithelial expression levels of sLe^x and/or sLe^a were even higher in most patients (9 of 12) who had metastatic compared with primary lesions. We show that endothelia in primary lesions express more sLe^x than in normal tissue and that metastatic lesions express even higher amounts of sLe^x compared with primary lesions. The expression of P- and E-selectin was also greatly enhanced in tumor-bearing tissue compared with normal tissue. Our data support the hypothesis that while they are circulating in the blood, sLe^x- and/or sLe^a-expressing carcinoma cells have a higher probability for extravasation at sites where the endothelium expresses E- and P-selectin and for generation of new metastases. Int. J. Cancer 74:296-300, 1997. © 1997 Wiley-Liss, Inc.

We describe here an assay that employs a highly sensitive nonradioactive method, time-resolved fluorometry, for measuring the activity of the enzyme GDP-Fuc:NeuNAcα2–3Galβ1–4GlcNAc-R (Fuc to GlcNAc) α1,3-fucosyltransferase (α1,3FT). In this assay, a neoglycoprotein substrate of α1,3FT is immobilized on a microtiter plate. Incubation with the fucose donor GDP-fucose and enzyme source converts the acceptor NeuNAcα2–3Galβ1–4GlcNAc-R to the product NeuNAcα2–3Galβ1–4(Fucα1–3)GlcNAc-R, which is quantified using a product-specific (antisialyl Lewis x) primary antibody and europium chelate-labeled secondary antibody. In the development of the assay, we used extracts of α1,3FT-transfected insect cells as the specific enzyme source. The reaction product formation was proportional to time of incubation (0–2 h) and the extract added (0.1–10 μU of enzyme) and was dependent on the GDP-fucose and glycoconjugate acceptor. We have also demonstrated with different cultured cancer cell lines that this time-resolved immunofluorometric assay allows rapid measurement of α1,3FT activity from a large number of crude cell lysate samples. Our results indicated that cell lines which expressed more sialyl Lewis x determinant on their surfaces had higher levels of α1,3FT activity. The advantages of this new assay are high sensitivity and a wide linear range of measurement. The assay is expected to be useful in the determination of regulation mechanisms of sialyl Lewis x-synthesizing α1,3-fucosyltransferases.

We have demonstrated that IFN‐γ, a potent peptide mediator in inflammatory responses, operates via the protein kinase C dependent transduction pathway in the induction of class II MHC antigens on rat microvascular endothelial cells. Stimulators of protein kinase C, like PMA, replaced IFN‐γ in the induction of MHC class II on endothelial cells in a dose‐dependent manner. Selective enzyme inhibitors of protein kinase C, H‐7 as well as sphingosine down‐regulated the IFN‐γ induced class II expression in a dose‐dependent manner. Addition of cAMP or cGMP in the culture, had no effect on the class II expression on the endothelial cells. Transient rise of cytosolic Ca2+by calcium ionophore A23187, or a calmodulin antagonist W‐7, had no effect on the IFN‐γ induced class II expression.

Leukotrienes are potent mediators of local microvascular environment. Leukotriene B 4treatment of cultured endothelium increases the binding of lymphocytes to endothelial cell monolayers within minutes. This effect is dose-dependent and reversible upon removal of the leukotriene. Pretreatment of lymphocytes slightly decreases the binding and pretreatment of both lymphocytes and endothelium with leukotriene B 4prior to the adherence assay did not alter the binding. These results suggest that leukotriene B 4regulates exclusively the vascular side, but not the white cell side of this interaction.

Acute graft-versus-host disease (aGVHD) of the liver was studied with fine-needle aspiration biopsies of thirty-four bone marrow transplant recipients. White cell differentials of liver FNABs and simultaneously taken blood samples were performed, and the increment and corrected increment methods were used to quantitate the inflammatory reaction in the liver. Biopsies taken before transplantation were used as the baseline. During aGVHD, the percentage of lymphoid cells and monocytes increased in the liver. The appearance of immunological blasts, together with a high proportion of activated lymphocytes in the FNABs, were typical findings during aGVHD. In patients with apparent pro longed liver graft-versus-host disease small lymphocytes were the predominating cell type. After initiating corticosteroid treatment, the number of blasts and the proportion of activated lymphocytes decreased. There was no significant difference in the proportions of CD4-and CD8-positive lymphoid cells in FNABs during or after aGVHD.

We have isolated the white cells from the bone marrow, spleen, and blood of a rat recipient of a bone marrow allograft and the inflammatory leukocytes from the recipient skin, lung, gut, and liver (the parenchymal target organs for acute graft-versus-host disease (aGVHD)) and compared the number of immunoglobulin-synthesizing and releasing cells in these cell populations to corresponding compartments of a syngeneic graft recipient. Bone marrow transplantation was associated in the early phase with marked immunoglobulin production in the cells of bone marrow, spleen, and blood of the allograft recipient; as, however, a similar response occurred in the syngeneic graft recipient we conclude that this is related to reconstitution rather than to aGVHD. Later, during aGVHD, the number of immunoglobulin releasing cells decreased significantly in the spleen and bone marrow of the allografted animal. In clear contrast, in the liver—but not in skin, lung, or gut—very few immunoglobulin-releasing cells were observed in the syngeneic graft recipient, whereas in the allograft recipient a very strong and significantly higher immunoglobulin synthesis and release was seen coinciding with the inflammatory episode of aGVHD in the liver.

The distribution of white cell subclasses in different lymphoid (bone marrow, spleen, and blood) and parenchymal (liver, skin, lungs, and gut) target organs was studied after bone marrow transplantation in the rat. BN rats were irradiated and transplanted with 60–80x106Lew (allogeneic) or BN (syngeneic) bone marrow cells. The recovery of lymphocytes was somewhat elevated in the bone marrow and spleen, slightly decreased in the blood, and markedly higher in the liver and skin in the allograft compared with the syngeneic graft recipient. A mild lymphocytic bronchitis was present in the lungs of the allografted animal, and the gut was hypocellular throughout the observation period. The total recovery of different lymphocyte subclasses; pan T, T helper, T suppressor-killer, class-II-positive cells, and surface-Ig-positive B cells in the different lymphoid organs—i.e., bone marrow, spleen, and blood—was similar in allogeneic compared with syngeneic graft recipients. In the liver and skin, which are the major target organs of acute graft-versus-host disease (aGVHD) in the rat, there was a massive infiltration of different T cell subclasses; high numbers of B cells were also seen in the liver. There was no difference in the T helper/T suppressor-killer ratio in the lymphoid organs or the liver of allograft compared with syngeneic graft recipients; in the skin and lungs the ratio was reduced more in the allograft compared with syngeneic graft recipient, whereas in the gut the situation was the opposite. These observations emphasize regional differences in the structure of inflammation in the different parenchymal target organs of aGVHD in the rat.

The α4β1integrin VLA‐4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol‐12‐myristate‐13‐acetate (PMA) leads to a selective decrease in the VLA‐4 α‐chain expression, both at the RNA and protein level. Meanwhile the expression of β1and that of α5another α‐chain associating with β1was seen to increase. The decrease of α4expression was restricted to monocytic cells, and was not observed on other VLA‐4‐positive cells tested (MOLT‐4 T cells and HOS sarcoma cells). The down‐regulation of the VLA‐4 α‐chain was followed by a decreased binding capacity of the cells to recombinant VCAM‐1. This data indicates that while previous findings show that the integrin‐dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.

We have demonstrated that IFN-γ, a potent peptide mediator in inflammatory responses, operates via the protein kinase C dependent transduction pathway in the induction of class II MHC antigens on rat microvascular endothelial cells. Stimulators of protein kinase C, like PMA, replaced IFN-γ in the induction of MHC class II on endothelial cells in a dose-dependent manner. Selective enzyme inhibitors of protein kinase C, H-7 as well as sphingosine down-regulated the IFN-γ induced class II expression in a dose-dependent manner. Addition of cAMP or cGMP in the culture, had no effect on the class II expression on the endothelial cells. Transient rise of cytosolic Ca 2+by calcium ionophore A23187, or a calmodulin antagonist W-7, had no effect on the IFN-γ induced class II expression.

Acute cardiac allograft rejection is characterized by infiltration of leukocytes into tissue parenchyma, but the site of entry and endothelial adhesion molecules involved are not yet defined. Lymphocyte binding to frozen sections prepared from day-3 rejecting cardiac allografts was significantly increased compared with sections made from normal hearts (number of bound lymphocytes, 983±216 per mm2vs. 309±121, respectively, P<0.001) or syngeneic grafts. The bound lymphocytes were located exclusively only on the top of the capillary structures and not on any other sites on the heart vasculature. We further wanted to analyze which of the cloned endothelial adhesion molecules and their counterreceptors would be involved in the increased lymphocyte binding. Lymphocyte pretreatment with

We have investigated the reasons why thymectomized, bone marrow—reconstituted (B) rats do not reject their allografts, by comparing the structure of inflammation and functions of inflammatory cells in nonrejecting allografts to rejecting allografts in normal control recipients. The results demonstrate that B recipients mount a specific cellular response towards the graft. The response in B recipients differs from that in normal controls by a smaller intensity of inflammation, fewer blast cells, and activated mononuclear phagocytes in the inflammatory infiltrate, as well as a delay in the appearance of specific donor-directed lytic activity in the graft. B rats also have fewer blast cells and an inverted CD4/8 ratio in the spleen. There is no obvious absence of any given cell type or cellular function in the graft inflammatory infiltrate. In light of these results no cell type responsible for allograft nonrejection can be pinpointed.

Leukotrienes are potent mediators of local microvascular environment. Leukotriene B4treatment of cultured endothelium increases the binding of lymphocytes to endothelial cell monolayers within minutes. This effect is dose‐dependent and reversible upon removal of the leukotriene. Pretreatment of lymphocytes slightly decreases the binding and pretreatment of both lymphocytes and endothelium with leukotriene B4prior to the adherence assay did not alter the binding. These results suggest that leukotriene B4regulates exclusively the vascular side, but not the white cell side of this interaction.

In the present experiments the cDNA coding for a truncated form of the β1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67–74-kDa fusion protein. Typically a yield of 750 μg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, converting the acceptor to the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Accordingly, we call the recombinant β1,6-GlcNAc transferase cIGnT6 to emphasize its action atcentral rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitroexpression of I-branching enzyme, in combination with the previously cloned enzymes, β1,4galactosyltransferase and β1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

A brief incubation of lymphocytes with either PMA, stimulating protein kinase C, or with dibutyryl-cAMP, leading to protein kinase A activation, led to increased lymphocyte penetration through intact endothelial monolayers in vitro. The PMA-induced penetration could be dose-dependently down-regulated with a protein kinase C inhibitor, H7. Similarly HA 1004, being mainly a protein kinase A inhibitor, decreased the dibutyryl-cAMP induced penetration. Treatment of lymphocytes with PMA and cAMP did not alter the expression of CD44 homing receptors on lymphocytes. Stimulation of lymphocytes with dibutyryl-cGMP or calcium ionophore had no effect on lymphocyte penetration. These results suggest that activation of both protein kinases A and C is important in the lymphocyte binding to endothelium.

A brief incubation of lymphocytes with either PMA, stimulating protein kinase C, or with dibutyryl‐cAMP, leading to protein kinase A activation, led to increased lymphocyte penetration through intact endothelial monolayers in vitro. The PMA‐induced penetration could be dose‐dependently down‐regulated with a protein kinase C inhibitor, H7. Similarly HA 1004, being mainly a protein kinase A inhibitor, decreased the dibutyryl‐cAMP induced penetration. Treatment of lymphocytes with PMA and cAMP did not alter the expression of CD44 homing receptors on lymphocytes. Stimulation of lymphocytes with dibutyryl‐cGMP or calcium ionophore had no effect on lymphocyte penetration. These results suggest that activation of both protein kinases A and C is important in the lymphocyte binding to endothelium.

We have developed an assay for α2,3-sialidase (EC 3.2.1.18) which employs a biotinylated carbohydrate–polyacrylamide conjugate as substrate for the enzyme. The solution-phase sialidase reactions are followed by a selective capture of biotinylated neoglycoconjugates onto a microtitration plate coated with streptavidin. The amount of the reaction product formed is then rapidly and easily quantified using a product-specific primary antibody and europium chelate-labeled secondary antibody. This method combines the advantages of solution-phase enzymatic reaction and suitability for high-throughput screening typical of solid-phase assays. The assay gives a detectable signal with 0.4% of substrate sites desialylated. We have demonstrated the utility of the assay by measuring α2,3-sialidase activity from crude lysates of cultured rat endothelial cells by using biotinylated sialyl Lewis x glycoconjugate as substrate. Endothelial sialidase(s) showed up to 250-fold higher activity toward soluble compared to immobilized substrate. Product formation detected with an anti-Lewis x antibody was linear in the range of 0.1–4 μg/ml of protein in endothelial cell lysate. High sensitivity of the assay was achieved by using solution-phase enzyme reaction and time-resolved fluorometric detection. The same assay format used here is easily adapted to detect activities of several different glycosidases or glycosyltransferases by using appropriate substrates and antibodies.

Leukocyte traffic into lymph nodes and sites of inflammation is guided by L-selectin. Experiments performed in vitro and with gene-deleted mice suggest that CD34 recognizes L-selectin if decorated by 6-sulfo sialyl Lewis x (sLex) saccharides and the MECA-79 epitope. However, very little is known about glycosylation of human L-selectin ligands. We report here on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of N- and O-linked oligosaccharide fractions from human tonsillar endothelial CD34. All detected O-glycans were sialylated; some were also monosulfated or monosulfated and monofucosylated. If a given CD34-glycan may carry all requirements for L-selectin recognition, that is, both 6-sulfo-sLex and MECA-79 epitopes, only one O-glycan fraction, O-9, SA2Hex3HexNAc3- Fuc1(SO3)1, meets the criteria. A candidate structure is SAα2-3Galβ1-4(Fucα1-3)(6-sulfo)GlcNAcβ1-3Galβ1-3(SAα2-3Galβ1-4GlcNAcβ1-6)GalNAc. However, if sulfo sLex glycans are supplemented with separate sulfated, nonfucosylated O-glycans, saccharides in O-6, O-8, or O-9, putatively carrying MECA-79 epitopes, could form multiglycan binding epitopes for L-selectin.

Leukocyte traffic into lymph nodes and sites of inflammation is guided by L-selectin. Experiments performed in vitro and with gene-deleted mice suggest that CD34 recognizes L-selectin if decorated by 6-sulfo sialyl Lewis x (sLex) saccharides and the MECA-79 epitope. However, very little is known about glycosylation of human L-selectin ligands. We report here on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of N- and O-linked oligosaccharide fractions from human tonsillar endothelial CD34. All detected O-glycans were sialylated; some were also monosulfated or monosulfated and monofucosylated. If a given CD34-glycan may carry all requirements for L-selectin recognition, that is, both 6-sulfo-sLex and MECA-79 epitopes, only one O-glycan fraction, O-9, SA2Hex3HexNAc3- Fuc1(SO3)1, meets the criteria. A candidate structure is SAα2-3Galβ1-4(Fucα1-3)(6-sulfo)GlcNAcβ1-3Galβ1-3(SAα2-3Galβ1-4GlcNAcβ1-6)GalNAc. However, if sulfo sLex glycans are supplemented with separate sulfated, nonfucosylated O-glycans, saccharides in O-6, O-8, or O-9, putatively carrying MECA-79 epitopes, could form multiglycan binding epitopes for L-selectin.

Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50–2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3′-phosphate 5′-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.