Ethinylestradiol in combined hormonal contraceptive has a broader effect on serum proteome compared with estradiol valerate: a randomized controlled trial.

STUDY QUESTION: Does an estradiol-based combined oral contraceptive (COC) have a milder effect on the serum proteome than an ethinylestradiol (EE)-based COC or dienogest (DNG) only? SUMMARY ANSWER: The changes in serum proteome were multifold after the use of a synthetic EE-based COC compared to natural estrogen COC or progestin-only preparation. WHAT IS KNOWN ALREADY: EE-based COCs widely affect metabolism, inflammation, hepatic protein synthesis and blood coagulation. Studies comparing serum proteomes after the use of COCs containing EE and natural estrogens are lacking. STUDY DESIGN, SIZE, DURATION: This was a spin-off from a randomized, controlled, two-center clinical trial. Women (n = 59) were randomized to use either EE + DNG, estradiol valerate (EV) + DNG or DNG only continuously for 9 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were healthy, young, white volunteer women. Serum samples were collected before and after 9 weeks of hormonal exposure. Samples from 44 women were available for analysis (EE + DNG n = 14, EV + DNG n = 16 and DNG only n = 14). Serum proteins were analyzed by quantitative, discovery-type label-free proteomics. MAIN RESULTS AND THE ROLE OF CHANCE: Altogether, 446 proteins/protein families with two or more unique peptides were detected and quantified. The number of proteins/families that altered over the 9-week period within the study groups was 121 for EE + DNG and 5 for EV + DNG, while no changes were detected for DNG only. When alterations were compared between the groups, significant differences were detected for 63 proteins/protein families, of which 58 were between the EE + DNG and EV + DNG groups. The most affected functions during the use of EE + DNG were the complement system, acute phase response signaling, metabolism and the coagulation system. The results were validated by fetuin-B and cortisol-binding globulin ELISA and sex hormone-binding globulin immunoassay. LARGE SCALE DATA: Data are available via ProteomeXchange with identifiers PXD033617 (low abundance fraction) and PXD033618 (high abundance fraction). LIMITATIONS, REASONS FOR CAUTION: The power analysis of the trial was not based on the proteomic analysis of this spin-off study. In the future, targeted proteomic analysis with samples from another trial should be carried out in order to confirm the results. WIDER IMPLICATIONS OF THE FINDINGS: The EE-based COC exerted a broader effect on the serum proteome than the EV-based COC or the DNG-only preparation. These results demonstrate that the effects of EE in COCs go far beyond the established endpoint markers of estrogen action, while the EV combination is closer to the progestin-only preparation. The study indicates that EV could provide a preferable option to EE in COCs in the future and signals a need for further studies comparing the clinical health outcomes of COCs containing EE and natural estrogens. STUDY FUNDING/COMPETING INTEREST(S): Funding for this researcher-initiated study was obtained from the Helsinki University Hospital research funds, the Hospital District of Helsinki and Uusimaa, the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Medical Association, the University of Oulu Graduate School, the Emil Aaltonen Foundation, the Swedish Cultural Foundation in Finland, the Novo Nordisk Foundation, Orion Research Foundation and the Northern Ostrobothnia Regional Fund. The funders had no role in study design, data collection and analysis, publishing decisions or manuscript preparation. T.P. has received honoraria for lectures, consultations and research grants from Exeltis, Gedeon Richter, MSD, Merck, Pfizer, Roche, Stragen and Mithra Pharmaceuticals. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. O.H. occasionally serves on advisory boards for Bayer AG and Gedeon Richter and has designed and lectured at educational events for these companies. The other authors have nothing to disclose. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02352090. TRIAL REGISTRATION DATE: 27 January 2015. DATE OF FIRST PATIENT'S ENROLMENT: 1 April 2015.

Thromboinflammatory changes in plasma proteome of pregnant women with PCOS detected by quantitative label-free proteomics.

Polycystic ovary syndrome (PCOS) is the most common endocrinological disorder of fertile-aged women. Several adverse pregnancy outcomes and abnormalities of the placenta have been associated with PCOS. By using quantitative label-free proteomics we investigated whether changes in the plasma proteome of pregnant women with PCOS could elucidate the mechanisms behind the pathologies observed in PCOS pregnancies. A total of 169 proteins with ≥2 unique peptides were detected to be differentially expressed between women with PCOS (n = 7) and matched controls (n = 20) at term of pregnancy, out of which 35 were significant (p-value < 0.05). A pathway analysis revealed that networks related to humoral immune responses, inflammatory responses, cardiovascular disease and cellular growth and proliferation were affected by PCOS. Classification of cases and controls was carried out using principal component analysis, orthogonal projections on latent structure-discriminant analysis (OPLS-DA), hierarchical clustering, self-organising maps and ROC-curve analysis. The most significantly enriched proteins in PCOS were properdin and insulin-like growth factor II. In the dataset, properdin had the best predictive accuracy for PCOS (AUC = 1). Additionally, properdin abundances correlated with AMH levels in pregnant women.

Higher mortality of adults with asthma: A 15-year follow-up of a population-based cohort.

BACKGROUND: Higher all-cause mortality in asthmatics has been shown previously. Polysensitization is associated with higher morbidity among asthmatic children, and allergic rhinitis and/or allergic conjunctivitis (AR/AC) are associated with higher morbidity in adult asthmatics. Little is known about the effect of AR/AC and other factors on mortality among adult asthmatics. The aim was to study mortality and its risk factors in adults with and without asthma. METHODS: We randomly selected 1648 asthmatics with age over 30 years from national registers and matched the asthma sample with one or two controls. Baseline information was obtained by a questionnaire in 1997, and the study population was linked with the death certificate information of Statistics Finland from 1997 to 2013. Overall and cause-specific survival between the groups was compared in several adjusted models. RESULTS: During a mean follow-up period of 15.6 years, 221 deaths among 1052 asthma patients and 335 deaths among 1889 nonasthmatics were observed. Cardiovascular diseases were the main cause of death in both groups. Asthma was associated with increased all-cause mortality (adjusted HR 1.25; 95% CI 1.05-1.49, P = .011) as well as mortality from chronic obstructive pulmonary disease (HR 12.0, 4.18-34.2, P < .001) and malignant neoplasms of respiratory organs (HR 2.33, 1.25-4.42, P = .008). Among asthmatics, smoking was associated with increased all-cause mortality, and self-reported AR/AC was associated with decreased mortality. Among nonasthmatics, smoking, and obesity were associated with increased all-cause mortality, whereas female gender showed an association with a decreased risk. CONCLUSIONS: Increased mortality among adult asthmatics was largely explained by the development of COPD, malignant respiratory tract neoplasms, and cardiovascular diseases. Smoking cessation is important for reduction in total mortality in both asthmatic and nonasthmatic adults. AR/AC was associated with decreased mortality only in asthmatics. Thus, studies in other populations of larger size are needed to explore further the nature of this association.

Low lymphatic vessel density associates with chronic rhinosinusitis with nasal polyps.

OBJECTIVES: Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP) and antrochoanal polyps (ACP) are different upper airway inflammation phenotypes with different pathomechanisms. In order to understand the development of tissue edema, the present study aimed to evaluate lymphatic vessel density in CRSsNP, CRSwNP and ACP. MATERIALS AND METHODS: 120 retrospective nasal and maxillary sinus specimens were stained immunohistochemically with a von Willebrand factor polyclonal antibody recognizing vascular and lymphatic endothelium, and with a podoplanin monoclonal antibody recognizing lymphatic endothelium. Vessels were studied by microscopy in a blinded fashion, and the vessel density and the relative density of lymphatic vessels were calculated. Patient characteristic factors and follow-up data of in average 9 years were collected from patient records. RESULTS AND CONCLUSION: In the nasal cavity, the low absolute and relative density of vessels and of lymphatic vessels was associated with CRSwNP and ACP tissues compared to control inferior turbinate. This was observed also in the inflammatory hotspot area. In the maxillary sinus, lower absolute and relative density of lymphatic vessels associated with the CRSwNP phenotype. High lymphatic vessel density in polyp tissue associated with the need for revision CRS-surgery. As a conclusion, low density of lymphatic vessels distinguished patients with CRSwNP not only in the hotspot area of polyp tissue, but also in maxillary sinus mucosa. Yet, higher lymphatic vessel density seems to associate with polyp recurrence. Further studies are still needed to explore if formation of nasal polyps could be diminished by intranasal therapeutics affecting lymphangiogenesis.

Birth decade affects the sensitization pattern and asthma risk in Finnish adult population.

We have previously shown that sensitizations to several types of allergens distinguish subjects with and without adult-onset asthma in Finland. The aim was to analyze how age affects sensitization and asthma risk. We used previous population-based case-control data (N=456) from Finnish adult asthma patients with one or two matched controls. Asthma was diagnosed based on a typical history of asthmatic symptoms and lung function tests. Allergic sensitization was determined by skin prick test (SPT) to 17 aeroallergens. Information on demographics was obtained by a questionnaire. Sensitization to more than one allergen type and the number of positive SPT reactions associated with younger age and asthma. Atopic subjects aged 65 and above were characterized by sensitization to only one to two allergens, with very few animal danders and without an association with asthma. Multiple sensitizations and animal dander sensitization are more common among Finnish asthmatic adults aged under 56 than among older asthmatics. Cohort studies are needed to understand timing of host-environmental interactions behind this.

Sensitization pattern affects the asthma risk in Finnish adult population.

BACKGROUND: There is a large global variation in the sensitization pattern and its association with allergic diseases. In temperate and tropical urban environments, mite monosensitization can be the predominant cause of allergic airway diseases, whereas in other environments, polysensitization is more typical. Sensitization to mite allergens associates with asthma. However, it is suggested that mite sensitization might play a minor role in Northern Europe. The aim of the study was to analyze how sensitization pattern affects the asthma risk in Finnish adults, with a special focus on mites. METHODS: A population-based case-control data (N = 523) from Finnish adult asthma patients with one or two matched controls were used. Asthma was diagnosed based on a typical history of asthmatic symptoms and lung function tests. The allergic sensitization was determined based on skin prick test (SPT) of five mites, three molds, and nine other aeroallergens. Information on demographics was obtained by a questionnaire. RESULTS: The proportion of sensitization to any allergen was 55% in the asthma group and 39% in the control group (P = 0.001, OR 2.06, 95% CI = 1.35-3.14). Sensitization to animal dander, pollen, or Aspergillus fumigatus was associated with asthma. Polysensitization to more than one allergen types and the number of SPT-positive reactions associated with asthma, whereas sensitization to only one allergen type was not associated with asthma. CONCLUSIONS: The large number of sensitizations to several types of allergens distinguishes subjects with asthma. Mite sensitization had little independent association with asthma in Finland.

Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug.

Malignant mesothelioma is a form of cancer that is highly resistant to conventional cancer therapy for which no major therapeutic advances have been introduced. Here, we identify gremlin-1, a known bone morphogenetic protein inhibitor crucial for embryonic development, as a potential therapeutic target for mesothelioma. We found high expression levels of gremlin-1 in the mesothelioma tumor tissue, as well as in primary mesothelioma cells cultured from pleural effusion samples. Downregulation of gremlin-1 expression by siRNA-mediated silencing in a mesothelioma cell line inhibited cell proliferation. This was associated with downregulation of the transcription factor slug as well as mesenchymal proteins linked to cancer epithelial-to-mesenchymal transition. Further, resistance to paclitaxel-induced cell death was associated with high gremlin-1 and slug expression. Treatment of gremlin-1-silenced mesothelioma cells with paclitaxel or pemetrexed resulted in efficient loss of cell survival. Finally, our data suggest that concomitant upregulation of fibrillin-2 in mesothelioma provides a mechanism for extracellular localization of gremlin-1 to the tumor microenvironment. This was supported by the demonstration of interactions between gremlin-1, and fibrillin-1 and -2 peptides as well as by colocalization of gremlin-1 to fibrillin microfibrils in cells and tumor tissue samples. Our data suggest that gremlin-1 is also a potential target for overcoming drug resistance in mesothelioma.

The multi-faceted role of allergen exposure to the local airway mucosa.

Airway epithelial cells are the first to encounter aeroallergens and therefore have recently become an interesting target of many studies investigating their involvement in the modulation of allergic inflammatory responses. Disruption of a passive structural barrier composed of epithelial cells by intrinsic proteolytic activity of allergens may facilitate allergen penetration into local tissues and additionally affect chronic and ongoing inflammatory processes in respiratory tissues. Furthermore, the ability of rhinoviruses to disrupt and interfere with epithelial tight junctions may alter the barrier integrity and enable a passive passage of inhaled allergens through the airway epithelium. On the other hand, epithelial cells are no longer considered to act only as a physical barrier toward inhaled allergens, but also to actively contribute to airway inflammation by detecting and responding to environmental factors. Epithelial cells can produce mediators, which may affect the recruitment and activation of more specialized immune cells to the local tissue and also create a microenvironment in which these activated immune cells may function and propagate the inflammatory processes. This review presents the dual role of epithelium acting as a passive and active barrier when encountering an inhaled allergen and how this double role contributes to the start of local immune responses.

Predictors of post-operative response to treatment: a double blind placebo controlled study in chronic rhinosinusitis patients.

BACKGROUND: In the majority of CRS patients suffering from primary or recurrent CRS, topical glucocorticoids are highly effective. A subset of CRS patients, however, does not respond to (topical) glucocorticoids and requires surgical intervention. Although surgery is highly effective in those individuals, recurrence of disease is observed in some. In this study we describe our search for one or more predictors predicting the response to surgery in combination with peri-operative oral glucocorticoids in CRS patients. METHODS: Thirty-five inferior turbinate specimens were randomly selected from a larger group of CRS patients requiring FESS for persistent disease that either responded favorably or demonstrated recurrent disease. Tissue biopsies were taken at the time of surgery and compared for inflammatory markers, endothelial cell markers, and various leukocyte subsets using immunohistochemistry. RESULTS: Compared to non-responders, the baseline level of lamina propria activated eosinophils is significantly increased in CRS patients responding to surgery in combination with peri-operative oral glucocorticoids treated or not treated post-operatively with topical glucocorticoids. No significant differences were observed for all other studied parameters. Post-operative treatment with FPANS 100 µg q.i.d. was significantly associated with response to treatment. A trend towards association was observed for increased numbers of eosinophils at baseline. CONCLUSION: Our data suggest that CRS patients with higher levels of eosinophils are less likely to suffer from post-operative recurrent sinonasal disease when treated post-operatively with FPANS 100 µg q.i.d.

Endothelial L-selectin ligand expression in nasal polyps.

BACKGROUND: L-selectins on leukocytes and their counter-receptors on endothelial cells have been shown to be involved in leukocyte recruitment in chronic rhinosinusitis without nasal polyps (NP). OBJECTIVES: The purpose of this study was to evaluate the expression level of functionally active endothelial L-selectin ligands in NP obtained from patients with NP of different etiology [simple NP, antro-choanal polyps (ACP) and cystic fibrosis (CF) NP] and inferior turbinate specimens of healthy controls and to compare these levels to the presence of various leukocyte subsets. METHODS: Nasal polyp specimens and healthy nasal mucosa specimens were obtained from patients undergoing surgery and were immunohistochemically stained with monoclonal antibodies detecting CD34, sialyl Lewis x (sLe(x)) of sulfated extended core 1 lactosamines and various leukocyte subsets. RESULTS: All NP are characterized by a decrease in the number of CD34+ vessels. The number of eosinophils and the percentage of vessels expressing endothelial sulfated sLe(x) epitopes is upregulated in all groups of simple NP. Tissue eosinophilia is increased in those patients with increased disease severity (acetyl salicylic acid intolerance), but the percentage of endothelial sulfated sLe(x) epitopes is not. Results on CF NP are similar to those observed for simple NP. Antro-choanal polyps, on the contrary, are characterized by low numbers of tissue eosinophils and relatively few vessels expressing endothelial sulfated sLe(x) epitopes. CONCLUSIONS: Our results suggest that functionally active L-selectin ligands might play a role in guiding leukocyte traffic into NP in patients with simple NP and CF NP but not ACP.

A network analysis of the single nucleotide polymorphisms in acute allergic diseases.

BACKGROUND: Genetics of acute allergies has focused on identifying single nucleotide polymorphisms (SNPs) within genes relevant in the pathogenesis. In this study, we begin a systems biology analysis of the interconnectivity and biological functions of these genes, their transcripts and their corresponding proteins. METHODS: The literature (Pubmed) was searched for SNPs within genes relevant in acute allergic diseases. The SNP-modified genes were converted to corresponding proteins and their protein-protein interactions were searched from six different databases. This interaction network was analysed with annotated vocabularies (ontologies), such as Gene Ontology, Reactome and Nature pathway interaction database. Time-series transcriptomics was performed with nasal epithelial cells obtained from allergic patients and their healthy control subjects. RESULTS: A total of 39 genes with SNPs related to acute allergic diseases were found from a literature search. The corresponding proteins were then hooked into a large protein-protein interaction network with the help of various databases. Twenty-five SNP-related proteins had more than one interacting protein and a network contained 95 proteins, and 182 connections could be generated. This network was 10-fold enriched with protein kinases and proteins involved in the host-virus interaction compared with background human proteome. Finally, eight of the 95 nodes on our network displayed nasal epithelial transcriptomal regulation in a time-series analysis collected from birch allergic patients during the spring pollen season. CONCLUSIONS: Signal transduction with special reference to host-virus interactions dominated in the allergy-related protein interaction network. Systems level analysis of allergy-related mutation can provide new insights into pathogenetic mechanisms of the diseases.

Time-series nasal epithelial transcriptomics during natural pollen exposure in healthy subjects and allergic patients.

BACKGROUND: The role of epithelium has recently awakened interest in the studies of type I hypersensitivity. OBJECTIVE: We analysed the nasal transcriptomics epithelial response to natural birch pollen exposure in a time series manner. METHODS: Human nasal epithelial cell swabs were collected from birch pollen allergic patients and healthy controls in winter season. In addition, four specimens at weekly intervals were collected from the same subjects during natural birch pollen exposure in spring and transcriptomic analyses were performed. RESULTS: The nasal epithelium of healthy subjects responded vigorously to allergen exposure. The immune response was a dominating category of this response. Notably, the healthy subjects did not display any clinical symptoms regardless of this response detected by transcriptomic analysis. Concomitantly, the epithelium of allergic subjects responded also, but with a different set of responders. In allergic patients the regulation of dyneins, the molecular motors of intracellular transport dominated. This further supports our previous hypothesis that the birch pollen exposure results in an active uptake of allergen into the epithelium only in allergic subjects but not in healthy controls. CONCLUSION: We showed that birch pollen allergen causes a defence response in healthy subjects, but not in allergic subjects. Instead, allergic patients actively transport pollen allergen through the epithelium to tissue mast cells. Our study showed that new hypotheses can arise from the application of discovery driven methodologies. To understand complex multifactorial diseases, such as type I hypersensitivity, this kind of hypotheses might be worth further analyses.

Modelling of Bet v 1 binding to lipids.

As birch pollen allergen enters epithelium of allergic patients via lipid rafts and caveola we began to analyse its putative amphiphilic and lipid ligands on atomic level using molecular modelling and computational ligand docking. We carry out 3D modelling docking with both experimentally verified Bet v 1 ligands as well as larger lipid molecules for which experimental affinity studies were not available. The results suggest that the hydrophobic cavity of Bet v 1 has different binding sites for different ligands and groups of ligand type-specific amino acids can be defined. Bet v 1 proteins may also be able to bind and transport more complex amphiphilic molecules like ceramides and sphingomyelins known to be enriched on caveolae/lipid rafts. Furthermore, the suggested binding mode, where the hydrophobic tail groups of lipids locate inside Bet v 1, while the polar head group may remain solvent accessible, would allow Bet v 1 to bind glycolipids, e.g. gangliosides, also rich on caveolae/lipid rafts. Taken together, this in silico work suggests that Bet v 1 bind to amphiphilic and lipid ligands present on the caveolae/lipid rafts and thus could provide a molecular mechanism for the pollen entry to epithelial tissue of allergic patients.

Birch pollen allergen Bet v 1 binds to and is transported through conjunctival epithelium in allergic patients.

BACKGROUND: Previous work in type-I pollen allergies has mainly focused on lymphocytes and immune responses. Here, we begin to analyse with a systems biology view the differences in conjunctival epithelium obtained from healthy and allergic subjects. METHODS: Transcriptomics analysis combined with light and electron microscopic analysis of birch pollen allergen Bet v 1 located within conjunctival epithelial cells and tissues from birch allergic subjects and healthy controls was carried out. RESULTS: Bet v 1 pollen allergen bound to conjunctival epithelial cells within minutes after the exposure even during the nonsymptomatic winter season only in allergic, but not in healthy individuals. Light- and electron microscopy showed that Bet v 1 was transported through the epithelium within lipid rafts/caveolae and reached mast cells only in allergic patients, but not in healthy individuals. Transcriptomics yielded 22 putative receptors expressed at higher levels in allergic epithelium compared with healthy specimens. A literature search indicated that out of these receptors, eight (i.e. 37%) were associated with lipid rafts/caveolae, which suggested again that Bet v 1 transport is lipid raft/caveola-dependent. CONCLUSIONS: We show a clear difference in the binding and uptake of Bet v 1 allergen by conjunctival epithelial cells in allergic vs healthy subjects and several putative lipid raft/caveolar receptors were identified, which could mediate or be co-transported with this entry. The application of discovery driven methodologies on human conjunctival epithelial cells and tissues can provide new hypotheses worth a further analysis to the molecular mechanisms of a complex multifactorial disease such as type-I birch pollen allergy.

Interplay between coagulation and inflammation in open and endovascular abdominal aortic aneurysm repair--impact of intra-aneurysmal thrombus.

AIMS: Our aim was to compare hemostatic and inflammatory mechanisms in abdominal aortic aneurysm (AAA) patients after open surgery (OPEN) and endovascular AAA repair (ENDO). SUBJECTS AND METHODS: From the 32 consecutive AAA patients recruited, 17 represented ENDO and 15 OPEN. The intra-aneurysmal thrombus was removed during OPEN, but stayed intact after ENDO. The preoperative volume of the intra-aneurysmal thrombus was calculated from computed tomography images. Markers of coagulation and inflammation were studied preoperatively, at one, two, three, four and seven days and at three months postoperatively. RESULTS: Preoperative upregulation of F 1+2, TAT and D-dimer was evident in both groups. The volume of intra-aneurysmal thrombus correlated with CRP (beta = 0.62, p = 0.001), IL-6 (beta = 0.60, p = 0.001) and PAI-1 ag (beta = 0.51, p = 0.007). Surgery further enhanced inflammation, coagulation and fibrinolysis. IL-6 increased in both groups, but the increases of CRP and PIIINP were higher in the OPEN group. Postoperative CRP correlated with the intra-aneurysmal thrombus volume in the ENDO group. At three months D-dimer (p < 0.05) was higher than preoperatively in the ENDO, in contrast to the OPEN group. CONCLUSION: Preoperatively both prothrombotic and fibrinolytic mechanisms are activated in patients with AAA. Intraluminal thrombus induces prothrombotic and inflammatory interactions, which persist after endovascular aortic aneurysm repair.

Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae.

Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O-antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H. pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.

Core 2 beta1,6-N-acetylglucosaminyltransferases and alpha1,3-fucosyltransferases regulate the synthesis of O-glycans on selectin ligands on oral cavity carcinoma cells.

Selectin-dependent cell binding has importance in the extravasation of blood-circulating tumor cells and in the generation of metastases. Cell surface glycoproteins decorated with sialylated, fucosylated epitopes, such as sialyl Lewis(x) (sLe(x)), are ligands for selectins. Not only terminal sLe(x) moieties but also proximal core structures contribute to the formation of binding epitopes for selectins. Core 2 beta1,6-N-acetylglucosaminyltransferases (C2GnT) and alpha1,3-fucosyltransferases (alpha1,3-FucT) have been suggested to be the rate-limiting enzymes in the synthesis of selectin ligands. We analyzed oral cavity epithelial carcinoma cell lines and showed their expression of RNA transcripts for C2GnT and alpha1,3-FucT, identified alpha1,3-FucT enzyme activities, and analyzed the cell surface sLe(x) expression levels. Neither the pattern of expressed enzymes nor the alpha1,3-FucT activity directly predicted the binding capacity of E-selectin. However, only the sLe(x)-expressing cell lines were capable of binding to E-selectin, but not to P-selectin, thus putatively promoting the selectin-mediated metastasis. These findings suggest that C2GnT in combination with alpha1,3-Fuc-T contribute to the selectin-mediated metastasis in oral cavity carcinomas.

Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography.

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.

Endothelial sulfated sialyl Lewis x glycans, putative L-selectin ligands, are preferentially expressed in bronchial asthma but not in other chronic inflammatory lung diseases.

Lymphocyte infiltrate is a hallmark of inflammatory responses. We have previously shown that de novo-induced endothelial sialyl Lewis x (sLex) expression guides lymphocytes in an L-selectin-dependent manner to sites of acute organ transplant rejections. In this research, we have analyzed five groups of chronic lung inflammations to determine the presence of properly glycosylated, i.e., sulfated, sLex-decorated, L-selectin ligands. Two anti-sLex (2F3 and HECA-452) and one anti-6- and/or 6'-sulfated and/or 6,6'-bisulfated (MECA-79) monoclonal antibodies (mAbs) were used. The control lung specimens did not express L-selectin ligands on endothelium. In contrast, the endothelial staining intensity and the number of positive peribronchial venules and capillaries with mAbs 2F3, HECA-452, and MECA-79 were significantly greater in bronchial biopsies from patients with asthma compared with normal specimens (P<0.003). However, no significant increase of peribronchial endothelial reactivity with these antibodies was observed in adult respiratory distress syndrome, chronic bronchitis, fibrosing alveolitis, and granulomatous inflammation compared with controls. These data suggest that sulfated sLex glycans, acting putatively as ligands for L-selectin, could be instrumental in lymphocyte extravasation into human peribronchial lung tissue during asthma, but not so important in several other inflammatory lung diseases.