Altered microheterogeneity at several N-glycosylation sites in OPSCC in constant protein expression conditions.

Protein glycosylation responds sensitively to disease states. It is implicated in every hallmark of cancer and has recently started to be considered as a hallmark itself. Changes in N-glycosylation microheterogeneity are more dramatic than those of protein expression due to the non-template nature of protein glycosylation. This enables their potential use in serum-based diagnostics. Here, we perform glycopeptidomics on serum from patients with oropharyngeal squamous cell carcinoma (OPSCC), compared to controls and comparing between cancers based on etiology (human papilloma virus- positive or negative). Using MS2, we then targeted glycoforms, significantly different between the groups, to identify their glycopeptide compositions. Simultaneously we investigate the same serum proteins, comparing whether N-glycosylation changes reflect protein-level changes. Significant glycoforms were identified from proteins such as alpha-1-antitrypsin (SERPINA1), haptoglobin, and different immunoglobulins. SERPINA1 had glycovariance at 2 N-glycosylation sites, that were up to 35 times more abundant in even early-stage OPSCCs, despite minimal differences between SERPINA1 protein levels between groups. Some identified glycoforms' fold changes (FCs) were in line with serum protein level FCs, others were less abundant in early-stage cancers but with great variance in higher-stage cancers, such as on immunoglobulin heavy constant gamma 2, despite no change in protein levels. Such findings indicate that glycovariant analysis might be more beneficial than proteomic analysis, which is yet to be fruitful in the search for biomarkers. Highly sensitive glycopeptide changes could potentially be used in the future for cancer screening. Additionally, characterizing the glycopeptide changes in OPSCC is valuable in the search for potential therapeutic targets.

Quantitative glycoproteomics of human milk and association with atopic disease.

The prevalence of allergic diseases and asthma is increasing rapidly worldwide, with environmental and lifestyle behaviors implicated as a reason. Epidemiological studies have shown that children who grow up on farms are at lower risk of developing childhood atopic disease, indicating the presence of a protective "farm effect". The Old Order Mennonite (OOM) community in Upstate New York have traditional, agrarian lifestyles, a low rate of atopic disease, and long periods of exclusive breastfeeding. Human milk proteins are heavily glycosylated, although there is a paucity of studies investigating the milk glycoproteome. In this study, we have used quantitative glycoproteomics to compare the N-glycoprotein profiles of 54 milk samples from Rochester urban/suburban and OOM mothers, two populations with different lifestyles, exposures, and risk of atopic disease. We also compared N-glycoprotein profiles according to the presence or absence of atopic disease in the mothers and, separately, the children. We identified 79 N-glycopeptides from 15 different proteins and found that proteins including immunoglobulin A1, polymeric immunoglobulin receptor, and lactotransferrin displayed significant glycan heterogeneity. We found that the abundances of 38 glycopeptides differed significantly between Rochester and OOM mothers and also identified four glycopeptides with significantly different abundances between all comparisons. These four glycopeptides may be associated with the development of atopic disease. The findings of this study suggest that the differential glycosylation of milk proteins could be linked to atopic disease.

Expression of Toll-like receptors in nasal epithelium in allergic rhinitis.

Toll-like receptors (TLRs) are important in barrier homeostasis, but their role in airborne allergies is not fully understood. The aim was to evaluate baseline and allergen-induced expression of TLR proteins in nasal epithelium during allergic rhinitis. Nineteen otherwise healthy non-smoking volunteers both allergic to birch pollen and non-allergic controls were enrolled. We took nasal biopsies before and after off-seasonal intranasal birch pollen or diluent challenge. The expression of epithelial TLR1-7, TLR9-10, and MyD88 proteins was immunohistochemically evaluated from the nasal biopsies. The TLR1-3 and TLR5-10 mRNAs were observed by RNA-microarray. Baseline epithelial expression of TLR proteins was wide and identical in controls and atopics. After off-seasonal intranasal birch pollen challenge, a negative change in the expression score of TLR1 and TLR6 proteins was detected in the atopic group. TLR mRNA expression was not affected by birch pollen challenge. Nasal epithelium seems to express all known TLRs. The mechanisms by which TLR1, and TLR6 proteins could affect pollen allergen transport need further studies.

Molecular mechanisms of nasal epithelium in rhinitis and rhinosinusitis.

Allergic rhinitis, nonallergic rhinitis, and chronic rhinosinusitis are multifactorial upper airway diseases with high prevalence. Several genetic and environmental factors are proposed to predispose to the pathogenesis of the inflammatory upper airway diseases. Still, the molecular mechanisms leading toward the onset and progression of upper airway diseases are largely unknown. The upper airway epithelium has an important role in sensing the environment and regulating the inhaled air. As such, it links environmental insults to the host immunity. Human sinonasal epithelium serves as an excellent target for observing induced early-phase events, in vivo, and with a systems biological perspective. Actually, increasing number of investigations have provided evidence that altered homeostasis in the sinonasal epithelium might be important in the chronic upper airway inflammation.

Allergy as an epithelial barrier disease.

The objective of this review is to focus on putative modified epithelial functions related to allergy. The dysregulation of the epithelial barrier might result in the allergen uptake, which could be the primary defect in the pathogenesis of allergic reaction. We review the literature of the role of respiratory epithelium as an active barrier, how allergens are transported through it and how it senses the hostile environmental allergens and other dangerous stimuli.

Allergen interactions with epithelium.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: Birch pollen allergen was rapidly and actively transported through the respiratory epithelium via caveolar-dependent mechanisms only in patients allergic to birch pollen but not their healthy controls. Transcriptomic analyses showed that whereas healthy individuals raised a strong epithelial response after intranasal allergen challenge, the allergic patients had a reduced response. Thus allergies could also be due to hyporeactive responses on the epithelial level. SUMMARY: Epithelium has emerged as an active and complex organ with mechanical, biochemical and immunological functions. The increasing awareness that epithelium interacts actively with allergens might provide new targets for the prevention and management of allergy.

Allergens are transported through the respiratory epithelium.

We used a top-down approach with a wide repertoire of wet laboratory and in silico techniques for analyzing the pathogenesis of early events within the type I allergic reactions. We could show a caveolar-dependent transport of the birch pollen allergen through the respiratory epithelium of allergic patients but not of their healthy controls. The application of discovery-driven methodologies can provide new hypotheses worth further analyses of complex multifactorial diseases such as type I allergy.

Network analysis of single nucleotide polymorphisms in asthma.

BACKGROUND: Asthma is a chronic inflammatory disease of the airways with a complex genetic background. In this study, we carried out a meta-analysis of single nucleotide polymorphisms (SNPs) thought to be associated with asthma. METHODS: The literature (PubMed) was searched for SNPs within genes relevant in asthma. The SNP-modified genes were converted to corresponding proteins, and their protein-protein interactions were searched from six different databases. This interaction network was analyzed using annotated vocabularies (ontologies), such as the Gene Ontology and Nature pathway interaction databases. RESULTS: In total, 127 genes with SNPs related to asthma were found in the literature. The corresponding proteins were then entered into a large protein-protein interaction network with the help of various databases. Ninety-six SNP-related proteins had more than one interacting protein each, and a network containing 309 proteins and 644 connections was generated. This network was significantly enriched with a gene ontology entitled "protein binding" and several of its daughter categories, including receptor binding and cytokine binding, when compared with the background human proteome. In the detailed analysis, the chemokine network, including eight proteins and 13 toll-like receptors, were shown to interact with each other. Of great interest are the nonsynonymous SNPs which code for an alternative amino acid sequence of proteins and, of the toll-like receptor network, TLR1, TLR4, TLR5, TLR6, TLR10, IL4R, and IL13 are among these. CONCLUSIONS: Protein binding, toll-like receptors, and chemokines dominated in the asthma-related protein interaction network. Systems level analysis of allergy-related mutations can provide new insights into the pathogenetic mechanisms of disease.

Caveolar transport through nasal epithelium of birch pollen allergen Bet v 1 in allergic patients.

BACKGROUND: Previous work in type I pollen allergies has focused on aberrant immunoresponses. OBJECTIVE: Our systems-level analyses explore the role of epithelium in early pathogenesis of type I allergic reactions. METHODS: We began top-down analyses of differences in human nasal epithelial cells and biopsy specimens obtained from patients with birch allergy and healthy control subjects in the resting state and after intranasal in vivo birch pollen challenges. Immunohistochemistry, immunotransmission electron microscopy, mass spectrometry, transcriptomics, and integration of data to a pathway were conducted. RESULTS: Bet v 1 allergen bound to epithelium immediately after in vivo birch pollen challenge during winter only in allergic individuals. It also travelled through epithelium with caveolae to mast cells. Sixteen unique proteins were found to bind to the Bet v 1 column only in lysates from allergic epithelial cells; 6 of these were caveolar and 6 were cytoskeletal proteins. The nasal epithelial transcriptome analysis from allergic and healthy subjects differed during the winter season, and these subjects also responded differentially to birch pollen challenge. Within this pollen-induced response, the gene ontology categories of cytoskeleton and actin cytoskeleton were decreased in allergic patients, whereas the actin-binding category was enriched in healthy subjects. Integration of microscopic, mass spectrometric, and transcriptomic data to a common protein-protein binding network showed how these were connected to each other. CONCLUSION: We propose a hypothesis of caveolae-dependent uptake and transport of birch pollen allergen in the epithelium of allergic patients only. Application of discovery-driven methodologies can provide new hypotheses worth further analysis of complex multifactorial diseases, such as type I allergy.

Endothelial L-selectin ligands in sinus mucosa during chronic maxillary rhinosinusitis.

RATIONALE: Chronic rhinosinusitis 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 organ-specific leukocyte infiltration into inflamed tissues. OBJECTIVES: The purpose of this study was to evaluate the endothelial expression of functionally active endothelial L-selectin ligands, sulfated sialyl Lewis x, in maxillary sinus mucosa from patients with chronic rhinosinusitis and from normal control subjects. METHODS: Maxillary sinus mucosa specimens (116) were obtained surgically and immunohistochemically stained with monoclonal antibodies detecting sialyl Lewis x or sulfated extended core 1 lactosamines. The severity of the inflammation was determined by intraoperative endoscopic findings, computed tomography scans, and histopathologic assessment of the specimens. MEASUREMENTS AND MAIN RESULTS: The percentage of vessels expressing endothelial sulfated sialyl Lewis x epitopes increased during chronic rhinosinusitis compared with uninflamed control tissue, especially in patients with additional allergic rhinitis, and decreased in specimens from aspirin-intolerant patients with preoperative oral corticosteroid treatment. In addition, the expression level of endothelial sulfated sialyl Lewis x epitopes and the number of mucosal eosinophils correlated with the severity of the inflammation, and decreased in specimens taken 9 months postoperatively compared with intraoperative samples, especially in patients with intranasal corticosteroid treatment. CONCLUSIONS: Our results suggest that functionally active L-selectin ligands might guide leukocyte traffic into maxillary sinus mucosa preferentially in patients with severe findings of chronic maxillary rhinosinusitis, thus leading to aggravation of the inflammation.

Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites.

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 novo induction 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.

A peptide mimic of selectin ligands abolishes in vivo inflammation but has no effect on the rat heart allograft survival1.

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.

Expression of cyclo-oxygenase-2 in human tongue carcinoma and its precursor lesions.

The expression of Cox-2 protein was studied by immunohistochemistry in normal oral mucosa and in mucosa with various lesions of oral leukoplakia, including hyperplasia and dysplasia of squamous epithelium and frank invasive squamous carcinoma. A gradient of Cox-2 staining was found: the expression of Cox-2 was lowest in normal epithelium, somewhat increased in hyperplastic epithelium, further increased in dysplastic epithelium, and highest in invasive squamous cell carcinomas. The presence of Cox-2 in squamous cell carcinomas of the oral mucosa and its precursor lesions indicate that Cox-2 could participate in the carcinogenic process of these oral malignancies.