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.

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.

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.

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.

Endothelial and epithelial expression of sialyl Lewis(x) in squamous carcinoma of the tongue.

In this retrospective study we describe the immunohistochemical expression pattern of sLe(x) epitopes in endothelial and epithelial cells of 59 squamous carcinomas of the tongue, and relate this to the relative survival rates of the patients. Endothelial sLe(x) expression was significantly elevated in malignant lesions compared to normal tissues, but did not have any prognostic value for the relative survival rate. In contrast, epithelial sLe(x) expression was decreased in carcinomas compared to normal tongue. The patients whose carcinoma showed only moderate epithelial HECA-452 reactivity had a significantly better relative survival rate than the patients with tumor specimens with neglible or very high HECA-452 reactivity. The epithelial staining with the two other anti-sLe(x) antibodies (CSLEX-1 and 2F3) did not correlate with the survival rates of tongue carcinoma patients.

Sialyl-Lewis(x/a)-decorated selectin ligands in head and neck tumours.

PURPOSE: E- and P-selectins, expressed on vascular endothelium, and their sialyl-Lewis(x )(sLe(x))- and/or sialyl-Lewis(a) (sLe(a))-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 sLe(x) and sLe(a) glycans and E- and P-selectins. RESULTS: Epithelial expression of sLe(x) and sLe(a) glycans was higher in benign than in malignant lesions in both epithelial and lymphoid tumours. On the other hand, endothelial expression of sLe(x), sLe(a), 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 sLe(x) and sLe(a )glycans acting on selectin ligands is linked to the development of head and neck tumours.

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands.

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLe(x), which facilitates their adhesion to endothelial E- and P-selectin.

Endothelial and epithelial expression of sialyl Lewis(x) and sialyl Lewis(a) in lesions of breast carcinoma.

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.