lactoferrin and Nasal-Polyps

Il profilo di espressione genica multipla rivela una disfunzione epiteliale nella rinosinusite cronica polipoide.. La rinosinusite cronica (CRS) è un disturbo infiammatorio eterogeneo risultante da una complessa interazione genetico-ambientale. Sebbene l’eziologia rimanga tuttora sfuggente, numerosi studi riportano alterazioni nell’espressione genica di diversi fattori implicati nell’ambito della risposta infiammatoria. Tuttavia, la gran parte di queste sono analisi isolate, non replicate, che prendono in considerazione un singolo gene alla volta. Inoltre, gli studi riguardanti analisi di espressione genica multipla, solitamente su mediatori infiammatori (es. citochine), spesso presentano risultati contrastanti, che in parte possono essere dovuti all’eterogeneità dei campioni o a metodologie analitiche di potenza limitata. In quest’ottica, il nostro obiettivo è stato di verificare simultaneamente l’espressione genica di un pannello di geni (AQP5, MUC5AC, CAV1, LTF, COX2, PGDS, TNFα, TGFβ1, MGB1) potenzialmente coinvolti nei meccanismi infiammatori della CRS. Nonostante la gran parte dei campioni sia stata esclusa dall’analisi a causa del deterioramento dell’RNA tissutale, siamo stati in grado di dimostrare una riduzione statisticamente significativa dell’espressione dei geni AQP5, CAV1, LTF e MGB1, in uno specifico sottogruppo di pazienti affetti da CRS nella variante con polipi nasali senza le tipiche comorbidità frequentemente associate (asma, allergia, intolleranza all’acido acetil-salicilico). Questi dati sembrano suggerire una disfunzione della barriera epitaliale nella CRS polipoide. Ulteriori studi saranno necessari per incrementare ulteriormente la nostra conoscenza sulla patogenesi della CRS. A tal proposito l’applicazione delle nuove e più potenti tecniche di sequenziamento, come la next-generation RNA sequencing, e la disponibilità di analisi bioinformatiche più complete potranno migliorare la caratterizzazione del transcriptoma negli endotipi della CRS.. Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder resulting from a complex gene-environment interaction. Although its aetiology remains elusive, numerous studies reported gene expression alterations of factors apparently implicated in all aspects of the inflammatory response. However, most investigations are limited, unconfirmed analyses of a single gene. Moreover, studies concerning multiple gene expression analyses, usually on inflammatory mediators (e.g. cytokines), show contrasting outcomes in part due to use of heterogeneous samples or methodologies with limited power. In this scenario, our goal was to simultaneously evaluate the expression of a panel of selected genes (AQP5, MUC5AC, CAV1, LTF, COX2, PGDS, TNFα, TGFβ1, MGB1) potentially involved in CRS inflammatory mechanisms. While most of the samples collected were excluded from the analysis because of poor quality RNA, we were able to demonstrate statistically significant downregulation of the AQP5, CAV1, LTF, MGB1 genes in a specific subset of polypoid CRS (patients without typical comorbidities), which might suggest relevant underlying epithelial dysfunction. Further studies are needed to enrich our knowledge on the pathogenesis of CRS. Forthcoming approaches might utilise next-generation RNA sequencing and comprehensive bioinformatics analyses to better characterise the transcriptome profiles of CRS endotypes.

Topics: Adolescent; Adult; Aged; Aquaporin 5; Caveolin 1; Chronic Disease; Cyclooxygenase 2; Cytokines; Epithelial Cells; Gene Expression; Gene Expression Profiling; Humans; Lactoferrin; Mammaglobin A; Middle Aged; Mucin 5AC; Nasal Polyps; Retrospective Studies; Rhinitis; Sinusitis; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Young Adult

Both up- and down-regulation of the Toll-like receptors (TLRs) and antimicrobial peptides (AMPs) of the sinonasal mucosa have already been associated with the pathogenesis of chronic rhinosinusitis with (CRSwNP) or without (CRSsNP) nasal polyps. The objective of this study was to determine the expression of all known TLR and several AMP genes and some selected proteins in association with allergy, asthma and aspirin intolerance (ASA) in CRS subgroups. RT-PCR was applied to measure the mRNA expressions of 10 TLRs, four defensins, lysozyme, cathelicidin and lactoferrin (LTF) in sinonasal samples from patients with CRSsNP (n = 19), CRSwNP [ASA(-): 17; ASA(+): 7] and in control subjects (n = 12). Protein expressions were detected with immunohistochemistry (n = 10). Statistical analysis was done with the Kruskal-Wallis ANOVA, Mann-Whitney U, and Student t test. TLR2, TLR5, TLR6, TLR7, TLR8, TLR9, β-defensins 1 and 4, cathelicidin and LTF mRNA expressions were significantly (p < 0.05) increased in CRSwNP, whereas only TLR2 and LTF were up-regulated in CRSsNP compared to controls. There was no statistical difference in respect of allergy, aspirin intolerance and smoking between CRSsNP, ASA(-) and ASA(+) CRSwNP patients. TLR2, TLR3, TLR4, LTF, β defensin 2 and lysozyme protein expressions were found to be elevated in macrophages of CRSwNP samples (p < 0.05). Gene expression analysis showed markedly different expressions in CRSwNP (6 out of 10 TLR and 4 out of 7 AMP genes were up-regulated) compared to CRSsNP (1/10, 1/7). The distinct activation of the innate immunity may support the concept that CRSsNP and CRSwNP are different subtypes of CRS. These findings were found to be independent from allergy, asthma, smoking, aspirin intolerance and systemic steroid application.

Topics: Adult; Antimicrobial Cationic Peptides; beta-Defensins; Case-Control Studies; Cathelicidins; Chronic Disease; Female; Humans; Hypersensitivity; Lactoferrin; Male; Middle Aged; Nasal Polyps; Rhinitis; RNA, Messenger; Sinusitis; Toll-Like Receptors; Young Adult

Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described.. 1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function.. Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array).. In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia.. Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model that could be useful both for studying the role of epithelium in CRSwNP while developing new therapeutic strategies, including cell therapy, for CRSwNP.

Topics: Cells, Cultured; Chemokines; Epithelial Cells; Humans; Immunohistochemistry; In Vitro Techniques; Lactoferrin; Models, Biological; Mucus; Nasal Mucosa; Nasal Polyps; Phenotype; Time Factors

Nasal polyps are strongly associated with a risk of chronic rhinosinusitis development as well as other obstruction including asthma and allergy. The following study tested the association of the 140A/G polymorphism of lactoferine (LF) encoding gene and the -33C/G polymorphism of osteoblast-specific factor-2 (OSF-2) encoding gene with a risk of chronic rhinosinusitis with nasal polyps in a Polish population. One hundred ninety five patients of chronic rhinosinusitis with nasal polyps as well as 200 sex, age and ethnicity matched control subjects without chronic sinusitis and nasal polyps were enrolled in this study. Among the group of patients 63 subjects were diagnosed with allergy and 65 subjects with asthma, respectively. DNA was isolated from peripheral blood lymphocytes of patients as well as controls and gene polymorphisms were analyzed by restriction fragments length polymorphism polymerase chain reaction (RFLP-PCR). We reported that the 140A/G LF (OR 4.78; 95% CI 3.07-7.24), the -33C/G OSF-2 OR 3.48; 95% CI 2.19-5.52) and the -33G/G OSF-2 (OR 16.45; 95% CI 6.71-40.30) genotypes were associated with an increased risk of chronic rhinosinusitis with nasal polyps among analyzed group of patients. Moreover, the group of patients without allergy or asthma indicated the association of the -33C/G (OR 3.72; 95% CI 2.24-6.19 and OR 15.11; 95% CI 5.91-38.6) and -33G/G (OR 3.73; 95% CI 2.24-6.19 and OR 14.07; 95% CI 5.47-36.16) genotypes of the OSF-2 as wells as 140A/G (OR 3.89; 95% CI 2.40-6.31 and OR 3.62; 95% CI 2.45-5.34) genotype of OSF-2 with an increased risk of chronic rhinosinusitis with nasal polyps. Finally, it was also found that the selected group of patients with allergy or asthma indicated a very strong association of the -33C/G (OR 2.40; 95% CI 1.23-4.69 and OR 2.40; 95% CI 1.23-4.69, respectively) and -33G/G (OR 16.01; 95% CI 5.77-44.41 and OR 17.90; 95% CI 6.53-49.05, respectively) genotypes of the OSF-2 as wells as 140A/G (OR 3.22; 95% CI 1.74-6.11 and OR 3.25; 95% CI 1.75-6.04, respectively) genotypes with an increased risk of chronic rhinosinusitis with nasal polyps. Thus, our results suggest that LF and OSF-2 gene polymorphisms may have deep impact on the risk of rhinosinusitis nasal polyps' formation which may also depend on asthma or allergy. Our results showed that the 140A/G polymorphism of LF gene and the -33C/G polymorphism of the OSF-2 gene may be associated with the risk of chronic rhinosinusitis with nasal polyps in a Polish pop

Topics: Adult; Aged; Alleles; Case-Control Studies; Cell Adhesion Molecules; Chronic Disease; Confidence Intervals; Electrophoresis, Agar Gel; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Lactoferrin; Male; Middle Aged; Nasal Polyps; Odds Ratio; Poland; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk Factors; Sinusitis

Chronic rhinosinusitis (CRS) is a disease characterized by inflammation of the nasal mucosa and paranasal sinuses. This inflammation may result in part from decreased epithelial barrier and innate immune responses, leading to frequent bacterial and fungal colonization. The objectives of this study were to investigate the expression of innate immune proteins of the palate lung and nasal epithelium clone (PLUNC) family in patients with CRS.. Nasal tissue samples were collected from control subjects and CRS patients with and without nasal polyps. Expression of the members of the PLUNC family was analyzed by real-time PCR. Expression of SPLUNC1 and LPLUNC2 proteins was analyzed by ELISA, immunoblot, and immunohistochemical analysis.. Levels of mRNA for most of the members of the PLUNC family were profoundly reduced in nasal polyps (NPs) compared to uncinate tissue from control subjects or patients with CRS. LPLUNC2 and SPLUNC1 proteins were decreased in NPs of patients with CRS compared to uncinate tissue from control subjects. Immunohistochemical data revealed that within submucosal glands of sinonasal tissues, SPLUNC1 and LPLUNC2 were differentially expressed, in serous and mucous cells, respectively. The decrease in the expression of these molecules is probably explained by a decrease in the number of glands in NPs as revealed by correlations with levels of the glandular marker lactoferrin.. Decreased SPLUNC1 and LPLUNC2 in NPs reflect a profound decrease in the number of submucosal glands. Decreased glands may lead to a localized defect in the production and release of glandular innate defense molecules.

Topics: Adolescent; Adult; Aged; Chronic Disease; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Glycoproteins; Humans; Lactoferrin; Male; Middle Aged; Nasal Mucosa; Nasal Polyps; Phosphoproteins; Rhinitis; Sinusitis; Young Adult

To study the local protective role of lysozyme(LZ) and lactoferrin(LF) in the uncinate process mucosa during chronic sinusitis.. Expression of LZ and LF was determined in 17 samples from normal subjects and 70 samples from chronic sinusitis patients with ABC immunohistochemical method. According to the presence or absence of nasal polyps, patients were divided into two groups.. Serous cells of submucosal glands displayed a strongly positive staining reaction to both LZ and LF in the normal uncinate process mucosa and mucosa from patients with chronic sinusitis. A positive though weak staining for LZ could also be found frequently within mucous cells of submucosal mixed glands and occasionally within goblet cells. In the mucosa from patients without nasal polyps, the staining reaction to LZ appeared to be intensified in goblet cells when compared with normal controls (P < 0.05). In patients with nasal polyps, the staining reaction to LZ appeared to be intensified in submucosal glands when compared with normal controls (P < 0.01) and patients without nasal polyps (P < 0.05). For LF, the staining reaction from patients with nasal polyps was stronger than that in normal controls (P < 0.01). The epithial cells stained negatively for LZ and LF.. It suggests that the observed increase in LZ and LF secreting activity of goblet cells and submucosal mixed glands may play a part role in the defense mechanism of uncinate process mucosa during the course of chronic sinusitis.

Topics: Adolescent; Adult; Child; Chronic Disease; Ethmoid Sinus; Female; Humans; Lactoferrin; Male; Middle Aged; Muramidase; Nasal Mucosa; Nasal Polyps; Sinusitis