Expression of VEGF, HGF, IL-6, IL-8, MMP-9, Telomerase in Peripheral Blood of Patients with Head and Neck Squamous Cell Carcinoma

Objectives

This study investigated the telomerase expression in peripheral blood mononuclear cells (PBMCs) and the relationship between the serum level of several soluble factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor, interleukin (IL)-6, IL-8, and matrix metallopeptidase-9 and the clinicopathological features of patients with head and neck squamous cell carcinoma (HNSCC).

Methods

Peripheral blood samples were collected from 50 HNSCC patients and 15 normal controls. The telomerase activity in the PBMCs was measured by Telomere Repeat Amplification Protocols. The serum levels of the soluble factors were analyzed by enzyme-linked immunosorbent assay.

Results

The expression of telomerase in the PBMCs of HNSCC patients was significantly correlated with the N and American Joint Committee on Cancer (AJCC) stages. The serum VEGF level was significantly higher in the patients with an advanced T stage, N stage and AJCC stage. Serum VEGF was significantly related with the expression of telomerase in the PBMCs. The telomerase expression and the VEGF expression were shown to be independent factors associated with poor survival.

Conclusion

The telomerase expression in the PBMCs and the serum VEGF level of HNSCC patients were significantly correlated with the N stage, the AJCC stage and the prognosis.     

Establishment and Characterization of an In Vitro Model for Cholesteatoma

Objectives

Experimental models are of importance to study the pathogenesis of middle ear cholesteatoma, however, they were not established until now. We aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue. HaCaT cell line was used as a "skin equivalent" and to compare the grade of homogeneity between cholesteatoma keratinocytes and HaCaT cells.

Methods

Primary keratinocytes were isolated from cholesteatoma tissue, co-cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air-exposed. The protein profile of cholesteatoma keratinocytes and HaCaT cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin (CK) 13 and 16. Tissue localization of CK 13 and 16 was accomplished with immunohistochemistry.

Results

Different protein profile and stronger expression of CK 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with HaCaT cells. Bigger stratification was observed in the 3D-in vitro systems when both cholesteatoma keratinocytes and HaCaT cells were respectively co-cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.

Conclusion

3D-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue.     

Expression of Bone-related Genes in Bone Marrow MSCs after Cyclic Mechanical Strain: Implications for Distraction Osteogenesis

Aim Understanding the response of mesenchymal stem cells （MSCs） to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis. Methodology In this study, a single period of cyclic mechanical stretch （0.5 Hz, 2,000 με） was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase （ALP） activity was examined. The mRNA expression of six bone-related genes （Ets-1, bFGF, IGF-Ⅱ, TGF-β, Cbfal and ALP） was detected using real-time quantitative RT-PCR. Results The results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfal expression became elevated later. The temporal expression pattem of ALP coincided perfectly with Cbfal. Conclusion The results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.     

Effect of Epigallocatechin-3-Gallate on PMA-Induced MUC5B Expression in Human Airway Epithelial Cells

Objectives

Among the inflammatory mediators, phorbol 12-myristate 13-acetate (PMA) is associated with the regulation of MUC5B expression in the airway epithelial cells. Epigallocatechin-3-gallate (EGCG) is the major component of green tea extract. The biological activity of EGCG includes reduction of cholesterol and antioxidant activity, as well as anti-inflammatory effect. However, the precise action mechanism of anti-inflammatory effect of EGCG in the airway epithelial cells has not been fully defined. This study investigates the effect and the brief signaling pathway of EGCG on PMA-induced MUC5B expression in the airway epithelial cells.

Methods

In NCI-H292 airway epithelial cells, the effect and signaling pathway of EGCG on MUC5B expression were investigated using real-time polymerase chain reaction analysis, enzyme immunoassay, immunohistochemical analysis, gelatin zymography assay, and immunoblot analysis.

Results

In NCI-H292 airway epithelial cells, PMA induced MUC5B expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK), and matrix metalloproteinase-9 (MMP-9) expression and protein activity. EGCG significantly decreased PMA-induced MUC5B expression, phosphorylation of p38 MAPK, and MMP-9 expression and protein activity. SB203580 (p38 MAPK inhibitor) significantly decreased PMA-induced MMP-9 expression. In addition, SB203580 and MMP-9 I (MMP-9 inhibitor) significantly decreased PMA-induced MUC5B expression.

Conclusion

These results suggest that EGCG down-regulates PMA-induced MUC5B expression through the p38 MAPK dependent MMP-9 signaling pathway in human airway epithelial cells.     

The Effect of Nano-Silver on Allergic Rhinitis Model in Mice

Objectives

Silver has long been known as a strong antimicrobial and disinfectant. Several types of nano-silver coated products have been developed. However, the antimicrobial and disinfectant characteristics of nano-silver have not been well studied. The aim of this study was to investigate the effect of nano-silver on allergic inflammation in a mouse model.

Methods

Female BALB/C mice were sensitized by intraperitoneal injection of ovalbumin (OVA) and aluminium hydroxide on days 0, 7, 14, and 21. Mice were challenged with intranasal instillation of OVA. Nano-silver was also administered nasally prior to intranasal instillation of OVA. Severity of allergic rhinitis was assessed according to nasal symptoms, serum OVA-specific IgE level, interleukin (IL)-4, IL-10, and interferon (INF)-γ levels in nasal lavage fluid. Hematoxylin-eosin stain and periodic acid-Schiff stain were performed for evaluation of histological change.

Results

Nano-silver attenuated manifestation of nasal symptoms in sensitized mice and inhibited production of OVA-specific IgE, IL-4, and IL-10, however, it had no effect on INF-γ level. In addition, the degree of inflammatory cell infiltration and goblet cell hyperplasia was attenuated by nano-silver.

Conclusion

These results suggest that nano-silver may effectively reduce allergic inflammation in a mouse model of allergic rhinitis. Through its properties as an anti-inflammatory agent, nano-silver may be a useful therapeutic strategy.     

Increase in the Level of Proinflammatory Cytokine HMGB1 in Nasal Fluids of Patients With Rhinitis and its Sequestration by Glycyrrhizin Induces Eosinophil Cell Death

Objectives

The nuclear protein high mobility group protein box 1 (HMGB1) is a proinflammatory mediator that belongs to the alarmin family of proinflammatory mediators, and it has recently emerged as a key player in different acute and chronic immune disorders. Several lines of evidence demonstrate that HMGB1 is actively released extracellularly from immune cells or passively released from necrotic cells. Because of the ability of HMGB1 to sustain chronic inflammation, we investigated whether the protein is present in nasal fluids of patients with different forms of rhinitis.

Methods

HMGB1 levels were evaluated in nasal fluids of healthy subjects or rhinitis patients who were treated or not treated with different treatments.

Results

We report that the level of HMGB1 was significantly increased in nasal fluids of patients with allergic rhinitis, patients with NARES (nonallergic rhinitis with eosinophiliac syndrome), as well as patients with polyps. We also found that a formulation containing the HMGB1-binding compound glycyrrhizin (GLT) reduced the HMGB1 content in nasal fluids of rhinitis patients to an extent similar to that with nasal budesonide treatment. We also found that among the cultured human leukocyte populations, eosinophils released higher amounts of HMGB1. Based on the ability of HMGB1 to sustain eosinophil survival and the ability of GLT to inactivate HMGB1, we report that GLT selectively killed cultured eosinophils and had no effect on neutrophils, macrophages, and lymphocytes.

Conclusion

Collectively, these data underscore the role of HMGB1 in rhinitis pathogenesis and the therapeutic potential of GLT formulations in treatment of chronic inflammatory disorders of the nasal mucosa.     

Nasal Obstruction and Palate-Tongue Position on Sleep-Disordered Breathing

Objectives

We wanted to evaluate whether the presence of nasal obstruction makes a change on the association between the modified Mallampati score and the severity of sleep-disordered breathing (SDB) and the sleep quality.

Methods

Polysomnography (PSG), the modified Mallampati score (MMS), the body-mass index, and a questionnaire about nasal obstruction were acquired from 275 suspected SDB patients. The subjects were divided into two groups according to the presence of nasal obstruction. The clinical differences between the two groups were evaluated and the associations between the MMS and PSG variables in each group were also assessed.

Results

Significant correlations were found between the MMS and many PSG variables, including the apnea-hypopnea index, the arousal index and the proportion of deep sleep, for the patients with nasal obstruction, although this was not valid for the total patients or the patients without nasal obstruction.

Conclusion

The severity of SDB and the quality of sleep are well correlated with the MMS, and especially for the patients with nasal obstruction. The MMS can give more valuable information about the severity of SDB when combined with simple questions about nasal obstruction.     

Modulators of Ion Transport in Nasal Polyps: An in situ Measurement of Short-Circuit Current

Objectives

To examine possible modulators of the ion transport through the apical membrane of the nasal polyps.

Methods

The study was conducted using the freshly-excised nasal polyps from the patients with chronic sinusitis. A voltage-sensitive vibrating probe technique was introduced to monitor the short-circuit current across the apical membrane of the polyp at 37℃.

Results

In the presence of amiloride, Adenosine 5''-triphosphate induced 4,4''-Diisothiocyanatostilbene-2,2''-disulfonic acidsensitive chloride current. Uridine 5''-diphosphate was less potent than Uridine 5''-triphosphate, and adenosine increased chloride secretion, which was blocked by the antagonist, 8-(p-sulfophenyl) theophylline on adenosine receptor. Based on the pharmacologic profiles, multiple purinergic receptors, including P2Y₂, P2Y₆, and P1 receptors, were functionally expressed. However, P2X receptor agonists (α,β-methyleneadenosine 5''-triphosphate and 2''- & 3''-O-[4-benzoyl-benzoyl] adenosine 5''-triphosphate), Cystic fibrosis conductance regulator (CFTR) activator (genistein), nitric oxide substrate (L-arginine), and nitric oxide donor (sodium nitroprusside) had no significant effect on the short circuit current.

Conclusion

Among tested drugs, P2Y receptor agonists were major modulators of ion transport in nasal polyps in situ.     

What is the Relationship between the Localization of Maxillary Fungal Balls and Intranasal Anatomic Variations?

Objectives

Although the mechanisms underlying the initiation and maintenance of inflammation in unilateral maxillary fungal balls (FBs) are poorly understood, the relationship between intranasal anatomy and maxillary FB is thought to play an important role. The aim of this study was to investigate the relationship between anatomic variations and FB.

Methods

We enrolled 140 patients who were composed of 56 patients with FB, 56 patients with unilateral chronic rhinosinusitis (CRS), and 28 patients with no sinus disease. Computed tomography scans were retrospectively analyzed to identify and compare the associated nasal anatomic abnormalities. To measure the volume of the nasal cavity and middle meatus, computed tomography scans were reconstructed into three-dimensional images.

Results

The relatively larger volume of the middle meatus was associated with the localization of the FB in contrast with the CRS. However, the nasal-cavity volume, nasal valve area, and nasal septal deviation were not significantly associated with localization of FB. The mean volumetric and areal measurements such as nasal cavity, middle meatus, and nasal valve in FB-ipsilateral sides were not significantly different from those in contralateral sides as well as other groups.

Conclusion

The middle meatus bears the major part of the inspiratory nasal airflow, and its volume may influence the occurrence of FB.     

CXCL12/SDF-1α Activates NF-κB and Promotes Oral Cancer Invasion through the Carma3/Bcl10/Maltl Complex

Aim To determine how SDF-1α/CXCR4 activates nuclear factor-kappa B （NF-κB） and promotes oral squamous cell carcinoma （OSCC） invasion.Methodology A lentivirus-based knockdown approach was utilized to deplete gene expression. NF-κB activation was evaluated by Western blot analysis and electrophoretic mobility shift （EMSA）. Results We show that the activation of NF-κB by CXCR4 occurs through the Carma3/Bcl10/Maltl （CBM） complex in OSCC. We found that loss of components of the CBM complex in HNSCC can inhibit SDF-1α induced phosphorylation and degradation of IκBα, while TNFα induced IKK activation remains unchanged. Further, we identified a role for novel and atypical, but not classical, PKCs in activating IKK through CXCR4. Importantly, inhibition of the CBM complex leads to a significant decrease in SDF-1α mediated invasion of OSCC. Conclusion The CBM complex plays a critical role in CXCR4-induced NF-κB activation in OSCC. Targeting molecular components of the NF-κB signaling pathway may provide an important therapeutic opportunity in controlling the progression and metastasis of OSCC mediated by SDF-1α.     

Effect of Botulinum Toxin Type A on a Rat Surgical Wound Model

Objectives

The tension on a wound is one of the important factors that determine the degree of fibrosis and scar formation. We hypothesized that local botulinum toxin type A (Botox) induced paralysis of the musculature subjacent to a surgical wound with a skin defect would minimize the repetitive tensile forces on the surgical wound''s edges, and this will result in a decreased fibroplastic response and fibrosis of the wound.

Methods

This is a prospective randomized experimental study. Two distinct surgical wounds were made to the dorsum of 15 adult rats, respectively. One of the 2 wounds was injected with Botox, and the other wound was used as a control, and this was done for all the rats'' wounds. We evaluated the wound size, the degree of fibrosis and inflammation, the blood vessel proliferation, the thickness of the wound and the expression of transforming growth factor (TGF)-β1 in the wounds.

Results

There were significant differences of wound size at the 3rd and 4th week between the Botox and control groups (P<0.05). The Botox group showed less infiltration of inflammatory cells than the control group at the 2nd week (P<0.05). The Botox group showed a smaller number of fibroblasts and less fibrosis than the control group at the 4th week (P<0.05). The Botox group showed much strong collagen density than the control group at the 8th week (P<0.05). For the immunohistochemical staining, there was a lower transforming growth factor (TGF)-β1 expression in the Botox group than that of the control group at the 4th week (P<0.05).

Conclusion

The wounds of the Botox-treated group showed a larger wound size, less infiltration of inflammatory cells and less fibrosis, a much greater amount of collagen and a lower expression of TGF-β1 than did the control group. Botox might be used to decrease the fibrosis of a surgical wound without damaging the epithelial growth in situations for which decreased fibrosis is necessary, such as for treating laryngeal, tracheal and nasal stenosis.     

Intersession repeatability of acoustic rhinometry measurements in healthy volunteers

Objectives

Acoustic rhinometry is a rapid, reliable and non-invasive technique for the evaluation of conditions associated with impaired nasal patency. This study aimed to examine the intersession repeatability of acoustic rhinometry measurements of unilateral and combined nasal parameters in a group of healthy volunteers.

Methods

Twenty healthy volunteers were studied. In each subject, acoustic rhinometry measurements were performed on five consecutive days, with multiple recordings. Five clinically relevant parameters were measured in each session and the intersession repeatability of these measurements was expressed in terms of mean coefficient of variation, intraclass correlation coefficient and inter-item correlations.

Results

Intraclass correlation coefficients showed a high, and greater repeatability over time for all the combined (mean) values compared to the unilateral values. All intraclass correlations for combined values were ≥0.80 confirming almost perfect agreement. All intraclass correlations and inter-item correlations were associated with P<0.001. The mean coefficient of variation was low (<10%) for all but the proximal minimum cross sectional area (MCA1) measurements.

Conclusion

Acoustic rhinometry provides highly repeatable measurements of nasal patency, which is best for combined (mean) nasal parameters. This property makes it suitable for use in the diagnosis and follow-up of conditions associated with nasal obstruction, either structural or functional.     

The Role of Platelet-Derived Growth Factor in the Pathogenesis of Sinonasal Polyps: Immunohistochemical Assessment in Epithelial,Subepithelial and Deep Layers of the Mucosa

Objectives

The aim of this study is to investigate the role of platelet-derived growth factor (PDGF) in the pathogenesis of sinonasal polyps.

Methods

Study group (groups 1-3) consisted of nasal polyp samples of patients with sinonasal polyps and the control group consisted of inferior turbinate samples of patients without nasal polyp. In group 1, 14 specimens from ethmoid sinus; in group 2, 10 specimens from nasal cavity; in group 3, 10 specimens from maxillary sinus; and in group 4 (control), 9 specimens from inferior turbinate were included. By immunohistochemical staining technique, the PDGF positivity index (PI) in mucosal layers and in the inflammatory cells were assessed at the epithelium (EP), subepithelial layer of lamina propria (SE), and deep paraglandular layer of the mucosa (D).

Results

Polymorphonuclear cell (PMNC)-percentage (%) values of ethmoid and maxillary sinus, and the PDGF PI from all cells of ethmoid sinus and nasal cavity were significantly higher than those of the control group. As mononuclear cell-% (MNC-%) increased, the PDGF_EP_basal PI, PDGF_SE_endothelial PI, and PDGF_D_endothelial PI decreased. As PMNC-PDGF PI increased, the PDGF_D_perivascular PI decreased and PDGF_D_endothelial PI increased. As PDGF-MNC PI increased, the PDGF_EP_apical PI, PDGF_SE_endothelial PI, and PDGF_D_endothelial PI decreased. As PDGF-all cells (PMNCs, MNCs, and fibroblasts) PI increased, the PDGF_EP_basal PI and PDGF_D_endothelial PI decreased, and the PDGF_D_perivascular PI increased.

Conclusion

We concluded that the PDGF systems play important roles in polyp pathogenesis. Fibroblast-derived PDGF may be more important than MNC-derived PDGF in polyp developing process. Increased perivascular-PDGF-PI in deep layers of the mucosa may result in sinonasal polyp formation by causing an increase in vascular permeability and extracellular edema, and thus promoting migration of inflammatory cells to extracellular area. Tissue oxygenization may be important for the initiation of PDGF release system. Because of this reason, nasal obstruction should be medically treated earlier, and, if necessary, by surgical approaches.     

Medialization thyroplasty using autologous nasal septal cartilage for treating unilateral vocal fold paralysis

Objectives

A persistent insufficiency of glottal closure is mostly a consequence of impaired unilateral vocal fold movement. Functional surgical treatment is required because of the consequential voice, breathing and swallowing impairments. The goal of the study was to determine the functional voice outcomes after medialization thyroplasty with using autologous septal cartilage from the nose.

Methods

External vocal fold medialization using autologous nasal septal cartilage was performed on 15 patients (6 females and 9 males; age range, 30 to 57 years). Detailed functional examinations were performed for all the patients before and after the surgery and this included perceptual voice assessment, laryngostroboscopic examination and acoustic voice analysis.

Results

All the patients reported improvement of voice quality post-operatively. Laryngostroboscopy revealed almost complete glottal closure after surgery in the majority of patients. Acoustic and perceptual voice assessment showed significant improvement post-operatively.

Conclusion

Medialization thyroplasty using an autologous nasal septal cartilage implant offers good tissue tolerability and significant improvement of the subjective and objective functional voice outcomes.