Comparing the nasal bacterial microbiome diversity of allergic rhinitis, chronic rhinosinusitis and control subjects.

PURPOSE: Until now, the microbiome of the nasal cavity and its contribution to nasal mucosal disease has remained poorly understood. The advent of cultivation-free molecular methods makes it possible to characterize the total microbiome of the nasal cavity. We sought to assess the microbial diversity and composition of the middle meatus in allergic rhinitis (AR) patients, chronic rhinosinusitis patients without polyps (CRSsNP) and a control population to determine the microbiota associated with the pathogenesis of AR and CRSsNP. METHODS: Microbial characterization was determined using 16S rRNA gene sequencing of 122 nasal swabs collected from patients with AR (n = 52) and CRSsNP (n = 37), and from healthy controls (n = 33). RESULTS: There was no difference in nasal microbiome richness and diversity among the three groups, and the dominant phyla were similar among three groups including Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. However, Spirochaetae abundance was significantly higher in AR than in the control group after FDR correction (FDR p = 0.021). At the genus level, although there was no statistical significance after FDR correction, there was a trend that Pseudomonas and Peptostreptococcaceae abundance were higher in AR than in controls (p = 0.005, p = 0.005) and CRSsNP (p = 0.023, p = 0.034); Lactobacillus abundance was lower in AR than in controls (p = 0.021); Moraxella abundance was lower in CRSsNP than in controls (p = 0.006); Haemophilus abundance was higher in CRSsNP than in AR (p = 0.003) but lower in AR than in controls (p = 0.018). CONCLUSION: These results suggested that microbial dysbiosis may play a role in the pathogenesis of heterogeneous nasal mucosal inflammation.

WITHDRAWN: Improved therapeutic effectiveness by combining recombinant p14ARF with antisense complementary DNA of EGFR in laryngeal squamous cell carcinoma.

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Improved therapeutic effectiveness by combining recombinant p14(ARF) with antisense complementary DNA of EGFR in laryngeal squamous cell carcinoma.

PURPOSE: The tumor suppressor p14(ARF) and proto-oncogene epidermal growth factor receptor (EGFR) play important roles in the development of laryngeal squamous cell carcinoma (LSCC). This study was aimed to determine whether combining recombinant p14(ARF) with antisense complementary DNA of EGFR could improve the therapeutic effectiveness in LSCC. MATERIALS AND METHODS: After human larynx cancer cells (Hep-2) were infected with recombinant adenoviruses (Ad-p14(ARF) and Ad-antisense EGFR) together or alone in vitro, the proliferation and cell cycle distribution of Hep-2 cells were detected by MTT assay and flow cytometer analysis, respectively. Furthermore, the antitumor effects of recombinant adenoviruses together or alone on Hep-2 xenografts were examined in vivo. The levels of p14(ARF) and EGFR expressed in Hep-2 cells and xenografts were determined by western blot assay. RESULTS: Ad-p14(ARF) combining with Ad-antisense EGFR markedly inhibited the Hep-2 proliferation compared with alone (P=0.001, P=0.002 respectively). Combination of Ad-p14(ARF) and Ad-antisense EGFR led to the proportion of Hep-2 cells in G0/G1 phases increased by up to 86.9%. The down-expression of EGFR protein and overexpression of p14(ARF) protein were observed in vitro and in vivo, and this effect was preserved when Ad-p14(ARF) was combined with Ad-antisense EGFR. Besides, Ad-p14(ARF) plus Ad-antisense EGFR significantly (P<0.05) increased the antitumor activity against Hep-2 tumor xenografts comparing with Ad-p14(ARF) or Ad-antisense EGFR alone. CONCLUSION: Combination Ad-p14(ARF) with Ad-antisense EGFR significantly increased the antitumor responses in LSCC. An effectively potential gene therapy to prevent proliferation of LSCC was provided.

Forkhead box protein 3 in human nasal polyp regulatory T cells is regulated by the protein suppressor of cytokine signaling 3.

BACKGROUND: In patients with persistent upper airway inflammation, the number of forkhead box protein 3 (Foxp3)(+) regulatory T (Treg) cells is reduced, but the regulation of Foxp3 expression in Treg cells is poorly understood. OBJECTIVE: We investigated the interaction between suppressor of cytokine signaling 3 (SOCS3) and Foxp3 expression in the airway mucosa. METHODS: Expression of SOCS3 and Foxp3 was measured in tissue from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and control tissue. Coexpression of SOCS3 and Foxp3 was evaluated in PBMCs and in tissue from patients with CRSwNP. We also switched off and overexpressed SOCS3 in tissue from patients with CRSwNP and in pancreatic carcinoma epithelial-like cell line (PANC-1) cells and examined the effect on Foxp3 expression. RESULTS: SOCS3 gene and protein expression was upregulated in inflammatory cells in airway mucosa, whereas Foxp3 gene and protein expression was downregulated. Mucosal Treg cells coexpressed both proteins. Switching off the expression of SOCS3 in human airway mucosa resulted in Foxp3 upregulation, whereas inducing it in PANC-1 cells led to Foxp3 downregulation. We also found that phosphorylation of signal transducer and activator of transcription (STAT) 3 was decreased in inflamed mucosa, and we hypothesized that SOCS3 was responsible. Phosphorylation of STAT3 increased on silencing SOCS3 expression in inflamed mucosa and decreased on SOCS3 plasmid transfection in PANC-1 cells. CONCLUSION: For the first time, we demonstrate that SOCS3 and Foxp3 are coexpressed in Treg cells in human nasal mucosa and that SOCS3 negatively regulates Foxp3 expression in human airway mucosa, possibly through phosphorylation of STAT3. Hence SOCS3 could be a potential target for restoring Foxp3 expression in Treg cells in patients with persistent mucosal inflammation.

[The correlation between nasal bacterial microbiome diversity and surgical prognosis for chronic sinusitis with nasal polyp].

Objective:To compare the nasal microbiota diversity between chronic rhinosinusitis with nasal polyp（CRSwNP） patients and controls, postoperative recurrent with non-recurrent CRSwNP, in order to provide new sight in CRSwNP treatment and prognosis. Method:Forty-eight patients with CRSwNP were recruited as the experimental group, and 33 patients who underwent FESS and had no sinus inflammatory disease, including nasal septum deviation,inverted papilloma, pituitary adenomas, chronic dacryocystitis,or optical canal fractures, were recruited as control group. High-throughput sequencing of 16S rRNA was used to detect the bacterial communities in the nasal secretion which was collected from middle meatus during the operation. The difference of the microbiota diversity between CRSwNP and controls was compared. Patients with CRSwNP were followed up for 1 year after surgery to observe whether they had relapsed or not, and nasal secretions were collected again for bacterial microbiota detection. The difference between postoperative and preoperative microbiota of the non-recurrent CRSwNP were compared, and the difference between postoperative and preoperative microbiota of the recurrent CRSwNP were compared. Result:One year after surgery, 12 cases of CRSwNP recurred（recurrent rate 25%）. The clinical history of the recurrent group was longer than that of the non-recurrent group（P=0.018）, and the preoperative CT score（P=0.001）, nasal polyp size score（P=0.004） and the severity of postnasal drip symptom（P=0.032） in the recurrent group were significantly higher than non-recurrent group. Comparing the preoperative nasal microbiota of CRSwNP with control, there was no significant difference about the richness, α diversity and ß diversity, but the relative abundance of Actinobacteria（FDR P=0.004） and Corynebacterium（FDR P=0.005） of CRSwNP were significantly lower than that of control. After operation, the relative abundance of Actinobacteria（FDR P＝0.012） and Corynebacterium（FDR P＝0.003） increased, while the Bacteroidetes（FDR P＝0.040） decreased in the non-recurrent CRSwNP; However, there was no change in the nasal bacterial microbiota in the recurrent group. Conclusion:CRSwNP was associated with nasal bacterial dysbiosis, and the postoperative improvement of dysbiosis was correlated with the prognosis of CRSwNP.

A CARE-compliant article: Extranasal glial heterotopia in a female infant: A case report.

RATIONALE: Nasal glial heterotopia is a rare type of neoplasm consisting of meningothelial and/or neuroglial elements. PATIENT CONCERNS: A 17-month-old female infant was evaluated for treatment for a congenital mass present since birth on the right side of the nasal dorsum. DIAGNOSES: The patient was preoperatively diagnosed with a congenital extranasal neoplasm. INTERVENTIONS: Surgery was performed under general anesthesia, and the mass was completely resected. The tissue was sent for histological examination, and the diagnosis was of extranasal glial heterotopia. OUTCOMES: The surgical outcome was good, and no surgical site infection was recorded. After 6 months of follow-up, the girl was asymptomatic with no recurrence. LESSONS: Surgical excision, a curative method used to address extranasal glial heterotopia, resulted in no recurrence during the clinical follow-up period. The potential for an intracranial connection must always be kept in mind when considering how to surgically treat a congenital midline mass to prevent the risk of cerebrospinal fluid leakage.

Combined p14ARF and antisense EGFR potentiate the efficacy of adenovirus-mediated gene therapy in laryngeal squamous cell carcinoma (LSCC).

The tumor suppressor p14(ARF) and protooncogene epidermal growth factor receptor (EGFR) play an important role in the development of laryngeal squamous cell carcinoma (LSCC). We explored the inhibition of proliferation and induction of differentiation in human larynx cancer cells (Hep-2) in vitro when p14(ARF) couples with antisense complementary DNA of EGFR to transfect into Hep-2 cells via the AdEasy-1 vector system. In vitro studies, using standard isobologram analyses, identified whether Ad-antisense EGFR is synergistic with Ad-14(ARF). To evaluate the cytotoxicity of these agents the gold standard clonogenic survival assay was used. Western blotting analyses, 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometer (FCM) analysis was used to detect protein expression, proliferation, and cell cycle distribution of Hep-2 cells, respectively. Meanwhile, empty vector and PBS were set as a control. The activity of proliferation of Hep-2 cells was inhibited markedly by infection of Ad-p14(ARF) combined with Ad-antisense EGFR compared with Ad-p14(ARF) or Ad-antisense EGFR alone (P = 0.001, P = 0.002, respectively), with Ad-sense EGFR (P = 0.0005), with vector control (Ad-Ctrl) (P = 0.0001), and with PBS (P = 0.0001). FCM revealed that the proportion in the G(0)/G(1) phases increased by up to 86.9% when Ad-p14(ARF) was associated with Ad-antisense EGFR to transfect Hep-2 cells. A weakened expression of EGFR protein and P14 (ARF) protein overexpression was observed. Our study in vitro indicated that association of Ad-p14(ARF) with Ad-antisense EGFR remarkably inhibited activity of proliferation and inducted differentiation of Hep-2 cells. Therefore, not only EGFR, but also p14(ARF), plays a major role in the genesis and in modulating cell growth and differentiation of LSCC, and their synergistic effect was obvious. An effective potential target of gene therapy to prevent LSCC proliferation was provided.

[Interference therapy of laryngeal squamous cell carcinoma by p16beta and EGFR-antisense cDNA in signal transduction].

OBJECTIVE: To test the effect of p163 and EGFR-antisense cDNA in signal transduction on Hep-2 laryngeal squamous cell carcinoma in vitro. METHODS: The Hep-2 laryngeal squamous carcinoma cells were transfected by recombinant adenovirus AdEasy-EGFR-antisense and AdEasy-p16beta in vitro. The inhibition of the EGFR expression and cell growth and changes of cell cycle, DNA content, apoptosis and ultramicrostructure of the Hep-2 cells were examined by MTT, Western blotting analysis, Flow cytometry analysis, Immunohistochemistry, and transmission electron microscope respectively. Results The proliferation of the Hep-2 cells was inhibited significantly by the infection of the Ad- Ad-p16beta or Ad-antisense EGFR. The infection also accelerated the apoptosis of the cancer cells. The proport of of cells in G0/G1 phases increased to more than 77.7%. The Ad-antisense EGFR-infected cells showed lower protein expression of EGFR. The P16beta protein over expression was observed in the Ad-p16beta-infected cells. CONCLUSION: The transfection of Ad- Ad-p16beta and Adantisense EGFR into Hep-2 cells leads to over-expression of Ad-pl6beta, and under-expression of EGFR, along with G1-phase arrest and apoptotic cell death. Both EGFR and Ad-p16beta play important roles in the genesis, growth and differentiation of the human laryngocarcinoma cells.

Effect of revision dacryocystorhinostomy under nasal endoscopy on recrudescent dacryocystitis.

OBJECTIVE: Patients with recurrent dacryocystitis are difficult to treat. We aimed to observe the role of dacryocystography in locating the site of obstruction and the effect of revision surgery in these cases of recurrent dacryocystitis. METHOD: We prospectively collected patients with recurrent dacryocystitis at West China Hospital of Sichuan University. Dacryocystography was performed before surgery. After revision endoscopy dacryocystorhinostomy (DCR), patients were followed-up regularly. Clinical features were recorded before and after operation, including the visual analog scale (VAS) score. Using the software of SPSS 13.0, VAS scores were compared between preoperation and postoperation by a Student's t test and repeated measure ANOVA. RESULTS: Twenty patients were collected; eight cases had a history of a one-time occurrence of DCR, and 12 cases had a history of two or more occurrence of DCR. Dacryocystography could show the site with the most lacrimal obstruction. During the operation, we could resect most of the lacrimal sac medial bone wall and expose the sac successfully. Follow-up showed no relapse occurrences and only one case had a slightly tearful eye subjectively but had enough big orificium fistulae and favorable mucosal epithelialization that it was similar to other cases. The VAS scores at follow-up decreased significantly compared with preoperation (P<0.05). CONCLUSION: For patients with recurrent dacryocystitis, dacryocystography could clarify the cause and exact site of the obstruction and provide information for further treatment. Through revision endoscope DCR, patients can effectively achieve enough drainage of the lacrimal sac accompanied with a significant improvement in symptoms and no observable complications.

The role of cysteinyl leukotrienes and their receptors in refractory nasal polyps.

Leukotriene signaling is essential in many diseases, including asthma, allergic rhinitis, atherosclerosis and inflammatory bowel disease. Nevertheless, the expression of cysteinyl leukotrienes (CysLTs) and its receptors (CYSLTRs) in different types of nasal polyps (NPs), and the role of their antagonist in the treatment of refractory chronic rhinosinusitis with nasal polyps (CRSwNP) are not well understood. The following study investigates the expression of CysLTs and CYSLTRs in different types of NPs, as well as the role of leukotriene receptor antagonist (montelukast) in refractory NPs. Our data showed that CysLTs and CYSLTRs were significantly elevated in CRSwNP group (p < 0.05), particularly in IL-5+NP patients, compared to patients with chronic rhinosinusitis but without NPs (CRSsNP) and the control group. Furthermore, montelukast have shown the ability to inhibit the expression of MUC5AC, TSLP, IL-4, IL-5, IL-13, and TGF-ß in NP explants after treatment with Staphylococcal Enterotoxins B (SEB). In addition, the patients treated by additional montelukast have better outcomes compared to those with INCS only. To conclude, our results demonstrate that the inhibition of CysLTs signaling by montelukast decreases the expression of cytokines and mucin in polyp explants, and in turn promotes the recovery in patients with refractory CRSwNP.

Distinct expression of NK2 homeobox 1 (NKX2-1) and goblet cell hyperplasia in nasal polyps with different endotypes.

BACKGROUND: Decreased expression of airway epithelial-specific transcription factor NK2 homeobox 1 (NKX2-1) was associated with allergic inflammation in asthma patients. However, the expression and role of NKX2-1 in nasal polyps (NPs) with different endotypes were undefined yet. METHODS: We examined the expression of key cytokines (interleukin [IL]-4 IL-5, IL-13, and IL-17A, etc.) and NKX2-1 in NPs with different endotypes and control tissues by immunohistochemistry staining, qualitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. RESULTS: We found 23% of chronic rhinosinusitis (CRS) with NP (CRSwNP) patients had IL-5+ eosinophilic NPs, 40.7% of NPs were key cytokines negative NPs (KCN NPs) with less eosinophil accumulation. The expression of NKX2-1 in IL-5+ NPs was significantly lower than KCN NPs and normal controls (p < 0.05). The expression of mucin 5AC (MUC5AC) and MUC5B, as well as goblet cells hyperplasia, were significantly elevated in IL-5+ NPs, which correlated with the decreased expression of NKX2-1 (p < 0.05). Moreover, "SAM pointed domain containing ETS transcription factor" (SPDEF) was significantly elevated, while expression of Forkhead Box A2 (FoxA2) was significantly decreased in IL-5+ NPs (p < 0.05). The expression of chemokine (C-C motif) ligand 17 (CCL17) and IL-4 was significantly increased in IL-5+ NPs, which was associated with eosinophil accumulation(p < 0.05). CONCLUSION: The downregulation of NKX2-1 in IL-5+ NPs may be associated with tissue eosinophilia and goblet cells hyperplasia.

[Construction of the recombinant adenovirus vector with EGFR antisense cDNA of controlling cell cycle].

OBJECTIVE: To recombine the adenovirus vector carrying EGFR sence/antisense cDNA which takes part in control of cell cycle. METHODS: The 1032 bp EGFR sence/antisense cDNA fragment was cloned into the shuttle plasmid pAdTrack-CMV. The resultant plasmid and the backbone plasmid pAdEasy-1 were transferred into E. coli BJ5183 for homologous recombination, and the recombinant adenoviruses were generated in cells. The recombinant adenoviruses were packaged and amplified in the 293 cells. Then the viral titer was detected by GFP. RESULTS: The recombinant adenovirus vector carrying EGFR sence/antisense cDNA to control the cell cycle was constructed successfully. The viral titers were 2.2 x 10(9) efu/mL and 2.5 X 10(9) efu/mL respectively. CONCLUSION: The recombinant adenovirus vector constructed by us could introduce EGFR antisense cDNA into the laryngeal squamous cell carcinoma line or tumor tissue, which would provide an experiment basis to study further the interfered mechanism of signal transdution and the therapies of laryngeal squamous cell carcinoma.

[Study in vitro on p16beta interfering the cell cycle signal conduction of laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the effect of p16beta interfering with the signal conduction of Hep-2 cell cycle in Vitro. METHODS: AdEasy Vector System was used to construct the recombinant adenovirus vector AdEasy-GFP-p1613. The recombinant adenovirus vector could introduce p16beta gene into HEK 293 cell. Then the purified recombinant adenovirus was used to infect Hep-2 cells in vitro. The protein expression of p16beta, proliferation inhibition, cell cycle arrest, DNA content, apoptosis ratio in Hep-2 cells were examined by MTT, Western blotting analysis, Flow cytometry assay, Immunocytochemistry respectively. RESULTS: The recombinant adenovirus Adeasy-p16beta was constructed successfully and higher titer recombinant adenovirus particle was got. When Adeasy-p16beta was transferred into Hep-2 cells, both the growth inhibition and P14 (ARF) protein overexpression in Hep-2 cells were visible effectively. The most Hep-2 cells were blocked in G1/G0 phase and the cell apoptosis ratio increased. CONCLUSION: The p16beta in Hep-2 cell infected by recombinant adenovirus, which lead to express P14 (ARF) protein effectively and to inhibit cell proliferative activation, effectively interfere the signal conduction mechanisms of culture Hep-2 laryngeal squamous cell carcinoma, and the growth of much cultured cells is blocked in G1/G0 phase.

Combination nonviral murine interleukin 2 and interleukin 12 gene therapy and radiotherapy for head and neck squamous cell carcinoma.

OBJECTIVES: To demonstrate that the combination of nonviral murine interleukin (mIL) 2 and mIL-12 gene therapy and external beam radiation therapy (XRT) have an enhanced therapeutic effect for the treatment of head and neck squamous cell carcinoma (HNSCC) in an orthotopic murine model and to elucidate the mechanism of action. DESIGN: A randomized, controlled study in a murine HNSCC model. INTERVENTIONS: Tumors were established in the floor of the mouth in C3H/HeJ immunocompetent mice with the SCC VII cell line. These tumors were directly injected with single lipid-formulated mIL-2 or single polymer-formulated mIL-12 or a combination of them and with phosphate-buffered saline or vector without mIL-2 and mIL-12 gene as controls. Then the local tumor was radiated twice with a dose of 1 Gy the next day and injected again 4 days later. Antitumor responses, cytokine expression, and natural killer cell and cytolytic T-lymphocyte activity were assayed. Meanwhile, tumor sizes were measured before and after treatment and compared among the different treatment groups and the controls. RESULTS: The combination mIL-2 + mIL-12 + XRT demonstrated a significant increase in antitumor effects compared with single therapy or controls. Increased expression levels of primary and secondary cytokines were found in the group treated with mIL-2 + mIL-12, and this effect was preserved when mIL-2 and mIL-12 treatments were combined with XRT. Combination therapy significantly increased antitumor effects, T-lymphocyte infiltration of CD4(+)and CD8(+), and the numerous necroses compared with monotherapy. CONCLUSIONS: Combination mIL-2 and mIL-12 gene therapy and XRT generates potent antitumor immune responses against HNSCC and significantly increases necrosis (apoptosis) in an orthotopic murine model of HNSCC. The nonviral mIL-2 and mIL-12 gene delivery system was well tolerated. Further optimization of treatment strategy for patients with HNSCC is warranted as well as consideration for human clinical trials.

[Combination of adenovirus p16(INK4A) gene therapy and ionizing radiation for laryngeal squamous cell carcinoma].

OBJECTIVE: To assess the anti-tumor effect of p16(INK4A) gene transfer and the combined effect of this gene therapy and ionizing radiation in the treatment of laryngeal squamous cell carcinoma. METHODS: A Complete p16(INK4A) gene was inserted into a replication-defective recombinant adenovirus, and the tumor cells were infected with Adenovinus-p16 (Ad-p16) and irradiated with X-rays. Confirmation of P16(INK4A) protein expression after Ad-p16 infection was performed by Western blotting. The therapeutic effects were evaluated both in vitro and in vivo. RESULTS: The replication-defective recombinant adenovirus could direct a high level of P16INK4A protein expression in laryngeal squamous cell carcinoma. Studies both in vitro and in vivo demonstrated that Ad-p16 treatment significantly inhibited the cell growth and the established tumors in mude mice when compared with the control treatments (P < 0.05). The combination of Ad-p16 and radiation group resulted in greater inhibition of tumor growth, compared with any other treatment groups and controls (P < 0.05). No significant difference was observed between Adenovirus-LacZ (Ad-LacZ) and PBS groups. CONCLUSION: These results demonstrate a significant antitumor effect of Ad-p16 against human laryngeal squamous cell carcinoma and also demonstrate for the first time a combined effect of radiation and Ad-p16 gene transfer in p16 deficient human laryngeal squamous cell carcinoma.

[A double-blind RCT of the hemostatic effects of local packing of Nasopore combined with hemocoagulase injection for postoperative management of FESS].

OBJECTIVE: To compare the hemostatic effects of local packing of Nasopore combined with hemocoagulase injection and local packing of Nasopore combined with saline injection for postoperative management of functional endoscopic sinus surgery by a double-blind, randomized control clinical trial. METHOD: Sixty-eight cases of chronic sinusitis needed functional endoscopic sinus surgery were randomly divided into the experimental group of 40 cases and control group of 28 cases, respectively. For the experimental group, 1 U of hemocoagulase dissolved in 0.5 ml saline was injected into Nasopore which was packed into the nasal cavity after operation. For the control group, 0.5 ml of saline was injected. The postoperative bleeding of the two groups were scored by visual analogue scale. RESULT: There was statistically significant difference between the bleeding VAS scores assessed 6 hours and the ones assessed 1, 2 and 3 days after the operation in the control group (P < 0.05). There was the statistically significant difference between the bleeding VAS scores of experimental group and control group assessed 6 h after the operation (P < 0.05). CONCLUSION: The hemocoagulase may improve the hemostatic effect of Nasopore 6 hours after the operation by combined injection with Nasopore as nasal cavity packing.

Inhibition of ataxia-telangiectasia mutated by antisense oligonucleotide nanoparticles induces radiosensitization of head and neck squamous-cell carcinoma in mice.

Ataxia-telangiectasia-mutated (ATM) is a radiosensitization gene. In the present study, we investigated the efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing ATM antisense oligonucleotides (ASOs) for the radiosensitization of head and neck squamous-cell carcinoma in mice, using the SCCVII cell line. Nanoparticles containing ATM ASOs were prepared with PLGA by using a double-emulsion solvent evaporation method. The results showed that the nanoparticles were suitable for intracellular uptake, and ATM ASOs inhibited ATM expression when delivered by using nanoparticles or lipofectin, but not in their free form. Meanwhile, we found that ATM reduction sensitized SCCVII cells in vitro and tumors in vivo to irradiation. In conclusion, biodegradable PLGA nanoparticles, used as a delivery carrier, enhanced intracellular uptake of ATM ASOs into SCCVII cells and the inhibitory effect of ATM ASOs. These results demonstrated that antisense ATM therapy, using PLGA nanoparticles, might provide a therapeutic benefit to patients undergoing radiation therapy for head and neck squamous-cell carcinoma.

Multifrequency tympanometry in adults with otitis media with effusion.

We compared the diagnostic values of multifrequency tympanometry (MFT) and conventional 226-Hz tympanometry in adults with otitis media with effusion (OME) and discuss whether the resonant frequency (RF) can be used as a reliable method in adults with OME. Prospective study was designed to compare the normal hearing group and the group with OME. In the OME group (n = 85), conductive or mixed hearing loss was found, air-bone gap was more than 10 dBHL, and acoustic reflex was not elicited. In the normal hearing group (n = 36), pure tone threshold was less than or equal to 15 dBHL and air-bone gap was less than 10 dBHL. Levene's test was used to compare the difference between the OME group and the normal hearing group on day 1, day 15, day 30, day 90, respectively. The relationship among multifrequency tympanometry, 226-Hz tympanometry and acoustic reflex test in ears recovering from OME was also investigated. A statistically significant decrease in RF value was found in ears with OME compared to normative data. In follow-up visits, both the RF values and the percentage of type A tympanograms increased while the percentage of type B and C tympanograms decreased. A high agreement between middle ear resonant frequency test and acoustic reflex test in ears recovering from OME was found. The resonant frequency test provides more detailed information than the 226-Hz tympanometry. Multifrequency tympanometry may be a more sensitive and objective diagnostic tool in adults with OME.

Signal transduction-related gene transfer leads to inhibition of proliferation and induction of differentiation in laryngeal squamous cell carcinoma in vitro.

The aim of this study is to target the interference therapy of signal transduction which is a novel therapeutic strategy in laryngeal squamous cell carcinoma (LSCC). We successfully constructed recombinant adenoviruses Ad-p14ARF, and Ad-antisense EGFR using AdEasy-1 vector System. Clonogenic cell assay, western blotting assay, 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometer (FCM) assay, and immunocytochemical technique were designed to examine the inhibition of proliferation, protein expression of p14ARF and EGFR and induction of differentiation, respectively. Furthermore the synergistic effect of Ad-p14ARF and Ad-antisense EGFR on Hep-2 cell was examined. We successfully used AdEasy-1 vector system to construct recombinant adenoviruses Ad-p14ARF and Ad-antisense EGFR. The activity of proliferation of Hep-2 cells was inhibited markedly by infecting Ad-p14ARF or Ad-antisense EGFR by comparing Ad-sense EGFR (P=0.005) with vector control (Ad-Ctrl) (P=0.005) and with PBS (P=0.003). This effect, combining Ad-antisense-EGFR with Ad-p14ARF became more noticeable than alone (P=0.01, P=0.02, respectively). P14 ARF protein overexpression, EGFR protein down expression, and inhibition of proliferation were observed in Hep-2 cells infected by either Ad-p14ARF or Ad-antisense EGFR. FCM revealed that the proportion of apoptosis cells transfected by Ad-p14ARF and Ad-antisense EGFR increased more obviously than the control. The proportion of (Hep-2 cells in) G0/G1 phases was increased by up to 78.5, 77.7, and 86.9% in Ad-antisense EGFR, Ad-p14ARF, and Ad-antisense EGFR+Ad-p14ARF, respectively. Our findings demonstrated that not only EGFR but p14ARF also plays a major role on the genesis and in modulating the cell growth and differentiation of human laryngocarcinoma. They efficaciously blocked the signal transduction of human laryngocarcinoma cell, and may therefore, be an effective potential target of gene therapy to prevent human laryngocarcinoma cell proliferation.

[Study on interference therapy induced by epidermal growth factor receptor-antisense cDNA in signal transduction of laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate effect of interference therapy induced by epidermal growth factor receptor (EGFR)-antisense cDNA in signal transduction of Hep-2 laryngeal squamous cell carcinoma in vitro. METHODS: AdEasy Vector System was used to construct the recombinant adenovirus vector sense/antisense-pAdEasy-EGFR. The recombinant adenovirus vector introduced EGFR-sense/antisense cDNA fragment into HEK293 cell. The purified recombinant adenovirus sense/antisense-pAdEasy-EGFR transfected Hep-2 cells in vitro. The inhibition of EGFR protein expression and proliferation of Hep-2 cells, the changes of cell cycle and DNA content in Hep-2 cells were examined by MTT, Western blot analysis, flow cytometry essay, and immunocytochemistry respectively. RESULTS: The higher titre sense and antisense mRNA expression recombinant adenovirus containing 1,032 bp EGFR-cDNA was constructed and prepared successfully. When antisense-pAdEasy-EGFR was transferred into Hep-2 cells the inhibition of cell proliferation and EGFR protein expression in Hep-2 cells were investigated effectively. CONCLUSION: The antisense-pAdEasy-EGFR effectively interfere the Hep-2 signal transduction pathway and induce apoptosis which results in inhibiting proliferation of laryngeal squamous cell carcinoma.