Effects of topical Amphotericin B on expression of cytokines in nasal polyps

Objective—Although chronic rhinosinusitis (CRS) is one of the most frequently reported chronic diseases its etiology is not well understood. Recently, fungi have been proposed to influence the chronicity of rhinosinusitis. If fungi do play an important role then topical antifungal treatment may improve the inflammatory process of CRS. Therefore, in this study we measured inflammatory cytokine levels in nasal polyps after intranasal antifungal irrigation.

Material and Methods—Nasal polyps were collected before and 4 weeks after treatment with 100 mg/l topical amphotericin B (n=16), 50 mg/l topical amphotericin B (n=14) or normal saline (n=11). The cytokine—IL-5, IL-8, interferon-γ, RANTES—protein content of polyp homogenates were determined by means of ELISA.

Results—Nasal polyps were found to contain large amounts of cytokines (IL-5, IL-8 and RANTES) compared with normal inferior turbinates. After 4 weeks of treatment with topical agents, IL-5 levels tended to decrease in comparison with those of the other cytokines, but this difference was not statistically significant.

Conclusions—Topical amphotericin B treatment and nasal saline irrigation both influence the expression of nasal polyp cytokines. Topical nasal irrigation may influence the inflammatory process of CRS.     

Effect of nasal antifungal therapy on nasal cell activation markers in chronic rhinosinusitis

OBJECTIVE: To examine the effect of nasal antifungal treatment on eosinophil cationic protein (ECP) and tryptase levels in samples of nasal lavage fluid from patients with chronic rhinosinusitis and nasal polyps. DESIGN: Prospective double-blind placebo-controlled clinical trial. SETTING: Tertiary surgical center. PATIENTS: Subjects with severe chronic rhinosinusitis and nasal polyps. Of 120 screened patients, 76 were eligible. Six patients withdrew because of minor adverse events, and 10 dropped out for other reasons. In total, 60 patients completed the study according to the study protocol. INTERVENTIONS: Nasal treatment with amphotericin B or saline control for 8 weeks. MAIN OUTCOME MEASURES: Nasal lavages were performed before and after treatment. Fungal elements were assessed by culture and with different polymerase chain reaction assays. Levels of ECP and tryptase were determined by fluorescent enzyme immunoassay. RESULTS: No correlation between cell activation markers and fungus detection was observed before treatment (all P>.20). Nasal amphotericin B treatment had no effect on levels of ECP (P = .17) or tryptase (P = .09) in nasal lavage samples. Moreover, successful fungus eradication, defined as fungus detection before but not after treatment, did not influence nasal ECP or tryptase levels (all P>.40). CONCLUSION: Neither topical amphotericin B therapy nor fungal state before and after treatment had any significant influence on activation markers of nasal inflammatory cells in chronic rhinosinusitis.     

Amphotericin B nasal spray has no effect on nasal polyps

Nasal polyps and chronic rhinosinusitis are the products of an inflammatory process. Recently, fungal involvement has been thought to stimulate the development of polyps, and administration of antifungal agents was therefore considered a potential treatment. Several studies have been published indicating amphotericin B as an effective treatment for nasal polyps and chronic rhinosinusitis. The aim of our investigation was to evaluate the efficacy of intranasal applied amphotericin B on the growth of nasal polyps in a three-month, prospective, open trial. Our results show that nasal amphotericin B spray is not effective for nasal polyps and may even cause deterioration.     

Amphotericin B irrigation for the treatment of chronic rhinosinusitis without nasal polyps: a randomized, placebo-controlled, double-blind study

BACKGROUND: Fungus-driven inflammation is proposed to play an important role in the pathogenesis of chronic rhinosinusitis (CRS). Previous studies tested the efficacy of intranasal antifungal agents for patients with nasal polyps. The purpose of this study was to evaluate the efficacy of intranasal amphotericin B (AMB) in patients who have CRS without nasal polyps (CRSsNP). METHODS: Patients diagnosed with CRSsNP were enrolled in this study. They were assigned randomly to receive irrigation with AMB solution (20 mg of AMB in 500 mL of normal saline) or placebo (yellowish dye in 500 mL of normal saline) for 4 weeks. The outcome measures included the Chinese version of the Rhinosinusitis Outcome Measure 31 (CRSOM-31), nasal endoscopy, and bacterial and fungal cultures. RESULTS: Seventy patients were enrolled and 64 patients completed this study. There was significant improvement in the AMB group (n = 32) both in endoscopic (p = 0.013) and CRSOM-31 scores (p < 0.0001). The placebo group (n = 32) showed significant improvement in CRSOM-31 scores (p < 0.0001). CRSOM-31 scores were significantly lower in the AMB group than in the placebo group after 2-week treatment (p = 0.018) and remained lower after 4-week treatment, although the difference was not significant (p = 0.091). There were no significant differences in endoscopic scores and bacterial or fungal culture rates between two groups after treatment. CONCLUSION: Our results showed that AMB irrigation improved symptoms and endoscopic scores but did not show superiority to saline irrigation alone in patients who have CRSsNP.     

Activity of nasal amphotericin B irrigation against fungal organisms in vitro

BACKGROUND: The role of fungi in chronic rhinosinusitis has been described in recent reports. Controversy exists on the use of topical amphotericin B therapy as a treatment modality for this condition. The effect of various concentrations of amphotericin B nasal irrigation on actively growing fungi was studied in vitro. METHODS: Ten species of fungi commonly found in the nasal cavity were grown on growth media plates. Each fungi was exposed to 20 mL of amphotericin B nasal irrigation at concentrations of either 100, 200, or 300 microg/mL or sterile water two times daily for 6 weeks. Each plate was subcultured on a weekly basis to examine for any viable fungi. RESULTS: Fungi growth was not arrested in the 100-microg/mL amphotericin B and sterile water groups at the end of 6 weeks. Use of the 300-and 200-microg/mL amphotericin B solutions showed failure of the subcultured fungi to grow at 5 and 6 weeks, respectively. CONCLUSION: Nasal amphotericin B irrigation is ineffective in killing fungi in vitro at a concentration of 100 microg/mL over a 6-week period. Concentrations of 200 and 300 lig/mL successfully prevented fungi growth at the conclusion of the study. The current concentration of commercially available topical amphotericin B (100 microg/mL) seems ineffective in eradicating fungi in vitro.     

IL-4 and TNF-alpha increased the secretion of eotaxin from cultured fibroblasts of nasal polyps with eosinophil infiltration

BACKGROUND: Nasal polyposis is considered a subgroup of chronic rhinosinusitis (CRS). Eosinophils are the most common inflammatory cells in nasal polyp and the degree of the tissue eosinophilia is correlated with the probability of the recurrence of nasal polyps. However, the mechanism by which eosinophils are selectively recruited in nasal polyp remains to be clarified. In the present study, fibroblasts were isolated from nasal polyps of patients with eosinophil-rich nasal polyps (Enp) and those with non-eosinophilic nasal polyps (NEnp) and the secreted levels of eotaxin, regulated upon activation normal T expressed and presumably secreted (RANTES), and vascular cell adhesion molecule-1 (VCAM-1) from the cultured fibroblasts were determined. The levels were compared between Enp and Nenp. The role of those chemokines and adhesion molecules in the pathogenesis of nasal polyp is discussed. METHODS: Fibroblasts isolated from nasal polyps of five patients with CRS with Enp and four patients with CRS with NEnp were cultured and stimulated with 10 ng/ml of tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) for 24 hours. After stimulation, culture supernatants were collected and concentrations of eotaxin, RANTES, and VCAM-1 were quantified by Enzyme linked immunosorbent assay (ELISA). RESULTS: TNF-alpha enhanced the secretion of VCAM-1 and RANTES by fibroblasts derived from both NEnp and Enp, but did not affect the release of eotaxin. IL-4 increased the secretion of VCAM-1 and eotaxin but not that of RANTES. Furthermore, TNF-alpha and IL-4, when added together, induced a synergistic effect on the secretion of VCAM-1 and eotaxin. The effect of IL-4 and IL-4 plus TNF-alpha on eotaxin release was more marked for Enp fibroblasts compared with NEnp fibroblasts. CONCLUSIONS: The results suggest that eotaxin plays an important role in the selective recruitment of eosinophils in Enp. Nasal fibroblasts in Enp are more sensitive than those in NEnp regarding eotaxin release induced by the stimulation with IL-4 and co-stimulation with TNF-alpha and IL-4. This difference might be associated with the pathogenesis of nasal polyposis having marked accumulation of eosinophils.     

慢性鼻窦炎伴鼻息肉与哮喘的相关性机制及治疗策略研究进展

慢性鼻窦炎伴鼻息肉(CRSwNP)和哮喘作为上下呼吸道最具代表性的慢性炎症性疾病,二者在发病过程中常相互并存,临床诊治棘手。目前对CRSwNP和哮喘的相互作用机制尚未阐明。欧洲鼻窦炎鼻息肉诊疗指南(EPOS 2012)对治疗CRSwNP伴或不伴哮喘已给出具体的指导意见,其常规治疗包括鼻用糖皮质激素、生理盐水洗鼻等,并进一步根据CRSwNP的特点、症状严重程度等进行后续的联合治疗。据报道,鼻窦手术和药物治疗CRSwNP对合并支气管哮喘的转归有益。但CRSwNP合并哮喘患者经药物与手术治疗后的疗效判定尚缺乏足够的随机对照试验证据。     

Eosinophil chemoattractants and related factors in nasal polyps

OBJECTIVE: In vitro studies and animal experiments have shown that cytokines and chemokines are closely related to eosinophil migration, activation, and survival. It remains controversial, however, whether some chemokines or cytokines are actually responsible for the accumulation of eosinophils in nasal polyp tissues. We studied cytokines and chemokines in nasal polyp tissues taken from patients with chronic rhinosinusitis to clarify the pathogenesis of eosinophil accumulation. MATERIALS AND METHODS: Nasal polyp tissues obtained from 20 patients with chronic rhinosinusitis were studied. Concentrations of interleukin (IL-) 5, IL-13, eotaxin, regulated upon activation in normal T cell expressed and secreted (RANTES), and thymus and activation-regulated chemokine (TARC) in homogenates of polyp tissues were measured by ELISA. Nasal polyp tissues were stained by hematoxillin and eosin and were immunostained by an antibody against EG2. The numbers of eosinophils and immunopositive cells for EG2 in the submucosal layer were counted using a microscope. RESULTS: No significant differences were seen in the numbers of eosinophils and EG2-positive cells, or in the concentration of IL-5, eotaxin, TARC, RANTES in nasal polyp tissues between patients with and without atopic predisposition. Significant positive correlations existed, however, between the number of eosinophils and IL-5, eotaxin, and TARC concentration. IL-13 concentration was below detection in all patients. CONCLUSION: We hound that IL-5, eotaxin, and TARC may play an important role in the accumulation of eosinophils in nasal polyps regardless of the presence of atopic predisposition.     

Interferon-α action in cytokine profile in eosinophilic nasal polyp cultures

IntroductionChronic rhinosinusitis is currently classified into two types: chronic rhinosinusitis without nasal polyps and chronic rhinosinusitis with nasal polyps. In the West, approximately 80% of chronic rhinosinusitis with nasal polyps cases are characterized by a predominantly eosinophilic cell infiltrate and a Th2 cytokine pattern.ObjectiveTo evaluate the effect of Interferon-α on cytokine levels of the eosinophilic nasal polyp cell culture supernatant.MethodsCell cultures were performed based on nasal polypoid tissue samples collected from 13 patients with eosinophilic chronic rhinosinusitis with nasal polyps. Polyps were considered eosinophilic according to the histopathological examination. Cell cultures were stimulated with 3000 IU of interferon-α. Before and after the stimulus, concentrations of Interferon-γ, tumor necrosis factor αand IL 2, 4, 6 and 10, using cytometric bead array, were assessed.ResultsCell samples from eosinophilic nasal polyps from 13 patients were included in the study. Twenty-four hours after interferon-α stimulation, eosinophilic nasal polyp culture supernatants showed significantly decreased IL-4 concentrations and increase in interferon-γ, IL-10 and IL-6 concentrations compared to controls. There were no significant differences in tumor necrosis factor -α and IL-2 concentrations.ConclusionWe demonstrated that interferon-α in vitro alters the pattern of cytokines in cell cultures of eosinophilic nasal polyps. Analysis of these alterations suggests that interferon-α promotes a rebalancing of inflammatory profiles in cell cultures, favoring the expression of Th1 and regulatory cytokines over Th2 cytokines.     

Eotaxin synthesis by nasal polyp fibroblasts

Nasal polyps is a chronic inflammatory disease of the upper airway characterized by structural abnormalities including stromal fibrosis. Fibroblasts are a rich source of cytokines and inflammatory mediators and are thought to play an important role in the development of fibrosis. In addition, there is considerable evidence for the participation of eosinophils in the pathophysiology of nasal polyps. Although increased numbers of eosinophils are present in nasal polyps, the mechanisms responsible for their selective accumulation are not completely clear. Eotaxin is a chemokine that promotes the selective recruitment of eosinophils. Thus, it may be an important molecule for the recruitment of eosinophils in nasal polyps. The purpose of this study was to investigate whether nasal polyp fibroblasts synthesize eotaxin after stimulation with lipopolysaccharide, IL-1beta or TNF-alpha. Using primary nasal polyp tissue-derived fibroblast lines, we demonstrated that LPS, IL-1beta and TNF-alpha induced the gene expression and protein production of eotaxin in nasal polyp fibroblasts. This responsiveness to LPS, IL-1beta and TNF-alpha was time- and dose-dependent. These findings support the hypothesis that fibroblasts could play an important role in the recruitment of eosinophils in nasal polyps through the production of eotaxin.     

Increased expression of acidic mammalian chitinase in chronic rhinosinusitis with nasal polyps

BACKGROUND: Chitin is an abundant polysaccharide found in fungi, insects, and parasitic nematodes. Innate immune host defense against chitin-containing pathogens include production of chitinases. In human lower airways, acidic mammalian chitinase (AMCase) is produced in epithelial cells via a Th2-specific, IL-13-dependent pathway, and may act as an inflammatory mediator in asthma. The role of AMCase in chronic rhinosinusitis (CRS) has not been studied previously. METHODS: Eleven controls and 22 subjects with medically recalcitrant CRS were prospectively enrolled before undergoing endoscopic sinus surgery. RNA was extracted from surgically obtained ethmoid mucosa, and real-time PCR was used to determine expression of AMCase, eotaxin, and IL-13. Subjects were followed for at least 6 months postoperatively to assess for polyp recurrence. Based on the presence or absence of polyps, the subjects were classified as either recalcitrant or responsive to therapy. RESULTS: AMCase mRNA was detected in the sinus mucosa of 72% of control subjects and in 72% of patients with eosinophilic CRS with nasal polyps (CRSwNP). The expression of AMCase was significantly greater in recalcitrant CRSwNP than it was in treatment-responsive CRSwNP. There was no significant difference in IL-13 expression between these two groups. CONCLUSION: AMCase may be an important mediator in the pathogenesis of Th2 inflammatory diseases of the respiratory tract. Failure of medical and surgical therapy in CRSwNP is associated with significantly increased expression of AMCase, but not the Th2 cytokines IL-13 and eotaxin. Additional studies are needed to determine the potential of AMCase as a therapeutic target in CRSwNP.     

布地奈德联合生理盐水鼻腔冲洗对慢性鼻-鼻窦炎的疗效及对鼻腔分泌物IL-6、IL-8、TNF-α的影响

目的 探讨布地奈德联合生理盐水鼻腔冲洗对慢性鼻-鼻窦炎(CRS)的疗效及对鼻腔分泌物白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子(TNF-α)的影响。 方法 研究对象选自2016年2月至2017年5月收治的128例CRS患者,采取随机数字表将其分成两组,每组64例。对照组给予生理盐水鼻腔冲洗,观察组在生理盐水中加入布地奈德混悬液,比较两组临床疗效、治疗前后鼻腔分泌物IL-6、IL-8、TNF-α水平变化情况以及患侧鼻腔Lund-Kennedy评分、鼻窦CT评分变化情况。 结果 经过4周治疗,观察组总有效率为93.75%(60/64),与对照组71.88%(46/64)相比明显上升(P<0.01)。治疗后两组患侧鼻腔Lund-Kennedy评分、鼻窦CT评分均较治疗前显著降低(P<0.01),但观察组评分下降更明显(P<0.01)。治疗后两组鼻腔分泌物IL-6、IL-8、TNF-α水平均较治疗前显著降低(P<0.01),但观察组各项炎性因子水平降低更明显(P<0.01)。两组治疗过程中均未出现明显不良反应。 结论 布地奈德联合生理盐水鼻腔冲洗治疗CRS可显著缓解鼻腔炎症反应,改善鼻腔充血、肿胀等症状,疗效确切。     

应用PCR技术研究真菌在鼻息肉及鼻窦炎发病中的作用

目的：探讨真菌在慢性鼻窦炎、鼻息肉患者中的致病作用。方法：采用PCR方法分别检测66例慢性鼻窦炎患者（鼻窦炎组）生理盐水冲洗前、后的鼻息肉及鼻窦黏膜的真菌,同时检测21例正常志愿者（对照组）的鼻腔分泌物中的真菌,应用Y。检验比较其差异性。结果：鼻息肉组真菌阳性率：息肉为89．2％,黏膜为89．4％,分别与对照组（66．7％）比较,差异均有统计学意义（均P〈0.05）;用生理盐水冲洗后,鼻息肉组真菌阳性率：息肉为7．14％,黏膜为2．63％,分别与冲洗前比较,差异均有统计学意义（均P〈0.05）。结论：真菌在鼻息肉和鼻窦炎的发病中可能发挥重要作用,PCR技术在真菌检测中有重要意义。     

Fungus culture of the nasal secretion of chronic rhinosinusitis patients: seasonal variations in Daegu, Korea

BACKGROUND: The role of fungi in the pathogenesis of chronic rhinosinusitis (CRS) is being increasingly recognized. The presences of fungal spores are a necessary condition for fungal sinusitis, and fungal spore counts vary widely, both geographically and seasonally. The aim of this study was to characterize fungal seasonal variation in the nasal cavity of CRS patients and to compare it with histologic types of nasal polyps. METHODS: One hundred eight patients with a clinical diagnosis of CRS with polyps were enrolled in this study for 1 year. Nasal secretions were obtained by aspiration and cultured to identify fungi. Nasal polyps were divided into four histologic types: eosinophilic, fibroinflammatory, seromucinous, and atypical. RESULTS: Positive cultures for fungi were obtained in 60 of 108 (63.0%) CRS patients, and 28 of 45 (62.2%) normal volunteers. Fungus culture rates were higher during summer and fall. Cladosporium, Aspergillus, Alternaria, and Penicilium were frequently isolated from CRS patients and normal volunteers. The most common histologic type was the fibroinflammatory type (60.2%), although in the summer and fall the proportion of the eosinophilic type increased. CONCLUSION: Fungi were commonly cultured during a hot and humid environment, during summer and fall, and cultured organism types were found to be seasonal dependent. Histologic types of nasal polyps appear to be associated with fungal culture rate, organism type, and seasonal variations.     

Biotherapy and treatment of adult primary chronic rhinosinusitis with nasal polyps: Cellular and molecular bases

The present article reviews the molecular and cellular mechanisms involved in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwCRSwNP) and underlying the action mechanisms of biotherapies. Biotherapy uses substances naturally produced by the organism or their specific antagonists targeting a proinflammatory mechanism. CRSwCRSwNP is a form of chronic rhinosinusitis (CRS), which is classically subdivided in to 2 types according to the presence of polyps. In recent years, the concept of endotypes emerged, with a more exhaustive definition of the types of CRS according to inflammatory mechanism, with a view to developing personalized treatments. CRSwNP pathophysiology is poorly understood. Polyps arise from a primary epithelial lesion in a context of chronic local inflammation, mainly type 2 in Europe, implicating eosinophils, IgE, Th2 cytokines (IL-4/IL-13, IL-5) and T and B cells. Biotherapy seems promising in CRSwNP. The present review details the various pathophysiological pathways underlying the action mechanisms of biotherapies, and the various published studies, assessing efficacy and mode of action in the treatment of CRSwNP.     

The pathogenesis of nasal polyposis by immunoglobulin E and interleukin-5 is completed by transforming growth factor-beta1

OBJECTIVES/HYPOTHESIS: Nasal polyps are benign mucosal protrusions into the nasal cavity of multifactorial origin and are characterized by chronic mucosal inflammation. The suggested multifactorial pathological mechanisms comprise several factors including cytokines and immunoglobulin E (IgE). The study was designed to examine the suggested roles of IgE, interleukin-5 (IL-5), and transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of nasal polyposis. METHODS: Nasal polyps (n = 34) and healthy nasal mucosa samples (n = 9) were taken during routine endonasal surgeries. Immunoglobulin E (n = 13), IL-5 (n = 22), and TGF-beta1 (n = 27) concentrations were measured with enzyme-linked immunosorbent assay technique in homogenized polyp tissue and in control mucosa. Atopic and nonatopic groups were selected and compared. Histomorphological examination and immunohistochemical analysis to detect IL-5 and TGF-beta1 were performed in five specimens. RESULTS: The level of tissue-bound IgE was significantly higher in polyps compared with control specimens and in atopic compared with nonatopic polyps, but between nonatopic polyps and control specimens the difference was not significant. However, significant correlation was found between tissue and serum IgE in the complete polyp (P =.001) and atopic polyps group (P =.05). Tissue IL-5 concentration was significantly higher in polyps compared with control specimens, in which it was below the limit (15 pg/mL), and there was no difference between atopic and nonatopic polyps. In atopic polyps there was significant correlation between tissue IgE and IL-5. Transforming growth factor-beta1 concentration proved to be significantly higher in control mucosa than in polyps, with no difference between atopic and nonatopic polyps. Immunohistochemical analysis revealed numerous IL-5-positive eosinophil cells and TGF-beta1 positivity in the lamina propria of polyp samples, but none in control specimens. CONCLUSIONS: High tissue TGF-beta1 quantity in healthy nasal mucosa without its active form on the cell surface and its low quantity in polyps may reflect its essential role in the inhibitory mechanisms of nasal polyposis. Interleukin-5 plays a key role in the eosinophil recruitment and activation, and both atopic and nonatopic pathways might activate this process. The main sources of IL-5 and TGF-beta1 are the eosinophils and macrophages. Immediate hypersensitivity, besides other mechanisms, might be related to atopic polyps, but the involvement of other, local allergic mechanisms in IgE production of nonatopic polyp tissue cannot be excluded.     

Notch通路在鼻息肉中的表达及其与调节性T细胞表达和嗜酸粒细胞浸润的相关性研究

目的:探讨Notch通路在鼻息肉中的表达及其与调节性T细胞（Treg细胞）表达和嗜酸粒细胞（Eos）浸润的相关性。方法:选择2012年11月至2018年8月期间在中山大学附属第三医院接受鼻内镜手术的慢性鼻窦炎（CRS）和鼻中隔偏曲的患者,分别作为CRS组和对照组。收集慢性鼻窦炎不伴鼻息肉（CRSsNP）患者（30例,男...     

鼻腔应用布地奈德对鼻息肉中IL-5表达的作用

目的 :观察鼻腔应用布地奈德治疗对鼻息肉中 IL - 5表达的影响 ,深入了解糖皮质激素类药物局部治疗对鼻息肉的作用机制。方法 :采用免疫组化 ABC法 ,观察经布地奈德治疗 6～ 8周和未经治疗的鼻息肉组织 (各 16例 )中 ,IL -5阳性细胞的浸润和分布状况。结果 :鼻息肉组织固有层中可见 IL - 5阳性细胞表达 ,且多为嗜酸性粒细胞 ,IL - 5阳性细胞密度与嗜酸性粒细胞的浸润程度密切相关 (y=14.782 + 2 .0 0 2 x,r=0 .6 48,P <0 .0 1)。经布地奈德治疗的鼻息肉组织中 IL - 5阳性细胞的浸润程度有下降的趋势 ,但与未治组织的差异未达显著性水平 (P >0 .0 5 )。结论 :1IL - 5的表达与嗜酸性粒细胞的浸润密切相关 ,嗜酸性粒细胞是鼻息肉组织中 IL- 5的来源之一 ;2鼻腔应用布地奈德 6～ 8周治疗可能抑制鼻息肉中 IL- 5表达。