Toll样受体在慢性化脓性中耳炎和中耳胆脂瘤中的差异表达及其意义

目的 检测Toll样受体2( toll-like receptor 2,TLR2)和Toll样受体4(toll-like receptor 4,TLR4)在慢性化脓性中耳炎、中耳胆脂瘤中的表达,探讨其在慢性化脓性中耳炎和中耳胆脂瘤发病中的作用.方法 选取耳硬化症患者(对照组)、慢性化脓性中耳炎患者(化脓性中耳炎组)、中耳胆脂瘤患者(中耳胆脂瘤组)各30例,应用实时定量聚合酶链反应( real-time PCR)、蛋白质印迹法( Western blot)、免疫组化检测TL R2/TLR4在正常外耳道皮肤,慢性化脓性中耳炎黏膜、肉芽组织,中耳胆脂瘤患者黏膜、肉芽组织及胆脂瘤囊壁中的表达,并比较表达程度的差异.结果 ①TLR2/TLR4mRNA及蛋白质在正常外耳道皮肤,慢性化脓性中耳炎中耳黏膜、肉芽组织,胆脂瘤的中耳黏膜、肉芽组织及胆脂瘤囊壁均有表达.②TLR2/TLR4 mRNA及其蛋白质在慢性化脓性中耳炎及胆脂瘤黏膜中的表达均高于正常外耳道皮肤(P值均＜0.05),在胆脂瘤囊壁中的表达低于正常外耳道皮肤(P值均＜0.05),两组黏膜中TLR2/TLR4的表达差异无统计学意义(P值均＞0.05).③TLR2/TLR4mRNA及蛋白质在两组中耳炎肉芽组织中的表达均高于正常外耳道皮肤(P值均＜0.01),且TLR2mRNA在中耳胆脂瘤肉芽组织中的表达高于慢性化脓性中耳炎(P＜0.05).④TLR2/TLR4阳性细胞主要分布在肉芽组织中,且明显多于正常外耳道皮肤,但胆脂瘤囊壁中的阳性细胞少于正常外耳道皮肤.结论 TLR2和TLR4在正常外耳道皮肤、慢性化脓中耳炎及中耳胆脂瘤中均有表达,提示中耳具有TLR2、TLR4参与调节的固有免疫系统,但二者的差异性表达也提示其在慢性化脓性中耳炎和中耳胆脂瘤的发病中所起的作用不同.     

TLR4/MyD88/NF-κB信号通路在嗅觉障碍小鼠模型中的表达变化

目的 探讨TLR4/MyD88/NF-κB信号通路在嗅觉障碍中的表达变化。方法 40只健康BALB/c小鼠分为观察组与对照组,各20只。通过定量逆转录PCR检测小鼠Toll样受体4(Toll-like receptors,TLR4)、髓样分化因子88(myeloid differentiation primary response gene 88,My D88)以及核转录因子κB(nuclear factor kappa B,NF-κB)水平;通过蛋白免疫印迹法（Western blot,WB）检测TLR4、My D88及NF-κB蛋白含量;免疫组化检测嗅觉标记蛋白（mouse olfactory marker protein,OMP）表达情况。结果 建模后观察组小鼠觅食时间长于对照组（P <0.05）;观察组小鼠TLR4、My D88以及NF-κB mRNA相对表达量明显高于对照组（P <0.05）;观察组小鼠TLR4、My D88以及NF-κB蛋白表达明显高于对照组（P <0.05）;观察组小鼠OMP阳性细胞数量明显低于对照组（P<0.05）。结论 TLR4...     

核因子κB在分泌性中耳炎动物模型鼓室黏膜中的表达

目的检测核因子κB(nuclear factor-κB,NF-κB)在分泌性中耳炎(secretory otitis media,SOM)动物模型鼓室黏膜中的表达,探讨NF-κB活化在SOM发病机制中的作用。方法应用灭活肺炎链球菌注入豚鼠中耳腔,制作SOM动物模型,采用免疫组织化学的方法检测致病鼠鼓室黏膜上皮在注入细菌后6h、1d、3d、7d及14d后NF-κBp65、TNF-α和IL-1β的表达及其关系。结果SOM动物模型的鼓室黏膜上皮中NF-κBp65在术后6h就有表达,24小时表达最强,随后表达明显下降,TNF-α和IL-1β的表达也出现相应波动。结论灭活肺炎链球菌致SOM的早期,NF-κB即被激活,在鼓室黏膜中出现过度表达,并与SOM发病中的重要致炎因子TNF-α和IL-1β的产生相关,提示NF-κB与SOM发病机制密切相关。     

Toll样受体2及其信号通路在大鼠面神经夹挫伤后的机制研究CSCD

目的 探究面神经损伤对大鼠Toll样受体2(TLR2)/核转录因子κB(NF-κB)信号通路的影响。方法 将面神经损伤大鼠随机分为三组，A组：正常对照组，B组：手术损伤后自然恢复的观察组，C组：手术后腹腔注射Pam3CSK4(TLR2激动剂)观察恢复。电镜比较3组面神经的表现；HE染色脑干组织观察神经元的凋亡，PCR观察TLR2、NF-κB p65 mRNA表达水平，免疫荧光观察TLR2、NF-κB的荧光表达。结果 面瘫造模顺利，电镜下面神经损伤明显，C组大鼠髓鞘较B组更为破碎；脑区内神经元也表现为胶质细胞变性更多；PCR显示A组TLR2及NF-κB p65 mRNA表达低于B组和C组；免疫荧光显示B组表达的TLR2、NF-κB荧光强于A组。结论 注射Pam3CSK4激活TLR2/NF-κB信号通路，加重炎症的表现使面神经功能障碍。     

重组活化基因1缺陷鼠和正常小鼠急性细菌性鼻窦炎模型的比较研究

目的　探讨C5 7BL/ 6J 重组活化基因 1(recombinantactivegene 1,Rag1)敲除鼠 (简称Rag1小鼠 )和C5 7BL/ 6 (C5 7)小鼠急性细菌性鼻窦炎的自然感染过程及二者之间的差别。方法　Rag1缺乏鼠和C5 7小鼠各 10只 ,采用鼻孔内接种肺炎链球菌T5 9,另外 4只接种大豆肉汤作对照。分别于接种2d(各 2只 )、5d(各 4只 )、10d(各 2只 )、14d(各 2只 )处死动物 ,对照组于第 5天处死。处死前作鼻腔灌洗培养 ,头颅石蜡包埋 ,连续切片 ,Luna染色 ,计算机辅助光镜观察 ,计算窦腔内中性粒细胞集落所占窦腔的百分率和每平方毫米窦腔黏膜中浸润的多形核白细胞数。结果　接种 5dRag1小鼠和C5 7小鼠窦腔感染均达到高峰 ,窦腔中白细胞集落和黏膜中白细胞数均明显高于对照组 (P <0 0 5 ) ,10d后C5 7小鼠窦腔感染逐渐减轻 ,14d后基本控制 ,而Rag1小鼠感染持续存在 ,与C5 7比较差异有显著性(P <0 0 5 ) ,14d后鼻灌洗液中仍培养出肺炎链球菌。结论　采用肺炎链球菌T5 9鼻内接种法成功地诱导出Rag1和C5 72种小鼠急性鼻窦炎 ,肺炎链球菌在C5 7小鼠鼻腔鼻窦内的感染可被完全、自主、快速控制 ,但Rag1小鼠则不能完全控制这种感染 ,并有演变成慢性炎症的倾向 ,提示T和B淋巴细胞依赖性免疫功能在清除细菌感染中起着关键的作用 ,基因敲除     

TLR_2、TLR_3和TLR_4在变应性鼻炎大鼠鼻黏膜组织中的表达

目的研究TLR2、TLR3和TLR4在实验性变应性鼻炎大鼠鼻黏膜组织中的表达。方法Wistar大鼠46只,随机分3组,即A组:模型组(20只),以卵清蛋白腹腔注射、雾化及滴鼻。B组:生理盐水对照组(20只),以生理盐水代替卵清蛋白腹腔注射、雾化及滴鼻。C组:空白对照组(6只),未予给药。用免疫组化方法检测3组大鼠鼻黏膜中TLR2、TLR3、TLR4的表达。结果A组大鼠成功激发为AR动物模型;3组鼻黏膜中均有TLR2表达;TLR3在3组鼻黏膜中均无明显表达;TLR4在实验组表达,在生理盐水对照组及空白对照组未见明显表达。三组间TLR2、TLR3的表达差异无统计学意义(P0.05);模型组与生理盐水组及空白对照组TLR4的表达差异有统计学意义(P0.05)。结论AR大鼠有TLR4的表达增高,表明TLR4可能参与了变应性鼻炎的发病。TLR2、TLR3在AR大鼠中的表达未见增高,其与变应性鼻炎的关系有待进一步研究。     

TLR4基因敲除小鼠对噪声性耳蜗损伤的反应

目的揭示Toll样受体(Toll-like receptor,TLR)基因缺失对噪声性耳蜗感觉细胞损伤,听觉功能障碍和耳蜗免疫活力的影响作用,探讨噪声性耳蜗损伤的免疫炎性机制。方法将TLR4基因敲除(TLR4 KO)小鼠接触持续噪声暴露(1-7kHz,120dB SPL)1小时,以噪声暴露的野生型(WT)小鼠为对照,检测噪声暴露前和噪声暴露后25天四个频率短纯音(4 kHz、8 kHz、16 kHz和32 kHz)诱发的小鼠双耳ABR阈值。噪声暴露后1天,25天处死动物,解剖取双侧耳蜗。采用白细胞共同抗原(CD45)荧光免疫抗体标记噪声暴露前和噪声暴露后1天耳蜗基底膜免疫细胞,鬼笔环肽(phalloidin)染色噪声暴露前和噪声暴露后25天耳蜗基底膜毛细胞纤毛、表皮板的丝状肌动蛋白。荧光显微镜下观察噪声暴露后TLR4 KO小鼠和WT小鼠耳蜗基底膜组织的巨噬细胞、单核细胞和毛细胞形态变化,计数耳蜗基底膜外毛细胞的缺失数目。结果噪声暴露后1天,WT和TLR4 KO小鼠耳蜗基底膜均呈现单核细胞渗入。噪声暴露后25天,不同频率短纯音诱发的TLR4 KO小鼠ABR阈移和耳蜗基底膜外毛细胞缺失数目均低于WT小鼠(F=71.590, df=1,90, P<0.001; Tukey test, P<0.001),(F=8.996, df=1,17, P=0.008; Tukey test, P=0.008)。结论噪声暴露后TLR4基因缺失没有阻止单核细胞渗入耳蜗基底膜,但减轻噪声暴露后耳蜗感觉细胞和听功能损伤的程度。     

Toll样受体在真菌性鼻及鼻窦炎的表达及意义

目的研究Toll样受体2(Toll—like receptor 2,TLR2)和Toll样受体4(Toll—like receptor 4,TLR4)在真菌性鼻及鼻窦炎(fungal rhinosinusitis,FRS)患者鼻黏膜中的表达,并探讨其在FRS发病中的可能作用。方法采用逆转录-聚合酶链反应和免疫组化SP法分别检测24例FRS、24例慢性鼻及鼻窦炎(chronic rhinosinusitis,CRS)和15例鼻中隔偏曲手术患者正常下鼻甲鼻黏膜中TLR2和TLR4 mRNA和蛋白的表达水平。结果TLR2和TLR4 mRNA在FRS组的表达水平(0.208±0.063和0.1 59±0.059)明显低于CRS组(0.505±0.097和0.406±0.102)和鼻中隔偏曲患者组(0.327±0.047和0.232±0.088,P均<0.05)。FRS组TLR2和TLR4蛋白的阳性表达率明显低于CRS组和鼻中隔偏曲组(P<0.05)。结论TLR2和TLR4在FRS中表达下调,提示Toll样受体介导的天然免疫在鼻黏膜抗真菌防御反应中作用下降,是引起鼻黏膜真菌清除率下降和真菌易感性增加的原因之一。     

L型电压门控钙通道α1D亚基在内耳毛细胞的分布及在听力中的作用

目的探讨L型电压门控钙通道α1D亚基在内耳毛细胞的分布及在听力中的作用。方法应用免疫细胞化学荧光标记观察L型电压门控钙通道α1D亚基在牛蛙毛细胞上的分布;利用听性脑干反应(ABR)检测携带不同L型电压门控钙通道α1D基因型(α1D / 、α1D /-和α1D-/-)小鼠的听力。结果L型电压门控钙通道α1D亚基主要密集分布于毛细胞侧膜和顶膜,在毛细胞的胞核区域和纤毛丛处没有阳性荧光染色。三种α1D基因型小鼠的ABR检测结果显示,α1D / 野生型小鼠的短声反应阈正常,为34.8±5.7dBSPL;α1D /-杂合子小鼠反应阈高于同窝生α1D / 野生型小鼠,为54.4±12.4dBSPL;α1D-/-小鼠呈现全聋,ABR在100dBSPL时仍无反应。结论L型电压门控钙通道的α1D亚基分布在牛蛙毛细胞的侧膜和顶膜,在内耳的听觉生理中具有重要作用。L型电压门控钙通道α1D亚基缺失或是数量的减少均可以影响小鼠听力。     

Toll-like Receptors 2 and 4 and Their Mutations in Patients with Otitis Media and Middle Ear Effusion

Objectives

Toll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion.

Methods

Middle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis.

Results

Among the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20±60.94 ng/mL, 21.59±7.96 ng/mL and 11.55±16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile).

Conclusion

TLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.     

The role of Toll-like receptor 4 in eliciting acquired immune responses against nontypeable Haemophilus influenzae following intranasal immunization with outer membrane protein

Objective

Acute otitis media (AOM) is one of the most common infectious diseases in children. Nontypeable Haemophilus influenzae (NTHi) is considered a major pathogen in AOM. Current treatment options depend mainly on the use of antibiotics, thus developing vaccines to prevent this disease is an urgent goal for public health. Outer membrane proteins (OMPs) are promising candidate targets for vaccination against NTHi.

Methods

We used C3H/HeJ (Toll-like receptor 4 [TLR4]-mutant) mice, which arose spontaneously and have a non-functional TLR4 protein, and normal wild-type (WT) C3H/HeN mice. These mice were immunized intranasally with OMP from NTHi to investigate the mechanism of acquired immunity via TLR4. We examined the kinetics of mucosal and systemic antibody secretion and the migration of antibody producing lymphocytes to the mucosa in both strains during the course of intranasal immunization.

Results

The mucosal and systemic immune responses against OMP from NTHi were elicited in both TLR4-mutant and WT mice. However, the mucosal IgA, and systemic IgG, and Th1 immune responses in WT mice were stronger than those in TLR4-mutant mice.

Conclusions

TLR4 plays an important role in relation to Th1 function for optimal development of the acquired immune responses to OMP administered intranasally. The variety of immune responses via TLR4 expression needs to be taken into consideration of individual vaccinations to prevent AOM.     

The effect of altered Toll-like receptor 4 signaling on cancer cachexia

OBJECTIVE: To determine whether mice unable to mount an intact inflammatory response because of a Toll-like receptor (TLR) pathway defect will develop less severe cancer cachexia. DESIGN: Prospective animal study. SETTING: Academic research center. SUBJECTS: Six- to eight-week-old, female C3H/HeJ mice (17-18 g) and age-, weight-, and sex-matched wild-type C3H/HeN mice, differing in that the HeJ mice have nonfunctional TLR4 due to a TLR4 double mutation (TLR4(d/d)). INTERVENTION: The mice were inoculated with equal numbers of SCCF-VII cells and housed in individual cages. MAIN OUTCOME MEASURES: Food intake, body weight, pretumor and posttumor body composition, circulating cytokines, and levels of a marker of muscle atrophy were analyzed. RESULTS: The wild-type HeN mice weighed less on average than the TLR4(d/d) mice (2.6 g vs 4.9 g) (P = .01). They consumed more food, had smaller tumors, and had less lean body mass and fat mass than the TLR4(d/d) mice. Interleukin 1beta level was significantly elevated in the tumor-bearing HeN mice (mean gain of 259 pg/mL) but not in the TLR4(d/d) mice (P = .03). Both mouse strains had evidence of muscle atrophy. CONCLUSIONS: In spite of increased food intake and smaller tumors, the wild-type HeN mice had more severe cachexia than the TLR4(d/d) mice. The impaired ability to secrete proinflammatory cytokines such as interleukin 1beta may protect these animals from developing severe cancer cachexia. This animal model represents a novel system in which the host contributions to cachexia may be further studied.     

Experimental recurrent pneumococcal otitis media. Protection and serum antibodies.

To elucidate immune mechanisms in otitis media, middle ear infection was induced in 12 rats by intratympanic inoculation of Streptococcus pneumoniae type 3, either the ipsilateral or the contralateral middle ear being re-challenged 4 weeks later. Otomicroscopy inspection confirmed the presence of acute otitis media (AOM) in all rats after the first challenge. After re-challenge, protection against AOM was noted both in the ipsilateral and contralateral ears. Serum antibody concentrations increased after the initial challenge, reaching a maximum at 4-7 days, but had decreased to pre-immune levels at re-challenge, after which no new increase was noted. Serum IgG-antibodies to pneumococcal type 3-polysaccharide were triggered following the initial induction of unilateral pneumococcal AOM, but mucosal immune mechanisms are argued to be a more probable explanation of the ipsilateral protection seen after re-challenge.     

TLR-9, NOD-1, NOD-2, RIG-I and immunoglobulins in recurrent otitis media with effusion

Objectives

Pattern recognition receptors (PRRs) induce appropriate immune responses after recognizing certain molecular characteristics of pathogens. It is not known, however, whether PRRs are expressed in middle ear infections and whether the expression of PRRs and immunoglobulins is correlated in recurrent otitis media with effusion (OME). We therefore investigated the expression of PRRs and immunoglobulins in children with OME.

Materials and methods

The study population consisted of 66 children with OME, of whom 27 had more than 4 episodes in 12 months or more than 3 episodes in 6 months (otitis-prone group), and 39 had fewer than 4 episodes in 12 months or 3 episodes in 6 months (non-otitis-prone group). The expression in middle ear effusion of Toll-like receptor (TLR)-9, nucleotide-binding oligomerization domain (NOD)-1, NOD-2, and retinoic acid-inducible gene (RIG)-I mRNA, as determined by real-time PCR, and the concentrations of IgG, IgA, and IgM, as determined by ELISA, were compared between the two groups.

Results

The levels of TLR-9, NOD-1 and RIG I mRNAs were significantly lower in the otitis-prone than in the non-otitis-prone group (p < 0.05 each). The concentrations of IgG, IgA and IgM in effusion fluid did not differ significantly between the two groups (p > 0.05), and there were no correlations between immunoglobulin concentration and the expression of PRPs (p > 0.05).

Conclusions

Decreased expression of PRRs may be associated with increased susceptibility to OME.     

Evaluation of importance of Toll-like receptor 4 in acute Streptococcus pneumoniae sinusitis in mice

OBJECTIVES: To investigate the effect of RC-527, a synthetic toll-like receptor 4 (TLR4) agonist, on stimulating the immune response before acute Streptococcus pneumoniae sinusitis in a mouse model, and to determine the importance of TLR4 in modulating the response to S. pneumoniae. Toll-like receptor 4 agonists have been shown to induce protective innate immune responses when administered before some bacterial or viral challenges in mice. DESIGN: We intranasally inoculated BALB/c, TLR4 complex-deficient C3H/HeJ, and wild-type C3H/HeOuJ mice with S. pneumoniae 24 hours after treatment with 10 or 1 microg of RC-527 or vehicle. Bacterial counts from nasal lavage culture and the cell markers GR1, CD11b, CD3, CD4, and CD8 in sinus tissue were quantified at postinoculation days 2, 5, and 14. MAIN OUTCOME MEASURE: Immune response induced by RC-527. RESULTS: Treatment with RC-527 induced an immune response through TLR4, as demonstrated by recruitment of phagocytes in uninfected wild-type C3H/HeOuJ mice, but not in TLR4 complex-deficient C3H/HeJ mice. The immune response was also demonstrated by a significant increase of CD3+, CD4+, and CD8+ T cells in infected and uninfected wild-type C3H/HeOuJ mice, but not in TLR4 complex-deficient C3H/HeJ mice. However, the enhancement of the immune response induced by the TLR4 agonist showed a limited effect on bacterial clearance. CONCLUSIONS: Our studies in mice suggest that stimulation of TLR4 plays a minor role in the overall response to S. pneumoniae infection of the upper airway, and stimulating this receptor before infection does not significantly enhance the immune response of immunocompetent mice to clear S. pneumoniae infection.     

Decreased Pattern-Recognition Receptor-Mediated Cytokine mRNA Expression in Obese Children With Otitis Media With Effusion

Objectives

To assess innate and humoral immune responses in middle ear effusion of obese pediatric patients with otitis media with effusion (OME).

Methods

We evaluated 219 children with OME, of whom 21 were obese and 198 were non-obese. We compared the expression in middle ear effusion of mRNAs encoding toll-like receptors (TLR) 2, 4, 5, and 9; nucleotide-binding oligomerization domains (NOD) 1 and 2; retinoic acid-inducible gene (RIG)-I; interleukins (IL)-6, -10, and -12; interferon (IFN)-γ; and tumor necrosis factor (TNF)-α mRNAs. We also compared the expression of immunoglobulins IgG, IgA, and IgM and the bacterial detection rate in the two groups.

Results

TLR2-mediated expression of IL-6 mRNA, TLR4-mediated expression of IL-6 and IL-10 mRNA, TLR5-mediated expression of IL-6, IL-10, and TNF-α mRNA, TLR9-mediated expression of IL-6 mRNA, and NOD2-mediated expression of IL-6, IL-12, and TNF-α mRNA were significantly lower in obese than in non-obese children (P<0.05). However, concentrations of IgG, IgA, and IgM in middle ear effusion were lower in obese than in non-obese children, but none of these differences was significant (P>0.05).

Conclusion

Mean body mass index was higher and pattern-recognition receptor-mediated cytokine mRNA expression was lower in obese than in non-obese children with OME.     

Influence of nasal allergic reactions on the clearance of middle ear effusion

In order to investigate the influence of nasal allergic reactions on the clearance of middle ear effusion, an animal model of nasal allergy and otitis media with effusion was produced in the same guinea pigs simultaneously by passive sensitization with serum of homologous animals containing IgE antibodies (for nasal allergy) and by inoculation of immunocomplex into the tympanic cavity (for otitis media with effusion). Usually, middle ear effusion appeared within 2 to 3 days and disappeared within 7 to 9 days after the inoculation of immunocomplex. Three days after the inoculation of immunocomplex, intranasal antigen challenge was performed three times daily and continued until the animals were killed. Disappearance of middle ear effusion appeared to be delayed in animals in which nasal allergic reactions were induced. Middle ear effusion was not found in those ears that were not inoculated with immunocomplex. Findings of the present study indicate that IgE-mediated allergic reactions of the mucous membrane lining the nose, nasopharynx, and eustachian tube constitute a factor indicative of a chronic state of disease, rather than a cause of otitis media with effusion.