TLR4基因沉默下调TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用

目的 研究单核巨噬细胞RAW264.7受烟曲霉孢子刺激时,TLR2和TLR4信号通路发挥的作用,以及沉默TLR4基因后,对TLR2信号通路的影响.方法 利用RNAi技术将TLR4-siRNA转染RAW264.7细胞24h后给予烟曲霉孢子刺激12h,将细胞随即分为正常组(N组)、正常+烟曲霉孢子刺激组(N+Af组)、正常+TLR4-siRNA组[TLR4(RNAi)组]、正常+TLR4-siRNA+烟曲霉孢子刺激组[ TLR4(RNAi) +Af组],RT-PCR和Western blot法检测细胞受烟曲霉孢子刺激后TLR2、TLR4、MyD88 mRNA及TNF-α蛋白的表达变化.结果 (1)TLR4基因沉默前:与N组比较,N+Af组TLR2、TLR4、MyD88 mRNA及TNF-α蛋白表达量均显著升高(P＜0.05).(2)TLR4-siRNA( 100nmoL/L)转染RAW264.7细胞,沉默效率达83％.(3)TLR4基因沉默后:与N组比较,TLR4( RNAi)组TLR2、MyD88 mRNA的表达量均显著降低(P＜0.05);与N+Af组比较,TLR4( RNAi)+Af组的TLR2、MyD88 mRNA和TNF-α蛋白的表达量均显著降低(P＜0.05);与TLR4( RNAi)组比较,TLR4(RNAi)+ Af组MyD88 mRNA表达量显著升高(P＜0.05),而TLR2 mRNA及TNF-α蛋白表达量却无显著变化(P＞0.05).结论 RAW264.7细胞受烟曲霉孢子刺激时,TLR2和TLR4信号通路被激活,通过释放促炎细胞因子TNF-α发挥抗烟曲霉孢子刺激作用;当沉默TLR4基因后,TLR2信号通路不能被很好地激活来抵抗烟曲霉孢子对细胞的刺激作用,沉默TLR4基因下调了TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用,可能TLR4较TLR2在抵抗烟曲霉孢子刺激时发挥更重要的作用.     

Stimulation by means of dendritic cells followed by Epstein-Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses

Adoptive immunotherapy with in vitro expanded antigen-specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocyte-derived dendritic cells (DC) as antigen-presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp-specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2-5x) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC-DC or autologous PPC-pulsed BLCL (PPC-BLCL). Lines from the DC/BLCL arm demonstrated Asp f16-specific cytotoxicity earlier and to a higher degree than lines generated with PPC-DC alone. The DC/BLCL-primed lines showed a higher frequency of Asp f16-specific interferon (IFN)-gamma producing cells but an identical effector cell phenotype and peptide specificity compared to PPC-DC-only-primed lines. Tumour necrosis factor (TNF)-alpha, but not IL-10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16-specific T cell responses and that a culture approach using initial priming with PPC-DC followed by PPC-BLCL may be a more effective method to generate Asp f16-specific T cell lines and requires less starting blood than priming with PPC-DC alone.     

Assessment of antibacterial and antifungal potential of Curcuma longa and synthesized nanoparticles: A comparative study

Curcumin nanoparticles were most recently considered in medical research because of their antibacterial properties. The main objective of the study was to develop the green synthesis and antibacterial activity of curcumin nanoparticles using Curcuma longa. The processing of curcumin nanoparticles was carried out after the collection, identification, and extraction of curcumin. The effect of a sample on the synthesis of nanoparticles, such as curcumin aqueous concentrations (5, 10, and 20 mg/ml) and curcumin nanoparticles (5, 10, and 20 mg/ml), and the antibacterial effect of these nanoparticles on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and the fungal strain Aspergillus niger. For examining antibacterial and anti-fungal activity disc diffusion method was performed, followed by the zone of inhibition. According to X-ray diffraction and scanning electron microscope analysis, nanoparticles have spherical shapes and size of 42.64 nm. Results showed that a high dose of 20 mg/ml curcumin nanoparticles have more antibacterial activity than curcumin extracts in E. coli as it showed the largest diameter of zone of inhibition as compared to other doses. Other bacterial and fungal strains also showed significant results but E. coli was most prominent. The biosynthesis of curcumin nanoparticles using an aqueous extract of C. longa is a clean, inexpensive, and safe method that has not been used any toxic substance and consequently does not have side effects. Since several pathogenic species have acquired antibiotic resistance, the combination of curcumin with various nanoparticles would be beneficial in the cure of pathogenic diseases.     

Immune responses to Aspergillus antigen in IL‐4‐/‐ mice and the effect of eosinophil ablation

BACKGROUND: Exposure to Aspergillus fumigatus allergens results in enhanced total serum IgE and peripheral blood eosinophils in mice. The associated pulmonary inflammation and immunologic responses are comparable to those detected in human allergic bronchopulmonary aspergillosis. Allergen-induced cytokines are thought to regulate the inflammatory and immune responses in these animals. METHODS: In the present study, we exposed C57BL/6 and BALB/c mice to A. fumigatus antigen. Both wild-type and IL-4 knockout phenotypes of animals of both strains were used. Some animals were also treated with anti-IL-5 or anti-IFN-gamma. Total serum IgE, Aspergillus species IgG subclass, peripheral blood eosinophils, and lung histology were studied. RESULTS: The results demonstrate similar lung inflammation in all wild-type and IL-4-/- animals exposed to A. fumigatus antigen. Similarly, in spite of the diverse immune response produced by the anticytokine treatment, no major differences were detected among any of the animal groups studied. CONCLUSIONS: It can be concluded that A. fumigatus exposure in an immunologically unaltered host is predominantly of a Th2 type, and that depletion of the Th2 cytokine leads to a similar lung inflammation but with a characteristic Th1 response, suggesting that the pathogenesis of allergic aspergillosis is the result of multiple induction pathways.     

Clinical utility of antifungal susceptibility testing in patients with fungal rhinosinusitis

PurposeTo determine the association between antifungal susceptibility test (AFST) results and in vivo therapeutic response in Indian patients with fungal rhinosinusitis.MethodsThe clinicoradiological, fungal culture, AFST, histopathology results and outcomes of 48 patients with fungal rhinosinusitis seen between 20132015 were analysed. Minimum inhibitory concentration (MIC) determination was performed for amphotericin B, itraconazole, voriconazole and posaconazole.ResultsForty patients had invasive and 8 had non-invasive fungal sinusitis. Rhizopus and Aspergillus species which comprised 46.9% each of isolates were mostly associated with acute invasive fungal rhinosinusitis and chronic granulomatous fungal rhinosinusitis respectively. All patients with non-invasive fungal rhinosinusitis had Aspergillus isolates.The Geometric Mean (GM) MIC for R. arrhizus of amphotericin B and posaconazole was 0.51 mcg/mL and 3.08 mcg/mL respectively and for A. flavus species for amphotericin B and voriconazole values were 1.41mcg/mL and 0.35 mcg/mL respectively.In patients with Aspergillus infections, while there was no association of MICs for azoles and outcome (p = 1), a strong association was noted between azole therapy and a good outcome (p = 0.003). In patients with Rhizopus infections, no association was found between MICs for amphotericin B and outcome (p = 1) and because of therapeutic complications, no association was found between amphotericin B therapy and outcome (p = 1).ConclusionNo significant association exists between in vitro (AFST) and in vivo responses despite low GM MICs for the drugs used in Aspergillus and Rhizopus infections. Therapeutic complications following conventional amphotericin B therapy confounds analysis. Clinical responses suggest that azoles are the drug of choice for Aspergillus infections.     

侵袭性肺曲霉菌病的MSCT表现

目的 探讨侵袭性肺曲霉菌病（IPA）的MSCT表现。方法 回顾性分析41例经临床及病理证实为IPA的MSCT表现。结果 35例血管IPA的MSCT主要表现为多发病灶25例,以肺野外周带分布为主26例;表现为结节或肿块影29例,伴晕征16例,出现空气半月征或空洞20例。6例气道IPA的MSCT主要表现为沿支气管分布的斑片影4例,以双肺中上野分布为主4例。15例血管IPA随访患者中,在出现晕征后的第2、4周分别出现空气半月征6例、空洞4例;5例气道IPA在随访1周时出现斑片结节融合4例,2周时支气管扩张4例,4周时4例支气管扩张加重。结论 血管IPA的MSCT多表现为肺野外周带多发结节或肿块影,常伴晕征或空气半月征或空洞;气道IPA多表现为沿支气管分布的斑片影,以中上肺野分布为主,常合并逐步加重的支气管扩张。IPA进展迅速,CT动态随访观察有助于诊断。     

原位肝移植术后侵袭性曲菌病的诊断和治疗

目的探讨原位肝移植术后侵袭性曲菌病的诊治。方法回顾性分析2000年1月至2005年1月完成的576例原位肝移植的临床资料,总结术后侵袭性曲菌病的防治经验。结果9例患者术后并发侵袭性曲菌病,疾病发生率为1．74％（9／576）,首发感染部位为肺部8例,中枢神经系统感染1例,发病时间在术后10d至2个月,术后持续或间断的低热可以是发病早期的主要症状。痰或其他分泌物的真菌镜检和培养是确诊的主要依据。二性霉素B脂质体是治疗的首选用药,对早期病例疗效满意,5例肺部感染患者痊愈,2例因肺部感染无法控制死亡,2例因并发多器官侵袭死亡。结论肝移植术后侵袭性曲菌病具有早期临床表现不典型和易于播散的特点,合理调整免疫抑制治疗方案及早期、足量和足程的抗真菌药物的使用是取得良好疗效的关键。     

霉菌性脊柱炎1例并文献复习

目的探讨霉菌性脊柱炎的临床表现、影像特征和病理特点,提高对霉菌性脊柱炎的认识和鉴别诊断能力。方法回顾性分析1例经手术病理证实的霉菌性脊柱炎病人的影像及病理资料并行文献复习。结果 CT检查显示胸4～胸7椎体广泛骨质硬化伴有周围软组织肿块,病变内部可见散在小片状骨质破坏区,邻近椎间隙无明显变窄;MRI显示胸4～胸8椎体可见斑片状异常信号影,T_1WI上呈低信号,T_2WI上呈稍高信号,抑脂序列呈高信号,邻近椎间盘未见明显异常,增强检查病变椎体、椎旁及硬膜外肿块呈明显强化。术后病理诊断为霉菌感染,血清曲霉菌半乳甘露聚糖检测为阳性。结论霉菌性脊柱炎罕见,其临床和影像表现无特异性,确诊需依赖于病理组织学检查。     

不同中药单体成分对两性霉素B耐药的烟曲霉抑菌活性的体外研究

目的研究4种中药单体成分对两性霉素B（AMB）耐药的烟曲霉菌的体外抗真菌效果,为进一步开发能抑制耐药烟曲霉的新药奠定研究基础。方法采用微量液基稀释法测出肉桂醛、柠檬醛、绿原酸、黄芩苷对9株临床分离的耐药烟曲霉菌的最低抑菌浓度（MIC）和最低杀真菌浓度（MFC）进行实验。结果肉桂醛对两性霉素B耐药的烟曲霉的MIC50、MIC50、MFC50和MFC90分别为128ug／ml、256u／ml、256ug／ml和1024ug／ml,柠檬醛对两性霉素B耐药的烟曲霉的MIC50、MIC90、MFC50和MFC90分别为2S6ug／ml、512ug／ml、512ug／ml和〉1024ug／ml,绿原酸、黄芩苷对两性霉素B耐药的烟曲霉的MIC50、MIC90、MFC50和MFC90均为〉1024ug／ml。结论肉桂醛、柠檬醛对AMB耐药的烟曲霉有抗真菌作用,绿原酸和黄芩苷在体外对AMB耐药的烟曲霉无抗真菌活性。