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.     

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.     

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.     

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.     

Itraconazole treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) patients is a potentially fatal inflammatory disease due to the dual-type immune response provoked by the fungal antigens. Despite serious side effects long-term treatment with corticosteroids is often required. Itraconazole has been reported to be a useful steroid-sparing agent. METHODS: In a retrospective follow-up of 21 CF patients from a total of 250 treated once or twice within a five-year study period (1994-98), 9 patients were treated with systemic glucocorticosteroids in combination with itraconazole and 12 patients were treated with itraconazole (200-600 mg/day) as monotherapy. RESULTS: During treatment the percentage of Aspergillus fumigatus (AF)-positive sputum cultures significantly reduced (P < 0.05); precipitating antibodies to AF decreased significantly in all patients (P < 0.05); forced expiratory volume (FEV1) increased to pre-exacerbation level; total IgE levels decreased in 42% of patients on monotherapy and in 56% on combination therapy. Specific IgE (radioallergosorbant; RAST) level decreased in 6 of 21 patients. Eleven patients had transient increased levels of alanine transaminase (ALAT). One patient had isolated increase in alkaline phosphatase and another in aspartate transaminase (ASAT). CONCLUSIONS: High dose itraconazole as monotherapy or in combination with systemic glucocorticosteroids seems effective in CF patients with ABPA. No hepatotoxicity was observed during long-term therapy.     

Antifungal properties of 5-hydroxytryptamine (serotonin) against Aspergillus spp. in vitro

This study shows that 5-hydroxytrypatmine (5-HT, serotonin) is fungicidal towards conidia and hyphae of clinical isolates of Aspergillus (A.) fumigatus, A. flavus and A. terreus. The minimal fungicidal concentrations for Aspergillus conidia and hyphae ranged between 14.68 to 117.5 mM and 29.37 to 235 mM during 24 and 48 h of incubation. Several serotonin receptor antagonists (5-HT2, 5-HT3) studied in vitro did not influence antifungal activity.     

Induction of innate immunity by Aspergillus fumigatus cell wall polysaccharides is enhanced by the composite presentation of chitin and beta-glucan

Chitin and β-glucan are conserved throughout evolution in the fungal cell wall and are the most common polysaccharides in fungal species. Together, these two polysaccharides form a structural scaffold that is essential for the survival of the fungus. In the present study, we demonstrated that Aspergillus fumigatus alkali-insoluble cell wall fragments (AIF), composed of chitin linked covalently to β-glucan, induced enhanced immune responses when compared with individual cell wall polysaccharides. Intranasal administration of AIF induced eosinophil and neutrophil recruitment, chitinase activity, TNF-α and TSLP production in mice lungs. Selective destruction of chitin or β-glucan from AIF significantly reduced eosinophil and neutrophil recruitment as well as chitinase activity and cytokine expression by macrophages, indicating the synergistic effect of the cell wall polysaccharides when presented together as a composite PAMP. We also showed that these cell wall polysaccharides induced chitin-specific IgM in mouse serum. Our in vivo and in vitro data indicate that chitin and β-glucan play important roles in activating innate immunity when presented as composite cell wall PAMPs.