Objective To explore the effects of prolonged inhalation of Aspergillus fumigatus ( Af)spores on epithelial cell injury and expression of epidermal growth factor receptor( EGFR) in airways of asthmatic rats. Methods 64 male Wistar rats were randomly divided into 8 groups, ie. chronic asthma group ( group A) , chronic asthma plus Af spores inhalation for 1 week ( group B) , 3 weeks ( group C) and 5 weeks ( group D) , chronic asthma plus saline inhalation for 5 weeks ( group E) , OVA-sensitized and salinechallenged group ( group F) , and OVA-sensitized and saline-challenged plus Af spores inhalation for 5 weeks ( group G) ( each n =8) . The airway resistance ( Raw) and change of Raw after acetylcholine provocation were detected using a computerized system. The concentrations of epidermal growth factor ( EGF) andtransforming growth factor alpha( TGF-α) in BALF were measured by ELISA. The extents of epithelial cell injury and goblet cell hyperplasia were evaluated on hematoxylin and eosin-stained( HE) and periodic acidschiff ( PAS) stained lung sections. The expression of EGFR in airway epithelia was demonstrated byimmunohistochemistry, and the level of EGFR protein in the rat lung tissues was measured by western blot.Results The concentration of EGF( pg/mL) ( 51. 72 ±8. 54, 68. 12 ±7. 85, 86. 24 ±9. 12, respectively)and TGF-α( pg/mL) ( 55. 26 ±9. 30, 75. 58 ±11. 56, 96. 75 ±14. 66, respectively) , detached/ inner perimeter of epithelium( % ) ( 11. 25 ±3. 12, 26. 45 ±5. 56, 28. 50 ±7. 50, respectively) , the ratio of goblet cell area to epithelial cell area ( % ) ( 16. 42 ±5. 24, 22. 64 ±6. 82, 36. 38 ±9. 21, respectively) , the integrated optical density ( IOD) of EGFR positive stain in airway epithelial cells ( 82 ±15,120 ±19, 165 ±21, respectively) , and the EGFR protein levels in lung tissues ( 0. 91 ±0. 26, 1. 61 ±0. 52, 2. 52 ±0. 78,respectively) in group B, C, and D were higher than those in group A, E, F and G( P lt; 0. 05 or P lt;0. 01) .The change rates of Raw( % ) ( 61. 91 ±5. 26, 84. 69 ±6. 38) in group C and D were higher than those in group A, E, F and G ( P lt; 0. 05 or P lt;0. 01) . The IOD of EGFR was positively correlated with detached/inner perimeter of epithelium( % ) and the ratio of goblet cell area to epithelial cell area( % ) ( r = 0. 692,P lt;0. 01; r = 0. 657, P lt; 0. 01, respectively) . Conclusion Prolonged inhalation of Aspergillus fumigatus spores can aggravate airway epithelial cell injury, up-regulate the expression of EGFR in airway epithelial cell and induce goblet cell hyperplasia, thus increase the airway responsiveness in rats with chronic asthma.
Objective To observe the levels of von Willebrand factor ( vWF) expressed by human umbilical vein endothelial cells ( HUVECs) infected by aspergillus fumigatus ( AF) alone or treatment with cytochalasin D, N-cadherin monoclonal antibody, dexamethasone, respectively, so as to explore the mechanism of angioinvasion in invasive aspergillosis. Methods An in vitro model of HUVECs infected by AF hypha was established. The experiment included six groups, ie. a sham control group, a TNF-αgroup, an AF hypha group, a cytochalasin D group, a N-cadherin antibody group, and a dexamethasone group. Cell supernatants were collected to detect the levels of vWF at 2 h, 6 h, 12 h, and 18 h by enzyme linked immunosorbent assay ( ELISA) . Results Compared with that of vWF at 2 h, the level was higher at 18 h in the sham controlgroup and the TNF-αgroup, and higher at 6 h, 12 h, and 18 h in the other groups( P lt; 0. 05) . Compared with the sham control group, the level of vWF in each experiment group increased at 2 h, 6 h, 12 h, and 18 h except that in the N-cadherin antibody group at 2 h ( P lt; 0. 05) . The level of vWF in TNF-α group was higher than that in the AF hypha group at 2 h, but lower at 18 h. ( P lt; 0. 05) . The level of vWF was not significantly different between the cytochalasin D group and the AF hypha group at each time point. The level of vWF was lower in the N-cadherin antibody group than that in the AF hypha group at 2 h and 6 h ( P lt;0. 05) . The level of vWF was not significantly different between the dexamethasone group and the AF hypha group at each time point. Conclusion HUVECs infected by AF hypha overexpress vWF. N-cadherinmonoclonal antibody can reduce the expression of vWF, but cytochalasin D or dexamethasone has no significant effect on it.
Objective To investigate the clinical manifestation and histopathologic changes of the fungal necrotizing retinochoroiditis. Methods Collecting 7 cases of fungal retinochoroiditis with severe immunodepression and loss of visual acuity.Seven removed eyeballs were stained with HE,PAS and silver methenamine,and observed by light microscopy,and in addition,2 of them examined by electron microscopy.Also fungal cultures of blood and affected tissues were performed. Results The chief clinical macnifestation included ciliary injection of conjunctiva,opaque aqueous fluid and vitreous and diffuse hemorrhage and greyt white opacity with retinal detachment in severe cases.Pathologic changes included hemorrhage in the retina,chorioretinal tissue necrosis,hyphae in the blood vessels,affected tissue and vitreous.Fungal culture of blood was positive in three cases.Culture of affected tissues was positive in all cases. Conclusions Eedogenous fungal infection of choroid and retina may be due to the severe immunodepression of the sufferers and usually causes chorioretinal tissue destruction and blind. (Chin J Ocul Fundus Dis, 1999, 15: 235-237)
Objective To investigate the correlation between persistent wheezing and positive result of sputum fungal culture in patients with chronic obstructive pulmonary disease ( COPD) . Methods The COPD patients who hospitalized in the respiratory department of Shanghai First People’s Hospital, Zhongshan Hospital and Huadong Hospital fromJanuary 2005 to December 2007 were analyzed retrospectively. Results Thirty-five cases were enrolled in the persistent wheezing group and 43 cases in the non-wheezing group. In the wheezing group, sputumfungal culture revealed positive yield in 32 cases while Aspergillus were isolated in 12 cases. In the non-wheezing group, sputum fungal culture revealed only 11 cases positive, and none of which were Aspergillus positive. Aspergillus distributions in the two groups were significantly different( P lt;0. 05) . There was also significant difference in the positive result of sputum fungal culture ( 91. 4% vs 25. 6%, P lt;0. 01) , while there was no significant difference in positive result of bacterial culture( 28. 6% vs 39. 5%, P gt; 0. 05) . In the wheezing group, the patients with antifungal treatment showed better prognosis than those without antifungal treatment( 81. 0% vs 36. 4% , P lt;0. 05) . Conclusion The persistent wheezing in the patients with COPD is correlated with the fungi, especially Aspergillus airway colonization.
ObjectiveTo explore the effects of Aspergillus fumigatus (A. fumigatus) on airway inflammation, airway responsiveness and total serum IgE in asthmatic rats. MethodsEighteen male Wistar rats were divided into three groups randomly, ie. a normal control group, an asthmatic model group, and an A. fumigatus group. The rats in the model group and the A.fumigatus group were sensititized and challenged with ovalbumin to establish asthmatic model. After establishment of asthmatic model, the rats in the A. fumigatus group were treated with chronic A. fumigatus spores inhalation. Subsequently, airway responsiveness/sensitivity to methacholine(Ach), levels of serum IgE and airway inflammation were assessed and compared among three groups. ResultsCompared with the asthmatic rats, the rats treated with A. fumigatus showed higher airway responsiveness (Penh/baselin value was significantly increased at the Mch concentration of 12.5, 25 and 50 mg/mL), increased inflammatory cells infiltration in pulmonary tissue slices and increased serum IgE level (P < 0.05). Most importantly, serum IgE level was detected in close relationship with PC100 which was defined as the dose of Mch causing 100% increase of enhance pause (Penh) value without Mch challenge (r=-0.873, P < 0.01). Serum IgE level was also closely related to the percentage of eosinophils in bronchoalveolar lavage fluid (r=0.937, P < 0.01). ConclusionsChronic A. fumigatus inhalation aggravates airway inflammation, bronchial hyperresponsiveness and serum IgE level in asthma. IgE may play an important role in facilitating the development of bronchial responsiveness and eosinophilic inflammation.
ObjectiveTo investigate the effects of chronic Aspergillus fumigatus (AF) exposure on the expression of platelet-derived growth factor (PDGF) and airway remodeling in the asthmatic rats. MethodsForty male Wistar rats were randomly divided into five groups (8 rats per group): group A (control group): sensitized with ovalbumin (OVA) and challenged with saline; group B (bronchial asthma group): sensitized and challenged with OVA to establish a chronic asthma model; groups C-E (bronchial chronic asthma + AF spores inhalation for 1 week, 3 weeks, or 5 weeks): the asthma rats were received intranasal inhalation of Aspergillus fumigatus spores for 1 week (Group C), 3 weeks (Group D), or 5 weeks (Group E). The concentrations of interleukin-5 (IL-5), interleukin-13 (IL-13), and transforming growth factor-beta (TGF-β) in bronchoalveolar lavage fluid (BALF) were measured using enzyme-linked immunosorbent assay (ELISA). Lung sections were stained with periodic acid Schiff (PAS) and Masson trichrome stain, and the images were analyzed morphometrically.The expression of PDGF were detected by immunohistochemistry and Western blot. ResultsThe levels of TGF-β (66.43 ± 2.78 ng/mL), IL-5 (33.49 ± 2.01 ng/mL), and IL-13 (50.78 ± 2.16 ng/mL) in BALF, as well as the extent of goblet cell hyperplasia (8.82 ± 1.95) and the degree of subepithelial collagen deposition (10.28 ± 2.48) in group B were higher than those in Group A (P<0.05). After inhalation of AF spores, the concentrations of TGF-β, IL-5, IL-13, along with the extent of goblet cell hyperplasia, and the degree of collagen deposition in the airways were increased progressively in group C, group D and group E (P<0.05). The expression of PDGF in the airways were upregulated in a dose-dependent manner. The extent of subepithelial collagen deposition and goblet cell hyperplasia in the airways were positively correlated with the expression of PDGF. ConclusionChronic exposure to Aspergillus fumigatus upregulates the expression of PDGF in the airways of bronchial asthma rats, thereby promoting airway remodeling.
Objective To explore the effects of prolonged Aspergillus fumigatus spores inhalation on airway inflammation and remodeling in rats with chronic obstructive pulmonary disease（COPD）.Methods Fifty Wistar rats were randomly divided into group A，B，C，D and E，(n=10 in each group) and group E was served as normal control.In group A，B，C and D，COPD models were established by intratracheal administration of lipopolysaccharide (LPS) combined with cigarette smoke exposure.The rats in group A，B and C were given intranasal inhalation of 1×106cfu spores，1×103cfu spores and 100 mL saline twice a week for consecutive 5 weeks，respectively，while the rats in group D were given no treatment.Bronchoalveolar lavage fluid(BALF) were collected for total and differential cell count，and interleukin-8（IL-8） and transforming growth factor-b(TGF-b) concentration measurement.The pathologic changes of lung tissue were observed by HE，PAS and Masson stainings.Results Pathological changes characteristic of COPD were found in group D.The total cell count，the percentage of neutrophile and lymphocyte in BALF in group A and B were higher than those in group C and D(all Plt;0.01).IL-8 and TGF-b in BALF in group A and B were higher than those in group C and D(all Plt;0.01).The pathologic score of airway inflammation in group A was higher than those in group B，C and D(all Plt;0.01)：The thickness of airway wall(WAt/Pbm) and airway smooth muscles(WAm/Pbm)，the collagen deposition in the total airway wall(WCt/Pbm) and in the outer airway wall(WCo/Pbm) and the percentage of goblet cells to epithelial cells in group A and B were higher than those in group C and D(all Plt;0.01).In group A and B，IL-8 was positively correlated with the percentage of neutrophile(r=0.856，Plt;0.01)，the pathologic score of airway inflammation(r＝0.884，Plt;0.01)，and the percentage of goblet cells to epithelial cells (r=0.702，Plt;0.05)，respectively.TGF-b was positively correlated with WAt/Pbm，WCt/Pbm，WCo/Pbm and the ratio of goblet cells to epithelial cells (r=0.706，Plt;0.05：r=0.802，Plt;0.01：r=0.876，Plt;0.01：r=0.713，Plt;0.05).Conclusion Prolonged inhalation of Aspergillus fumigatus spores can aggravate the airway inflammation and remodeling in rats with COPD.
ObjectiveTo investigate antifungal activity in vitro of single or combination of triazole and echinocandin against Aspergillus species. MethodsBased on EUCAST protocol,the susceptibilities of 62 isolates of Aspergillus spp. were determined for voriconazole (VRC),itraconazole (ICZ),caspofungin (CAS) and micafungin (MICA). For VRC and ICZ,MIC-0 and MIC-2 were determined. For CAS and MICA,minimum effective concentration (MEC) and MIC-2 were determined. The fractional inhibitory concentration (FIC) was used to evaluate the effect of combination of triazole and echinocandin. ResultsIndifference was found in 2 isolates of Aspergillus fumigatus in combination of ICZ and CAS or MICA by using MIC-0 endpoint. Synergy was found in all other isolates of Aspergillus spp.With MIC-2 and MEC endpoints,synergy for VRC and CAS,VRC and MICA,ICZ and CAS and ICZ and MICA was found in 16,21,11 and 14 isolates of Aspergillus fumigatus,9,13,9 and 11 isolates of Aspergillus flavus,0,2,1 and 1 isolates of Aspergillus niger,respectively. ConclusionThe in vitro sensitivity results of combination of triazole and echinocandin are different with different endpoints. Thus,the efficacy of combination of triazole and echinocandin can not predicted by in vitro sensitivity and should be further confirmed in invasive aspergillosis animal experiments.
Objective To explore the effects of Aspergillus fumigatus（A. fumigatus） spores on airway inflammation and responsiveness in asthmatic rats.Methods Seventy male Wistar rats were randomly divided into Ⅰ and Ⅱ groups（n=35 in each group），then Group Ⅰ and Group Ⅱ were subdivided into a normal control group（n=5），an asthma group（n=10），a spores-treated control group（n=10），and a spores-treated asthma group（n=10）.The rats were sensitized to ovalbumin（OVA） and challenged with aerosol OVA to establish the asthma model.The effects of A. fumigatus spores on asthmatic rats before and after OVA aerosol challenging were investigated in Group Ⅰ and Group Ⅱ，respectively.The parameters associated with bronchial epithelial damage were observed by total protein concentration in BALF measured by BCA method.Total and differential cell counts in BALF were also counted.The airway resistance and airway responsiveness were calculated by transpulmonary pressure and gas flow rate.Results In Group Ⅰ，the total protein in BALF in the asthma group treated with A. fumigatus spores before OVA challenging（Group CA） was increased remarkably compared to the asthma group（Group A1）[（1.125±0.254）μg/mL vs（0.825±0.173）μg/mL，Plt;0.01].The nonspecific airway resistances induced by different concentration of acetylcholine in Group CA ［（0.997±0.196）cm H2O?mL-1?s-1，（1.123±0.142）cm H2O?mL-1?s-1，（1.130±0.197）cm H2O?mL-1?s-1］were increased significantly compared to Group A1 [（0.655±0.089）cm H2O?mL-1?s-1，（0.687±0.048）cm H2O?mL-1?s-1，（0.821±0.043）cm H2O?mL-1?s-1]（all Plt;0.05）.In Group Ⅱ，however，the above parameters in the asthma group treated with A. fumigatus spores after OVA challenging（Group AC） were not dramatically increased compared with the asthma group（Group A2）（all Pgt;0.05）.The differences in the total and differential cell counts in BALF in Group CA were not remarkable compared to other subgroups in Group Ⅰ（all Pgt;0.05）.But the BALF neutrophil count in Group AC was increased obviously compared to Group A2 ［（2.488±0.420）×106 vs （0.936±0.459）×106，Plt;0.05］.Conclusion These data indicate that exposure to A. fumigatus spores before challenging causes aggravated epithelial damage and increased airway resistance in an asthma rat model.
ObjectiveTo investigate the role of Aspergillus in the severe refractory exacerbations of chronic obstructive pulmonary disease (COPD).MethodsThe clinical data of two COPD patients suffering from refractory acute exacerbations were analyzed and the relevant literature were reviewed.ResultsTwo patients were male, aging 72 and 64 years respectively. Both of them had a history of frequent acute exacerbations with severe COPD recently. Meanwhile, they received intravenous use of antibiotics repeatedly, one of them took oral corticosteroids to control wheezing, but failed. Their serum Aspergillus-specific IgG antibody was weakly positive. Besides traditional treatment, they received additional antifungal therapy, and the symptoms alleviated. There was no acute exacerbation in the half a year follow-up period after appropriate therapy.ConclusionsAspergillus colonization, sensitization, infection should be considered in patients with severe COPD. When Aspergillus-associated evidence are acquired, antifungal therapy will be unexpected helpful.