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BACKGROUND:Pulmonary aspergilosis is a disease caused by pulmonary fungal infection. Its diagnosis and treatment is usualy delayed because of nonspecific clinical symptoms, physicial sign and imaging changes as wel as uncertainties of histological and bacterial findings. Therefore, it is necessary to establish mouse models of invasive pulmonary aspergilosis to investigate the underlying pathological mechanism and novel therapeutic methods. OBJECTIVE: To establish mouse models of invasive pulmonary aspergilosis, detect the expression ofγ-interferon, Tol-like receptor 2 and Tol-like receptor 4, and discuss the mechanism of action underlying invasive pulmonary aspergilosis. METHODS:Seventy-five female BALB/c mice of clean grade, aged 6-8 weeks, were randomly and evenly divided into five groups: blank control group (group A), immunosuppressive model group treated with high concentrations of Aspergilus fumigatus spore suspension (group B), normal infection group treated with high concentration of Aspergilus fumigatus spore suspension (group C), immunosuppressive model group treated with low concentration of Aspergilus fumigatus spore suspension (group D), normal infection group treated with low concentration of Aspergilus fumigatus spore suspension (group E). First, mice in the groups B and D were intraperitonealy injected with cyclophosphamide to establish immunosuppressive models. The mice in the groups D, E (108 cfu/mL) and groups B, C (109 cfu/mL) were treated with 12 mL Aspergilus fumigatus spore suspension through the use of nebulizer. Mice in the group A were treated identicaly with sterile PBS. At 1, 3, 5 days of infection, the pathological change of lung tissue was observed, the mass concentration of γ-interferon in bronchoalveolar lavage fluid and the expression levels of γ-interferon mRNA and Tol-like receptor 2 and Tol-like receptor 4 mRNA and protein in the lung tissue were determined. RESULTS AND CONCLUSION:Abscess, spores and very severe bleeding and congestion, widenened alveolar septum and tracheal epithelial cel shedding and necrosis were observed in the mouse lung tissue in the group B. At 5 days of infection, the mass concentration of γ-interferon in bronchoalveolar lavage fluid and the expression ofγ-interferon mRNA in the lung tissue in the group B were significantly decreased compared with the group A (P < 0.05). Tol-like receptor 2 expression was strongly positive in the group B. Tol-like receptor 2 expression in the group C was significantly lower than that in the group B (P< 0.05). Tol-like receptor 4 expression was positive in the groups B and C, and its expression in the group C was significantly greater than in the group B (P < 0.05). The expression of Tol-like receptor 2, 4 mRNA in the mouse lung tissue of group B was significantly increased at 1, 3, 5 days of infection (P < 0.05). These results suggest that atomizing high concentration of aspergilus fumigatus spore suspension to immunosuppressive mice can establish stable invasive pulmonary aspergilosis models with typical pathological features. The infection of aspergilus fumigatus can activate tol-like receptor 2, 4 at the same time, and the pathological mechanism is closely related to organism’s immune defense function.
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BACKGROUND:MicroRNAs (miRNA) through regulating specific target gene mRNA expression play important roles in different processes of diseases. OBJECTIVE:To study the interaction of miRNA-21 with its target gene TLR4 in Hela cels. METHODS:The candidate target gene of miRNA-21 was determined according to miRNA analysis databases. The constructed recombinant adenovirus vector carrying pri-miRNA-21 gene was used, which could package and amplify viruses to transfect Hela cels. Then, the expression of fluorescent proteins was detected. Forty-eight hours after transfection of miRNA-21 or control, extracted proteins were used for detection of TLR4 protein using western blot assay. RESULTS AND CONCLUSION:Recombinant adenoviruses pAd/pri-miRNA-21 and pAd/neg at 100 MOI could successfuly infect Hela cels. Bioinformatic analysis suggested several possible binding sites between miRNA-21 and TLR4. The experimental results showed that miRNA-21 down-regulated TLR4 at protein levels, indicating that miRNA-21 can interfere with the expression of TLR4 target gene.
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This article expounds the molecular biological characteristics and signaling pathw ay of Tol -like receptor 2 and investigates its advances in research on the roles of neuronal and blood brain barrier damage after cerebral ischemia.
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BACKGROUND:Tol-like receptor (TLR) and its signaling pathway play an important role in autoimmune diseases, hypersensitivity, inflammation, apoptosis and transplant rejection; however, its effects on immune pathogenesis of Henoeh-Schonlein purpura in children have not been fuly elucidated. OBJECTIVE:To investigate the TLR2 expression in peripheral blood mononuclear cels in children with Henoeh-Schonlein purpura and its correlation with immune response. METHODS: Sixty-four children with Henoeh-Schonlein purpura were divided into two groups: non-renal damage group (n=36) and renal damage group (n=28). Meanwhile, another 30 healthy children subjected to health examination acted as control group. Flow cytometry and florescent quantitative PCR were employed to detect TLR2 protein and mRNA expression in peripheral blood mononuclear cels, respectively. ELISA was used to detect plasma interferon-γ and interleukin-4 levels and transforming growth factor β and interleukin-10 levels secreted from Treg cels. RESULTS AND CONCLUSION:Levels of interferon-γ and interferon-γ/interleukin-4 in the children with Henoeh-Schonlein purpura were significantly lower than those in the control group (P < 0.05), while the level of interleukin-4 was higher than the control group (P < 0.05). The expression of TLR2 protein and mRNA was significantly higher in the Henoeh-Schonlein purpura children than the healthy children (P < 0.05) and significantly higher in the renal damage group than the non-renal damage group (P < 0.05). Compared with the control group, the levels of interleukin-10 and transforming growth factor β were significantly higher in the children with Henoeh- Schonlein purpura (P < 0.05). These findings indicate that Henoeh-Schonlein purpura children have increased levels of TLR2 protein and mRNA in the peripheral blood mononuclear cels, and exhibit immune imbalance. TLR2 is involved in the pathogenesis of Henoeh-Schonlein purpura, and transforming growth factor β can be used to evaluate Treg immune response and provide reference for diagnosis, treatment of prognosis of Henoeh-Schonlein purpura children.
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BACKGROUND:Our previous studies have shown that mesenchymal stem cells play an immunomodulatory role in Th17 cells, but the mechanism of action remains to be elucidated, therefore, to explore the role of Tol-like receptor 1 in which wil provide possible experimental basis for the future potential of celltherapy strategies. OBJECTIVE:To investigate the role of Tol-like receptor 1 in the immunoregulatory function of mesenchymal stem cells on Th17 cells. METHODS:Separation of adherent bone marrow-derived human embryonic sources of mesenchymal stem cells, immunomagnetic separation of normal CD4+T cells. CD4+T cells were cultured alone or in combination with mesenchymal stem cells co-cultured 4d. Real-time quantitative polymerase chain reaction assay interleukin-17, Tol-like receptor 1 expression levels related genes;number of Th17 cells by flow cytometry. Bone marrow mesenchymal stem cells from human embryos were separated using adherence method, and co-cultured with human CD4+T cells from healthy donors using immunomagnetic separation method for 4 days. The expression of interleukin-17 and Tol-like receptor 1 mRNA was detected by real-time PCR, and the number of Th17 cells was observed by flow cytometry. RESULTS AND CONCLUSION:Tol-like receptor 1 mRNA was expressed in both CD4+T cells and mesenchymal stem cells. Level of interleukin-17 mRNA was significantly higher in mesenchymal stem cells co-cultured with CD4+T cells than in CD4+T cells cultured alone (relative value 3.59±0.11 vs. 1.14±0.08, P<0.01). Consistent with the expression of interleukin-17 mRNA, increased level of Tol-like receptor 1 mRNA was detected in mesenchymal stem cells co-cultured with CD4+T cells compared with CD4+T cells cultured alone (relative value 6.07±1.79 vs. 1.53±0.63, P<0.01). Furthermore, Flow cytometry analysis showed that the percentage of Th17 cells in the mesenchymal stem cells co-cultured with CD4+T cells was significantly higher than that in CD4+T cells cultured alone, (4.53±1.27)%vs. (2.39±0.80)%(P<0.01). Tol-like receptor 1 might be involved in the immunoregulatory regulation of Th17 cells by mesenchymal stem cells.
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BACKGROUND:Previous studies have found that embryonic bone marrow mesenchymal stem cel s can promote human Th17 cel proliferation, but the inherent regulatory mechanisms stil need to be elucidated. OBJECTIVE:To investigate the role of Tol-like receptor 3 in the immunoregulation of Th17 cel s by mesenchymal stem cel s. METHODS:Human CD4+T cel s from healthy donors were isolated by immunomagnetic bead method, and then cultured alone or co-cultured with embryonic bone marrow mesenchymal stem cel s for 4 days. The mRNA expression level of interleukin-17, Tol-like receptor 3 and MyD88 was detected by real-time PCR. RESULTS AND CONCLUSION:Compared with CD4+T cel cultured alone group, the mRNA level of interleukin-17 was significantly higher in the co-culture group (3.59±0.11 vs. 1.14± 0.08, P<0.01). Consistent with the expression of interleukin-17 mRNA, increased level of Tol-like receptor 3 mRNA was detected in the co-culture group compared with the CD4+T cel cultured alone group (3.10±1.60 vs. 0.94± 0.01, P<0.05). Furthermore, MyD88 in the co-culture group was significantly higher than that in CD4+T cel cultured alone group (2.29±0.05 vs. 1.85±0.31, P<0.01). Tol-like receptor 3 may be involved in the immunoregulation of Th17 cel s by embryonic bone marrow mesenchymal stem cel s, which provides experimental evidence for potential cel therapeutic strategy.
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BACKGROUND:Tol-like receptor 4 (TLR4) and its ligand, lipopolysaccharid, are closely associated with the occurrence and development of periodontitis. Meanwhile, the immunological properties of periodontal ligament stem cells (PDLSCs) play an important role in the reconstruction of periodontal tissue and cel-based therapy of periodontitis. However, the effect of TLR4 and lipopolysaccharid on the immunological properties of PDLSCs remains unclear. OBJECTIVE:To investigate the effect of TLR4 on the immunological characteristics of human PDLSCs. METHODS:PDLSCs were isolated by enzyme digestion method as previously reported, and were cultured in the medium containing 10 mg/L lipopolysaccharid, the ligand of TLR4 for 3 days. Using un-treated PDLSCs as controls, we then investigated whether lipopolysaccharid-treated PDLSCs could cause the proliferation of al ogeneic T lymphocytes as wel as the effect of lipopolysaccharid-treated PDLSCs on the mixed lymphocytes reaction and proliferation of lymphocytes induced by phytohemagglutinin. PDLSCs, peripheral blood mononuclear cells and phytohemagglutinin were co-cultured, and the concentration of prostaglandin E2 in the culture supernatant was examined. Then we added indomethacin, which is the inhibitor of prostaglandin E2, into the co-culture system of PDLSCs, peripheral blood mononuclear cells and phytohemagglutinin, and tested the proliferation of lymphocytes. RESULTS AND CONCLUSION:Lipopolysaccharid-treated PDLSCs did not lead to the proliferation of al ogeneic T lymphocytes just as un-treated PDLSCs, and could suppress the mixed lymphocytes reaction and proliferation of phytohemagglutinin-induced lymphocytes. However, the inhibitory ability of lipopolysaccharid-treated PDLSCs was significantly lower than that of un-treated PDLSCs. The levels of prostaglandin E2 were significantly elevated in the co-culture of PDLSCs, peripheral blood mononuclear cells and phytohemagglutinin. After adding of indomethacin, the PDLSCs-suppressed proliferation of lymphocytes restored to normal levels. Lipopolysaccharid weakened the immunosuppressive capacity of PDLSCs, which may be due to the decreasing secretion of prostaglandin E2.