| Objective:At present, the specific pathogenesis is not fully clear. Last several years, studies have identified EV71 has two other specific receptors—PSGL-1 and Annexin A2 on cell surface, except for SCARB2 identified by cell in vitro experiments. 2 articles were reported about PSGL-1 in human tissues, and there is no literature reported on Annexin A2. At the same time,studies have shown that TLR3 and TLR8 can identify the some double-stranded RNA viruses and single-stranded RNA viruses, respectively. However, not have seen in the human body tissues to explore the effect by TLRs and TLR/MyD88 downstream signal molecules in severe hand, foot and mouth disease caused by EV71 infection.Therefore, this study will be conducted by immunohistochemical method to detect whether PSGL-1 and the Annexin A2 provide a basis to invade human tissues when EV71 infection, to clarify the significance of TLR3 mRNA and TLR8 mRNA in EV71 infection, and to clarifythe role of TLR/MyD88 downstream signal transduction molecules—IRAK-1,TRAF-6, NF-κB p65 in inflammatory response caused by EV71 infection.Methods:The brain and lung tissue wax blocks from 9 cases which died of EV71 infection and 7 cases which died of noninflammatory diseases, were slected as the research objects. All families of cases had signed the autopsy informed consents, and this study had been passed and approved by hospital ethics committee. The expression of PSGL-1, Annexin A2, IRAK-1, TRAF-6and NF-κB p65 were tested by immunohistochemistry(IHC). The expression of TLR3 mRNA and TLR8 mRNA were tested by in situ hybridization(ISH). Using image analysis software to detect the integrated optical density(IOD) of pictures,then all experimental datas were analyzed by SPSS18.0 statistical software.Results:(1) EV71 virus receptors detection results: immunohistochemical method showed that both PSGL-1 and Annexin A2 were positive expressed in the lung and brain tissues of the EV71 group and the control group. PSGL-1 and Annexin A2 IOD values in the lung tissues of EV71 group were 125033, 9376,respectively; PSGL-1 and Annexin A2 IOD values in the lung tissues of control group were 60939, 4179, respectively. The expression differences of PSGL-1and Annexin A2 between the two groups were both statistically significant(P<0.05). PSGL-1 and Annexin A2 IOD values in the brain tissues of EV71 group were 49871, 39883, respectively; PSGL-1 and Annexin A2 IOD values in the brain tissues of control group were 9932, 3810, respectively. The expression differences of PSGL-1 and Annexin A2 between the two groups were both statistically significant(P<0.05).(2)TLRs detection results:(1)TLR3 mRNA expressed positively in part of cytoplasm of alveolar epithelial cells, bronchialwall epithelial cells, neutrophils, monocyte-macrophages in lung tissue from 4case in EV71 group(9 cases) and 3 cases in control group(7 cases) detected by in situ hybridization. 1 case of brain tissue in EV71 group(9 cases) had a few microglia detected by in situ hybridization which showed with weak positive signal. Immunohistochemical detection results showed that TLR3 were positive expression in 9 cases of lung, brain tissue of EV71 group.(2)TLR8 mRNA expressed positive signal in part of cytoplasm of bronchial wall epithelial cells,alveolar epithelial cells and neutrophils in lung tissues from 4 cases in EV71group(9 cases) and 1 case in control group(7 cases) detected by in situ hybridization. TLR8 mRNA expressed weak positive signal in part of cytoplasm of neurons, microglia and leukomonocyte in brain tissues from only 1 case in EV71 group(9 cases) and 2 cases in control group(7 cases) detected by in situ hybridization. Immunohistochemical detection results showed that TLR8 were positive or strong positive expression in 9 cases of lung, brain tissue of EV71 group.(3) The comparison of positive expression score between IHC and ISH methods: the positive expression score of TLR3, TLR3 mRNA in the lung tissue of EV71 group were 3.78±0.67, 1.11±0.93, detected by IHC and ISH methods respectively. The positive expression score of TLR3, TLR3 mRNA in the brain tissue of EV71 group were 3.33±0.5, 0.89±0.67, detected by IHC and ISH methods respectively. The positive expression score of TLR8, TLR8 mRNA in the lung tissue of EV71 group were 5.22±0.83, 1.44±0.53, detected by IHC and ISH methods respectively. The positive expression score of TLR8, TLR8 mRNA in the brain tissue of EV71 group were 4.56±0.73, 0.78±0.67, detected by IHC and ISH methods respectively. The positive expression scores had statistically significant difference between the two methods(P<0.05).(4)TLRs signalingmolecule detection results: immunohistochemical detection results showed that IRAK-1, TRAF-6 and NF-κB p65 were positive expressed in the lung and brain tissues of the EV71 group and the control group. IRAK-1, TRAF-6 and NF-κB p65 IOD values in the lung tissues of EV71 group were 15198, 10605, 78733,respectively; IRAK-1, TRAF-6 and NF-κB p65 IOD values in the lung tissues of control group were 123613, 56820, 5165, respectively. The expression differences of IRAK-1, TRAF-6 and NF-κB p65 between the two groups were both statistically significant(P<0.05). IRAK-1, TRAF-6 and NF-κB p65 IOD values in the brain tissues of EV71 group were 8324, 3240, 35855, respectively;IRAK-1, TRAF-6 and NF-κB p65 IOD values in the brain tissues of the control group were 20079, 7179, 5251, respectively. The expression differences of IRAK-1, TRAF-6 and NF-κB p65 between the two groups were statistically significant(P<0.05).Conclusions:(1)EV71 virus receptors( PSGL-1 and Annexin A2) exist in human lung and brain tissues, which prompt that EV71 virus could directly invade the brain and lung tissue.(2) TLR/MyD88 downstream signaling molecules—IRAK-1, TRAF-6 and NF-κB p65 may participate in the proinflammatory and suppression of inflammation reaction process in the severe hand, foot and mouth disease infected by EV71.(3) For the human brain and lung tissues fixed by10% formalin and embeded by paraffin after a long time, IHC method is more sensitive than ISH method to detect the expression of TLRs. |
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