The Toll-like receptors are a kind of pattern recognition receptors,which are expressed in a variety of cells,playing an important role in innate immunity and subsequent acquired immunity.At the same time,they are also involved in the immunity,proliferation,apoptosis and other physiological processes of the cells.Toll-like receptors are also expressed in hematological malignancies and involved in the progress of tumor development,immune escape,resistance,which play a double-edged sword role.In this paper,basic knowledge of TLR molecule and study on hematological tumor were mainly reviewed,aiming to provide a train of thoughts for the treatment of hematologic malignancies.

Objective:To investigate the effect and mechanism of targeted B7-H3 gene silencing on the tumorigenesis and metastasis of human hematological malignancy xenograft tumor in nude mice.Methods: Real-time fluorogentic quantitative PCR (qPCR) and flow cytometry (FCM) were used to detect the expression of B7-H3 in 13 strains of malignant hematologic cells.Then,U937,Maver and Z138 cells which expressed high level of B7-H3 were screened out.Targeted B7-H3 knockdown in U937,Ma-ver and Z138 was performed by lentivirus transduction and the effect of B7-H3 silencing in stable cell lines was tested by qPCR and FCM.Injecting the nine groups subcutaneously into the nude mice to establish xenograft models after dividing the U937,Maver and Z138 into non-infected control group (CON),B7-H3 knockdown group (KD) and negative non-targeted control infected group (NC),respectively,for detecting the tumorigenicity and metastasis in vivo.Furthermore,the expression of Ki-67 in xenograft tumors was detected by immunohistochemistry (IHC).The expression of metalloproteinase 2 (MMP-2) was detected by western blot.Results: The stable B7-H3 silencing cell lines of U937,Maver and Z138 were successfully established.Compared with the NC group,the KD groups of U937,Maver and Z138 had an obviously slower tumor growth.The average tumor inhibition rates at the end of observation period were 61.83％ (F=43.78,P<0.05),59.12％ (F=36.51,P<0.05) and 67.37％ (F=40.29,P<0.05);there was no significant difference in tumor volume growth between the NC group and the CON group (P>0.05).The liver distant metastasis of all the xenograft tumor models in nude mice was the most common and the rates of distant metastasis in KD groups were significantly lower than that of the corresponding NC groups.The Ki-67 indexes of the KD groups were significantly lower than those of the relative NC groups in three cell lines (U937: 40.3％±5.2％ vs.79.1％±6.3％,q=30.31,P<0.05,Maver: 35.2％±6.4％ vs.69.6％±5.1％,q=24.82,P<0.05;Z138: 38.4％±7.1％ vs.75.7％±4.8％,q=28.07,P<0.05);there was no significant difference in the expression of Ki-67 between the NC group and the CON group (P>0.05).The expressions of MMP-2 were also significantly lower in the KD groups than in the NC groups (U937: q=14.59,P<0.05;Maver: q=9.25,P<0.05;Z138: q=11.04,P<0.05);there was no significant difference in the expression of MMP-2 between the NC group and the CON group (P>0.05).Conclusion: Targeted B7-H3 gene silencing could inhibit the tumorigenesis and metastasis of human hematological malignancy xenograft tumor in nude mice.The mechanism may be related to the down-regulation of Ki-67 and MMP-2.

Objective To investigate the association between SLC10A1 gene mutations in c.800G>A mutation and c.356 +1098C >T mutation, and the susceptibility to HBV infection by mother-to-child transmission ( MTCT) .Methods Totally 306 individuals born to HBeAg-positive mothers with high load HBV and without receiving nucleotide analogues treatment, including 247 HBV-infected cases and 59 non-HBV-infected ones were enrolled from Southwest Hospital during May 2011 and July 2015.Blood samples were collected from all the subjects, then genomic DNA was extracted and c.800G>A mutation and c.356+1098C>T mutation of SLC10A1 were genotyped .Chi-square test (Pearsonχ2or continuity correctionχ2) was performed to identify the difference in genotypes between two groups.Results Among vaccinated individuals (55 HBV infected and 56 not infected), the frequency of genotype GA of c.800G>A mutation in non-infected ones was 14.3%(8/56), there was a tendency of increasing compared with HBV infected ones (5.5%, 3/55), but the difference was not statistically significant (χ2 =2.424, P =0.119). Similarly, the frequencies of genotypes CC, CT and TT of the c.356+1098C>T mutation in HBV infected ones were 20.0%(11/55), 47.3%(26/55) and 32.7%(18/55), while those in non-infected ones were 12.5% (7/56), 69.6% (39/56) and 17.9% (10/56), and the difference was not of statistical significance (χ2 =5.766, P=0.056).In all subjects (vaccinated and non-vaccinated), the frequency of genotype GA of c.800G>A mutation in non-HBV infected group had an increasing tendency compared with HBV-infected offspring (13.6% vs.6.9%), but the difference was not statistically significant (χ2 =2.010, P=0.156);the frequencies of genotype CC, CT and TT of c.356+1098C>T mutation in HBV infected patients were 20.2%(50/247), 49.8%(123/247) and 30.0%(74/247), while those in non-HBV-infected group were 11.9%(7/59), 69.5%(41/59) and 18.6%(11/59), and the difference was statistically significant (χ2 =7.436, P =0.024 ) .Within the HBV infected group, the frequencies of genotype GA of c.800G>A mutation were 5.5%(3/55) in vaccinated individuals and 7.3%(14/192) in non-vaccinated individuals, and the difference was not of statistical significance (χ2 =0.030, P=0.863);Similarly, the frequencies of genotype CC, CT and TT of c.356 +1098C >T mutation in vaccinated individuals were 20.0%(11/55), 47.3%(26/55) and 32.7%(18/55), while those in non-vaccinated individuals were 20.3%(39/192), 50.5%(97/192) and 29.2%(56/192), and the difference was not of statistical significance (χ2 =0.274, P=0.872).Conclusion c.356+1098C>T mutation in SLC10A1 may be associated with susceptibility to HBV infection of child born in HBeAg positive pregnant women infected with high load HBV.

The ORFK8.1 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant 0RFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 ug/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein's specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS),primary effusion lymphoma (PEL) and a proportion of cases of multicentric Castleman's disease (MCD). The ORF73 protein was cloned into pQE80L-orf73 and expressed in E.coli and purified. The expressed recombinant ORF73 was identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A protein of about 27 kDa was expressed as expected. Western Blotting showed that the purified recombinant ORF73 reacted with KSHV positive serum. The immunogenicity of the recombinant ORF73 was further analysed by ELISA and the optimal conditions were determined. The ORF73 ELISA was used to compare the KSHV seroprevalence between Hubei and Xinjiang Han people. The Hart people in Xinjiang have significantly higher KSHV seroprevalence than their counterparts in Hubei (6.7% vs 2.9%, P = 0.005).

Objective To explore the level of expression, clinical signiifcance of sB 7-H 3 in the bronchoalveolar lavage lfuid (BALF) of refractory Mycoplasma pneumoniae (MP) pneumonia (RMPP) in children and the relationship between sB7-H3 and various cytokines. Methods The BALF of forty-three hospitalized children with RMPP (RMPP group) were collected for the diagnosis and treatment. Thirteen cases were lavaged only once and the other thirty cases had collected the BALF twice. The BALF of iffteen hospitalized children with bronchial foreign body were collected as control group. The expression levels of sB 7-H 3 , IL-1β, IL-2 and IL-36 in the BALF were detected by enzyme-linked immunosorbent assay. The expression levels of sB 7-H 3 , IL-1β, IL-2 and IL-36 in the BALF at the acute phase were compared with control group and the group after treatment. Analyzed the correlation between the level of sB 7-H 3 and IL-1β, IL-2 , IL-36 in the BALF of RMPP children at acute stage. Results The levels of sB 7-H 3 , IL-1β and IL-36 in the BALF of the ifrst lavage group were higher than those of single lavage group and control group (all P<0 . 05 ). The levels of sB 7-H 3 , IL-1β, IL-2 and IL-36 in the BALF of single lavage group were higher than those of control group (all P<0 . 05 ). The levels of sB 7-H 3 , IL-1β, IL-2 and IL-36 in the BALF of the second lavage group were lower than those of the ifrst lavage group (all P<0 . 05 ).The levels of sB 7-H 3 , IL-2 in the BALF of the second lavage group were higher than those in the control group (both P<0 . 05 ), but the levels of IL-1β, IL-36 in the BALF showed no difference between the second lavage group and the control group (both P>0 . 05 ). The levels of sB 7-H 3 had positive correlation with the levels of IL-1β, IL-2 and IL-36 (all P<0 . 001 ). Conclusions sB 7-H 3 may control the secretion of IL-1β, IL-2 and IL-36 , and participate in immune response and lung injury after MP infection, which may lead to occurrence and development of RMPP.

Objective To analyze the correlation between response to hepatitis B virus (HBV) vaccine and cellular immunity and clinical characteristics in children with respiratory infection.Methods Nine hundred and sixty children in Department of Respiratory in Children's Hospital of of Soochow University,who were over 7 months old and had full course of HBV vaccination between January and December 2015 were enrolled in this study.Peripheral blood (1-2 mL) was collected,and antigen-antibody of HBV was detected by using enzyme-linked immunosorbent assay and PCR included HBV surface antigen,hepatitis B antibody,HBV e antigen,HBV e antibody,HBV core antibody,and HBV nucleic acid.According to the results,these children were divided into 4 groups:non response group,low response group,normal response group and high response group according to their responses to HBV vaccine.Cellular immunity was detected by using flow cytometry and patients' clinical data was collected.Results There was no statistical differences of CD3 + CD4 +,which were (3.43 ± 0.28) ％,(3.42 ± 0.30) ％,(3.43 ± 0.36) ％ and (3.52 ± 0.29) ％,among the four groups (F =0.520,P =0.669).CD3 + CD8 + in non response group was (3.18 ±0.28)％,which was significantly higher than that in low response group,normal response group and high response group [(3.08 ± 0.36)％,(3.05 ±0.34)％,(2.93 ±0.30)％],the differences were significant (all P＜0.05);CD4/CD8 in non response group (0.26 ± 0.43) were significantly lower than that in normal response group (0.40 ± 0.50),the differences were significant (P =0.001).There was no significant difference of CD3 +,CD3 + CD8 + and CD4/CD8 among low response group,normal response group and high response group (all P ＞ 0.05).CD3-CD19 + and CD19 + CD23 + level were lowest in non response group [(3.00 ± 0.57) ％,(2.25 ± 0.67) ％] and highest in high response group [(3.33 ± 0.45) ％,(2.57 ± 0.38) ％],the differences were significant (all P ＜ 0.05).Among the 4 groups,children in normal response group had the shortest average hospitalization days [(1.88 ±-0.31) d],which was significantly shorter than that in non response group,low response group and high response group [(1.96 ± 0.39) d,(1.95 ± 0.38) d,(1.96 ±0.15) d],the differences were significant (all P ＜0.05),there was no significantly difference of average hospitalization days among other 3 groups (all P ＞ 0.05).Proportion of severe pneumonia was significantly higher in non response group [6.1％ (22/363cases)] and high response group [13.3％ (2/15 cases)] compared to those in normal response group [2.6％ (7/274cases)],the differences were statistically significant (x2 =4.417,P =0.036;x2 =5.476,P =0.019).The total white blood cell number was lowest in non response group (F =4.695,P =0.003).Platelet number was increased with higher degree of response to HBV (F =6.598,P ＜ 0.001).Conclusions Cellular immunity is lower in respiratory infection children with non response or low response to HBV vaccine.After they have respiratory infection,children with non response to HBV vaccine may have a longer course of disease and worse condition.

Objective To study the characteristics of etiology of lobar pneumonia in hospitalized children.Methods Medical history and sputum specimens were collected from 1 179 hospitalized children with lobar pneumonia from January 2006 to December 2015.Multiple pathogenic joint detection combined with the history data were used for analysis.Seven kinds of common respiratory virus were detected by direct immunofluorescence.Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP) and human Bocavirus (hBoV) were detected by fluorescence quantitative polymerase chain reaction (PCR).Human Rhinovirus (HRV) and human Metapneumovirus (hMPV) were detected by reverse transcription PCR.Aspirates were cultured for bacteria.MP specific antibody IgG and IgM were tested by enzyme-linked immunosorbent assay (ELISA).Positive rates of each group were compared by χ2 test or Fisher exact test.Results Total etiology detection rate of lobar pneumonia in hospitalized children was 83.9% (989/1 179).The etiology detection rate of MP, virus, bacteria and streptococcus pneumoniae (SP) were 74.0%, 14.2%, 18.3% and 12.2%, respectively.The virus detection rate in 1-3 years old group was the highest, and that in ≥6 years old group was lower than other group (χ2=70.095, P<0.01).The MP detection rate increased with age (χ2=119.777, P<0.01).The bacteria detection rate in ≥6 years old group was significantly lower than those of <1 years old group, 1-3 years old group and 3-6 years old group (χ2=8.939, 14.319, 45.738, all P<0.01).The detection rates of total virus, MP, bacteria and mixed infection had no statistical difference in the four seasons (all P>0.05).The MP detection rate was above 70% in every season.The detection rates of SP and hBoV were basically the same in every season.The detection rate of HI was higher in spring, Pinf 3 and SA were higher in summer, HRV was higher in autumn, and respiratory syncytial virus (RSV) and moraxella catarrhalis (MC) were higher in winter.Conclusions Lobar pneumonia occurs more common in elder children.MP is the major pathogen of lobar pneumonia, and SP is the second.The MP detection rate increases with age.The pathogen detection rate varies with age, but the effect of seasonal factor is not obvious on pathogen detection in lobar pneumonia.

Objective To explore the non-bacteria pathogens of acute laryngitis in children. Methods The clinical data and sputum sample were collected from 325 patients hospitalized due to acute laryngitis in consecutive 10 years from January 2006 to December 2015 . The multiple non-bacteria pathogens were detected and analyzed with clinical data. Seven types of respiratory viruses were detected by direct immunolfuorescence. Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), and Boca virus (HBoV) were detected by lfuorescence quantitative PCR. The rhinovirus (HRV) and human metapneumovirus (hMPV) were detected by RT-PCR. Venous blood was collected within 24 h after hospitalization and 7-10 d after treatment. The MP antibody of IgG and IgM were detected by ELISA. Results The detection rate of non-bacteria pathogens was 46 . 2%in 325 children with acute laryngitis ( 150/325 ), including 76 cases ( 23 . 4%) of virus and 99 cases ( 30 . 5%) of MP. Virus detection rate in 1-3 year old children was obviously higher than in 0-1 year old children and over 3 years old children (χ2?=?9 . 527 , P=?0 . 009 ). With the increase of age, the detection rate of MP increased gradually (χ2?=?10 . 132 , P=?0 . 006 ). The detection rates of RSV and hBoV were higher in under 3-year-old children. The detection rates of virus in winter and spring were signiifcantly higher than those in summer and autumn (χ2?=?5.064, P=?0.024). The detection rates of MP in winter, spring, summer, and autumn was 13.1%, 25 . 0%, 38 . 2%, and 44 . 9%respectively, and the MP detection rates were increased gradually over seasons (χ2?=?4 . 438 , P=?0 . 035 ). The detection rate of RSV was higher in winter, and hBoV was higher in summer. Conclusion Acute laryngitis mainly occurred in children under 3-years-old children, and the detected non-bacteria pathogens were different among different ages and seasons. Virus was the major pathogens in young children, while MP was more common in older children.

ObjectiveTo study the clinical characteristics and laboratory ifndings ofMycoplasma pneumoniae (MP) and EBV infection in children and provide reference for clinical diagnosis and treatment.MethodsOne hundred and twenty two (122) hospitalized children with pathogen detection of MP and EBV double positive in hospitalized children with pneumonia from May 2013 to April 2014 (n=2213) were recruited as mixed infection group. In the mixed infection group, patients were further devided into high EBV mixed infection group if the EBV DNA copies were more than 1.0×104 copies/ml and the low EBV mixed infec-tion group when EBV DNA copies were less than 1.0×104 copies/ml. And another 45 hospitalized children with MP pneumonia were rectuited as control group. Clinical data and laboratory ifndings of all children were collected and analyzed.Results The mixed infection rate of MP and EBV was 5.51% (122/2213). As children getting older, the incidence of mixed infection was increased (χ2=84.08,P<0.001). And the mixed infection incidence in the lobar pneumonia group was signiifcantly higher than the bronchopneumonia group (χ2=37.44,P<0.001). The incidence of ALT and CK-MB elevated, lobar pneumonia, average fever days and hospitalization days in mixed infection with the high EBV copies group were signiifcantly higher than those in the low EBV copies group and the control group (bothP<0.05). The incidence of ALT and CK-MB elevated, average fever days and hos-pitalization days in mixed infection with the high EBV copies group were signiifcantly higher than those of the low EBV copies group and the control group (allP<0.05).ConclusionThe mixed infection of MP and EBV could aggravate the injury both in and out of the lung. Number of EBV copies plays an important role in the degree of injury both inside and outside the lung due to pneumonia with mixed infection of MP and EBV. When a patient with MP pneumonia complains with severe clinical symptoms and obvious injury outside the lung, EBV detection, especially quantitative detection of EBV DNA copies could be beneifcial for clinical diagnosis and treatment.