α4ß7high CD4+ T cells are prone to be infected by HIV-1 and associated with HIV-1 disease progression.

INTRODUCTION: To investigate the characteristics of ß7high CD4+ T cells during HIV-1 infection and the relationship between ß7high CD4+ T cells and HIV-1 disease progress. METHODS: This study enrolled 124 HIV-1-infected patients, including 80 treatment naïve patients (TNs), 41 patients who underwent antiretroviral therapy (ARTs), and three long-term no progression patients (LTNPs). Nineteen matched healthy subjects were included as controls (HCs). The characteristics and frequency of ß7high CD4+ T cells were analyzed using flow cytometry. An in vitro culture experiment was used to study HIV-1 infection of ß7high CD4+ T cells. Real-time polymerase chain reaction was performed to quantify HIV-1 DNA and CA-RNA levels. RESULTS: The frequency of ß7high CD4+ T in the peripheral blood was significantly decreased and negatively correlated with disease progression during chronic HIV-1 infection. A large proportion of ß7high CD4+ T cells showed Th17 phenotype. Furthermore, ß7high CD4+ T cells were preferentially infected by HIV-1 in vitro and in vivo. There were no significant differences of HIV-1 DNA, and CA-RNA levels between ß7high CD4+ T and ß7low CD4+ T subsets in HIV-1 infected individuals after antiviral treatment. CONCLUSION: The ß7high CD4+ T cells were negatively correlated with disease progression during chronic HIV-1 infection. ß7high CD4+ T cells are susceptible to infection with HIV-1 and HIV-1 latent cells.

Effect of different types of macrophages on hepatic fibrosis in Echinococcus Granulosus mice.

OBJECTIVE: The purpose of this study is to illustrate the therapeutic effect of which kind of polarized macrophages-based cell therapy in hepatic fibrosis caused by cystic echinococcosis. METHODS: The isolation culture and polarization induction of mouse bone marrow-derived macrophages (BMDM) are established in an in vitro environment. A model of Echinococcus granulosus infection is established by direct injection of the Echinococcus granulosus suspension into the left hepatic lobe. The macrophages are labeled in vitro and the localization of the returned macrophages in the liver of the mice is determined by in vivo tracing. Macrophages of different polarization types are injected into the successfully modeled mice through the tail vein, and the results of HE, Masson, Sirius Red, Desmin immunohistochemistry and Hyp content are inspected to evaluate by liver tissue. Liver pathology and changes in the degree of fibrosis. RESULTS: Bone marrow-derived macrophages have been successfully obtained and induced into M1 and M2 macrophages by different conditions; a model of Echinococcus granulosus infection was successfully established. Macrophages labeled in vitro were returned to the model through the tail vein and they can be located in the liver; a variety of experimental results show that compared with the PBS group, the degree of fibrosis in the M0 group and the M1 group have been reduced, with statistical difference, and the M1 is better than M0 in terms of the therapeutic effect. There is no significant change in the degree of fibrosis in the M2 group. CONCLUSION: Both M1 and M0 macrophages can alleviate liver fibrosis caused by persistent infection of Echinococcus granulosus, but the treatment effect of M1 macrophages is more significant. Cell therapy based on M1 macrophages may be a new idea for treating liver fibrosis caused by persistent infection of Echinococcus granulosus.

Inhibition of Asthma in OVA Sensitized Mice Model by a Traditional Uygur Herb Nepeta bracteata Benth.

Asthma is a chronic lung inflammation which affects many people. As current therapies for asthma mainly rely on administration of glucocorticoids and have many side effects, new therapy is needed. In this study, we investigated Nepeta bracteata Benth., a traditional Uygur Herb, for its therapeutics effect in OVA induced asthmatic mice model. Treatment of OVA sensitized asthma mice with extract from Nepeta bracteata Benth. demonstrated improved lung pathology, as well as reduced infiltration of eosinophil and neutrophil. Nepeta bracteata Benth. extract also contributed to the rebalance of Th17/Treg cell via decreasing the Th17 cell and increasing the Treg, which was corresponding with the inhibited Th17 cytokine response and increased IL-10 level. Moreover, the reduced TGF-ß level and Smad2/3 protein level also suggested that Nepeta bracteata Benth. extract could inhibit TGF-ß mediated airway remodelling as well. Taken together, these data suggested that Nepeta bracteata Benth. may be a novel candidate for future antiasthma drug development.

Bioinformatic prediction of epitopes in the Emy162 antigen of Echinococcus multilocularis.

The aim of the present study was to predict the secondary structure and the T- and B-cell epitopes of the Echinococcus multilocularis Emy162 antigen, in order to reveal the dominant epitopes of the antigen. The secondary structure of the protein was analyzed using the Gamier-Robson method, and the improved self-optimized prediction method (SOPMA) server. The T- and B-cell epitopes of Emy162 were predicted using Immune Epitope Database (IEDB), Syfpeithi, Bcepred and ABCpred online software. The characteristics of hydrophilicity, flexibility, antigenic propensity and exposed surface area were predicted. The tertiary structure of the Emy162 protein was predicted by the 3DLigandSite server. The results demonstrated that random coils and ß sheets accounted for 34.64 and 21.57% of the secondary structure of the Emy162 protein, respectively. This was indicative of the presence of potential dominant antigenic epitopes in Emy162. Following bioinformatic analysis, numerous distinct antigenic epitopes of Emy162 were identified. The high-scoring T-cell epitopes were located at positions 16-29, 36-39, 97-103, 119-125 and 128-135, whilst the likely B-cell epitopes were located at positions 8-10, 19-25, 44-50, 74-81, 87-93, 104-109 and 128-136. In conclusion, five T-cell and seven B-cell dominant epitopes of the Emy162 antigen were revealed by the bioinformatic methods, which may be of use in the development of a dominant epitope vaccine.

[Construction and expression of the Echinococcus granulosus recombinant BCG-EgG1Y162].

OBJECTIVE: To construct and express Echinococcus granulosus recombinant bacille Calmette-Guerin (BCG) strain rBCG-EgG1Y162. METHODS: The encoding gene of the antigen EgG1Y162 of E. granulosus was recombined with E. coli-Mycobacterium shuttle expression plasmid vector pMV361 by genetic engineering technique, and transformed into E. coli for amplification. The recombinant plasmid rpMV-EgG1Y162 was identified by PCR, double digestion with restriction enzymes, and sequence analysis. The confirmed rpMV-EgG1Y162 was transformed into BCG strain via electroporation technique to construct the recombinant rBCG-EgG1Y162. After identification by PCR and double digestion with restriction enzymes, the recombinant strain was cultured for about 2 weeks. In order to induce the expression of target protein, the rBCG was placed in 45 degrees C for 30 min. SDS-PAGE and Western blotting were used to analyze the expressive protein. RESULTS: The product of recombinant plasmid rpMV-EgG1Y162 was approximately 360 bp by PCR amplification and double digestion with restriction enzymes, consistent with the expected fragment length. Sequencing results showed that the inserted sequence was correct. The rBCG-EgG1Y162 grew well and the identification of PCR and enzyme digestion revealed accuracy. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (M(r)) of the protein was about 71 000. CONCLUSION: The E. granulosus rBCG-EgG1Y162 strain is constructed and expressed.

Transmembrane protein 166 expression in esophageal squamous cell carcinoma in Xinjiang, China.

OBJECTIVE: Transmembrane protein 166 (TMEM166) expression in esophageal squamous cell carcinoma (ESCC) and remote normal esophageal tissues was examined to assess any role in tumour biology. METHODS: TMEM166 mRNA expression in 36 cases with ESCC (36 tumour samples, 36 remote normal esophageal tissue samples) was detected by RT-PCR. TMEM166 protein expression was analysed in paraffin-embedded tissue samples from the same cases by immunohistochemistry. RESULTS: Semi-quantitative analysis showed TMEM166 mRNA expression in ESCCs to be significantly lower than in remote normal esophageal tissues (0.759 ± 0.713 vs. 2.622 ± 1.690, P=0.014). TMEM166 protein expression was also significantly reduced (69.4% vs. 94.4%, P<0.01). CONCLUSION: TMEM166 mRNA and protein expression demonstrated significant reduction in ESCCs compared with remote esophageal tissues, albeit with no correlation with tumour size, differentiation, stage, and lymph node metastasis, suggesting a role in regulating autophagic and apoptotic processes in the ESCC.

[Dynamic change of IL-10 and TGF-beta1 in the liver of Echinococcus multilocularis-infected mice].

OBJECTIVE: To observe the dynamic expression and function of IL-10 and TGF-beta1 in liver of BALB/c mice infected with Echinococcus multilocularis (Em). METHODS: Sixty female BALB/c mice were randomly divided into experiment group and control group. Mice in the experiment group were each injected with 0.2 ml Em protoscolex suspension (containing about 400 protoscoleces) , while those in control group received same volume of normal saline. At 2, 8, 30, 90, 180, and 360 d after infection, 5 mice from each group were sacrificed and liver specimens were collected for pathological examination and immunohistochemical detection for IL-10 and TGF-beta1. RESULTS: In mice of the experiment group, Em cysts in different sizes were found in the abdominal cavity and the liver tissue, which gradually enlarged with the time. HE staining showed infiltration of lymphocytes in liver tissue, pathological change between the cyst wall and hepatic cells. In the control, the liver lobules showed integrity and inflammatory cells were seen occasionally. The level of IL-10 expression in liver tissue of the infected mice increased with the time, and reached a peak [(1639 +/- 1.73) %] at 90 d post-infection and maintained a high level thereafter. The expression of TGF-beta1 also reached the highest level [(23.69 +/- 2.29) %] . Both were significantly higher than the control (P < 0.01), though a low level expression was found in the control at 90d post-injection. CONCLUSION: The expressions of IL-10 and TGF-beta1 both increase in the middle and late stages of the infection. Besides, their inhibited functions do not be helpful for clearing and controlling Echinococcus multilocularis infection in livers.

[Bioinformatics prediction of egA31 recombinant antigen epitopes of Echinococcus granulosus].

Specific primers were designed and synthesized based on the reported EgA31 gene of Echinococcus granulosus (GenBank Accession No. AF067807). Total RNA was extracted from E. granulosus and its EgA31 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was purified and cloned into plasmid pUCM-T, then transformed into Escherichia coli DH5alpha. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. The positive recombinant plasmid pUCM-T/EgA31 was confirmed by sequencing and homology comparison. Five parameters and methods were used to predict B-cell epitopes in amino acid sequence of EgA31. The amplified DNA fragment (636 bp) had an identity of 100% with the EgA31 gene sequence of E. granulosus. B-cell and T-cell epitopes of EgA31 were probably at or adjacent to 32-79, 79-95, 105-124 and 141-154 in its amino acid sequence.

[The effect of Uygur medicine Hyssopus officinalis L on expression of T-bet, GATA-3 and STAT-3 mRNA in lung tissue of asthma rats].

AIM: To study the Uygur medicine Hyssopus officinalis L on T-bet, GATA-3, STAT-3 mRNA levels of asthma rats in order to explore the mechanism of its treatment of asthma. METHODS: Rats were randomly divided into normal control group, asthma model group and dexamethasone group and water extract of Hyssopus officinalis L high and low dose group. The rats were sensitized with OVA, Al(OH)(3); and DPT vaccine and then challenged with inhalation of aerosolized OVA solution for Preparation of asthma model and the level of T-bet, GATA-3, STAT-3 mRNA were detected with RT-PCR. RESULTS: The normal control group and model group, model group and treatment group, the expression of T-bet, GATA-3 and STAT-3 mRNA in the lung tissue was statistically significant differences(P<0.05). Compared with model group, after treatment of Hyssopus officinalis L the expression of GATA-3 and STAT-3 mRNA of asthma rats significantly reduced (P<0.05), but the expression of T-bet mRNA was significantly higher (P<0.05).The expression of GATA-3 and STAT-3 mRNA of Hyssopus officinalis L high-dose treatment group was lower than the low-dose treatment group (P<0.05), but T-bet mRNA that was higher(P<0.05). The expression of T-bet mRNA has negative correlation with GATA-3 mRNA (r=-0.696), the expression of STAT-3 mRNA has correlation with T-bet mRNA and GATA-3 mRNA(r=-0.767, 0.772), P<0.05. CONCLUSION: Hyssopus officinalis L probably regulates the differentiation of Th1, Th2 and Th17 on transcription level to play the role of anti-inflammatory.

[Regulation on phenotypic and functional maturation of dendritic cells by apolipoprotein A-I].

AIM: to investigate the effects of apolipoprotein A-I (ApoA-I) on the peripherial blood dendritic cell (PBDC) and monocyte derived DC (MDDC) in vitro. METHODS: isolate PBDC or monocyte by cell isolation kit, monocyte were induced to MDDC by treated with GM-CSF plus IL-4 for 6 days, and then collect PBDC and MDDC treated them with apoA-I, LPS or TNF-α for 24 hours. Then check the cell surface marker and phagocytic capacity by flow cytometry. ELISA was used to detect the levels of cytokine secretion. T cells were stained with CFSE and T cell proliferation was assessed by flow cytometry. RESULTS: collect the PBDC and MDDC with high purity. In the presence of ApoA-I, the surface markers on MDDC, such as CD40, CD86 and MHC-II, were up-regulated which were detected by flow cytometry. CD83 expression on both PBDC and MDDC was remarkably increased. ApoA-I DC demonstrated decreased the phagocytic capacity. ApoA-I also stimulated MDDC to produce IL-12 and TNF-α. Furthermore, ApoA-I can induce considerable Th cell proliferation. CONCLUSION: ApoA-I can induce the maturation and activation of MDDC and PBDC, including the cytokine secretion, specific antigen presentation and T cell proliferation and decreasing the phagocytic capacity. Therefore, ApoA-I may attribute to the immune response in AS process.

[Determination of ten metal elements in Ziziphora clinopodioides Lam by microwave digestion-FAAS].

Comprehensive utilization of traditional Uighur medicine has been increasingly emphasized, and the relationship between metal elements and traditional Uighur medicine has attracted great attention, so it is quite important to determine the contents of traditional Uighur medicine. The Ziziphora clinopodioides Lam powder was digested with HNO3 by microwave digestion before determination. Ten metal elements in Ziziphora clinopodioides Lam were determined by FAAS. The work conditions, accuracy and precision of the method were studied. The linear correlations of standard curves are good (r = 0.999 0-0.999 8). The recovery (n = 6) is 95%-108%, and the RSD(n = 6) is 0.45%-1.53%. The results showed that there were comparatively rich metal elements, among which are comparatively high calcium, magnesium and potassium in Ziziphora clinopodioides Lam. The method offers traits of low detection limit, high sensitivity, speediness and exactness, and wasapplied to the determination of metal elements in samples with satisfactory results. It provided useful data for discussing the relationship between the content of the metal elements in Ziziphora clinopodioides Lam and clinical application of the Uighur medicine.

[Cloning and sequence analysis of the egG1Y162 gene of Echinococcus granulosus].

A pair of primers (egG1Y162) were designed according to the nucleotide sequence of Echinococcus multilocularis emY162 antigen gene. Using genomic DNA and cDNA from protoscoleces and adult worms of E. granulosus as templates, PCR was performed with the primers to obtain fragments of egG1Y162 gene. PUCm-T/egG1Y162 recombinant plasmids and PUCm-T/egY162 cDNA recombinant plasmids were constructed and identified by PCR, digestion with restriction enzyme and sequencing. The egG1Y162 antigen gene was amplified in protoscoleces and adult worms of E. granulosus. The size of the egG1Y162 gene was 1 648 bp and cDNA was 459 bp, and GenBank accession numbers were AB458258 and AB458259, respectively.

[Transient expression of Echinococcus granulosus Eg95 DNA vaccine and induction of immune response in mice].

OBJECTIVE: To detect the in vitro expression of pcDNA3-Eg95 and to observe the immunological effect of the Eg95 DNA vaccine in mice. METHODS: The eukaryotic recombinant plasmid pcDNA3-Eg95 was transfected into HeLa cells with liposome-mediated method. RT-PCR, ELISA and Western blotting were used to analyze the expression of Eg95 mRNA and Eg95 protein, respectively. The BALB/c mice were immunized by pcDNA3-Eg95. Anti-Eg95 IgG and IgG2a in murine serum were determined by ELISA. The proliferation activity of spleen T lymphocytes was tested using MTT assay. RESULTS: Using RT-PCR method, the expression of Eg95 mRNA was confirmed in vitro. The results of ELISA and Western blotting showed that there was a specific Eg95 protein, which can be specifically recognized by anti-sera of Eg95-prokaryotic-expressing protein in pcDNA3-Eg95 transfected HeLa cell lysis. The specific IgG was induced during the 3rd week and continued to increase until week 10. IgG2a was detected after 2 weeks and maintained a higher level till week 10. There was a significant difference of IgG2a level between pcDNA3-Eg95 immunized group and pcDNA3 control (P<0.01). In spleen T cell proliferation response, the stimulation index (SI) in pcDNA3-Eg95 group was higher than that of vector control (P<0.01). CONCLUSION: Eg95 DNA vaccine can induce significant cellular and humoral immune response in mice.

[Relationship between endometriosis and glutathione S-transferase M1, T1 genes of the Uygurs and Hans in Xinjiang].

OBJECTIVE: To examine the association between glutathione S-transferase (GST)M1, T1 null genotypes and endometriosis of the Uygurs and Hans in Xinjiang. METHODS: The polymerase chain reaction method was used to detect the presence or absence of the GSTM1 and GSTT1 genes in genomic DNA from the Uygurs (107 controls and 41 cases) and the Hans (105 controls and 80 cases) in Xinjiang. RESULTS: The frequencies of the GSTM1-null genotype, GSTT1-null genotype, combined GSTM1-null and GSTT1-null genotype in endometriosis of the Uygurs (51.2%, 36.6%, 24.4%) were not significantly different from those in controls (53.2%, 29.9%, 13.1%). Similarly, no statistically significant difference was observed in the frequency of GSTM1-null genotype in cases of endometriosis of the Hans (56.8%) compared with the controls (51.8%), but the frequencies of GSTT1-null genotype, combined GSTM1-null and GSTT1-null genotype in endometriosis of the Hans (73.7%, 42.5%) were significantly different from those in controls (44.3%, 22.8%). When comparing the Uygurs with the Hans, we found no significant difference in the frequencies of GSTM1-null genotype, GSTT1-null genotype, combined GSTM1-null and GSTT1-null genotype between the two control populations, neither in the frequencies of the GSTM1-null genotype, combined GSTM1-null and GSTT1-null genotype between the two endometriosis populations. However, the frequency of GSTT1-null genotype in cases of the Hans (73.7%) was significantly higher than that in cases of the Uygurs (36.6%). CONCLUSIONS: No evidence was found to suggest an association between GSTM1-null genotype and endometriosis in the Hans and Uygurs. An association was found between GSTT1-null genotype and endometriosis in the Hans, but not in the Uygurs. The two nationalities have different genetic predisposing factors to the development of endometriosis.

[Change of cytokines in mice with Echinococcus multilocularis infection].

OBJECTIVE: To observe the change of six cytokines in mice infected with Echinococcus multilocularis as part of the study on immunological mechanism in the infection. METHODS: Mice were infected by abdominal inoculation of echinococcus protoscoleces. The change of serum level of the cytokines IL-2, IFN-gamma, TNF-alpha, IL-4, IL-5 and IL-10 was determined by ELISA during the infection which lasted for 260 d. RESULTS: Compared with uninfected control, the levels of the cytokines all significantly increased in the 260 d. The level of IL-2 reached a peak after 80 d post-infection (p.i.), then decreased quickly after 140 d p.i., High level of TNF-alpha was detected after 40 d, compared to uninfected control, reached a peak at 100 d p.i., and decreased quickly after 140 d. The level of IFN-gamma reached a peak after 80 d p.i., and decreased slowly after 140 d p.i. The levels of IL-4, IL-5 and IL-10 remained lower before 80 d, and increased sharply after 100 days. The levels of IL-4 and IL-10 reached peaks at 100 d p.i., and that of IL-5 at 140 d p.i. CONCLUSION: The data suggest that the induction of Th2 antibody-mediated immunity (AMI) with a parallel expansion of Th1 cell-mediated inflammatory (CMI) responses are important mechanism of the host in defending against the metacestodes. Th1 CMI plays an important role at the early stage of infection, and Th2 AMI is important in the later stage of infection.

Cloning, homological analysis and construction of Eg95 Xinjiang strain DNA vaccine.

OBJECTIVE: To study the structure specificity of Echinococcus granulosus 95 (Eg95) gene and the open reading frame (ORF) of the full-length cDNA sequence in Xinjiang, northwestern China and construct Eg95 Xinjiang strain DNA vaccine. METHODS: Primers of Eg95 were designed on the basis of the sequence of Eg95 antigen cDNA. Genomic DNA was extracted from E.granulosus protoscoleces (sheep) in Xinjiang. The Eg95 gene and full-length Eg95 cDNA were amplified by PCR from the genomic DNA and protoscolex cDNA library of E.granulosus in Xinjiang, respectively. The Eg95 gene was cloned into pUCm-T plasmid and the Eg95 cDNA into eukaryotic expression plasmid pcDNA3 for the construction of full-length ORF DNA vaccine pcDNA3-Eg95/XJ. Both Eg95 gene and Eg95 cDNA were sequenced and analyzed by DNAman and NCBI/Blast program. RESULTS: DNA sequence analysis of Eg95 Xinjiang strain (Eg95/XJ) cDNA fragment indicated that the coding region of the full-length of Eg95/XJ was 471bp and that encoding a peptide with 156aa and the genomic DNA size was 1191bp. Homological comparison showed that the ORF of Eg95/XJ cDNA was identical to the cDNA sequence of Eg95 reported in the reading frame, but the genomic DNA was a new sequence, named Eg95/XJ and the multiple nucleotide differences, which were represented in Eg95/XJ gene in comparison with those of the New Zealand strain, occurred predominantly in the non-coding regions of the gene. The pcDNA3-Eg95/XJ positive clone was the exact recombinant plasmid and could be used as a DNA vaccine. CONCLUSION: pcDNA3-Eg95/XJ Xinjiang strain DNA vaccine is successfully constructed.

[Cloning and sequence analysis of Eg95 cDNA from different stages of Echinococcus granulosus in Xinjiang].

OBJECTIVE: To study expression and sequence differences of Echinococcus granulosus 95(Eg95) antigen cDNA from different stages of protoscolex, oncosphere and adult worm of E. granulosus from Xinjiang Uighur Aut. Reg. METHODS: In accordance with the sequence of Eg95 antigen cDNA, the primers of Eg95 were designed. Eg95 antigen cDNAs were amplified by PCR from protoscolex, oncosphere and adult worm cDNA libraries of E. granulosus, respectively and were cloned into pUCm-T plasmid, and sequenced. The sequences were analyzed by DNAman and GenBank/BLAST biosoftware. RESULTS: PCR results showed that Eg95 antigen cDNA was amplified from three stages of E. granulosus cDNA libraries. Sequencing analysis indicated that the Eg95 cDNA length was 402 bp, same as the reported data in GenBank. CONCLUSION: The Eg95 antigen cDNA was expressed in the different life-cycle stages of E. granulosus in Xinjiang and there was no nucleic acid sequence difference of Eg95 antigen among the protoscolex, oncosphere and adult worm of E. granulosus.