Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb) infection,remains a major global concern.The activation of anti-TB immune responses is mediated by several complicated mechanisms, among which the local immune microenvironment at the disease site plays a crucial role.In this review, we discuss the roles of anti-TB immunity, Mtb immune escape and the elements at the local site of infection.

Persisters are a sub-population of bacteria that can survive lethal concentrations of antibiotics.They contribute to recurrent or intractable chronic infections.Persisters can emerge as a result of multiple mechanisms which lead to drug tolerance.Unlike antimicrobial resistance which usually acquires genetic mutation, phenotypes of persisters are non-inheritable.Despite the distinct difference between persistence and resistance, recent studies have demonstrated that not only persistence can be observed in resistant mutants, but also persisters themselves can be regarded as an intermediate state which promotes the emergence of resistance.This review focuses on mechanisms of drug tolerance in persisters, phenomena of persistence in antimicrobial resistant bacteria and possible mechanisms of the conversion of persisters to resistant bacteria.Moreover, future research directions are also prospected is this review.

Leptospirosis is recognized as an important emerging zoonotic disease caused by pathogenic Leptospira spp.and has a serious impact on people′s health and animal husbandry.Therefore, it has attracted more and more attention.With the development of biotechnology, major breakthroughs have been made in the fields of pathogenicity, molecular epidemic features and evolution mechanism of Leptospira.In this review, we summarize progress in evolution and molecular typing of Leptospira at home and abroad in order to provide a reference for further research on molecular epidemiological surveillance and new vaccine development.

Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.

Objective To investigate the changes in myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) and T helper 17 (Th17) cells in peripheral blood of patients with HPV infection and cervical diseases including ectopic cervical columnar epithelium (CE), cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CC), and to analyze the roles of MDSC, Treg and Th17 cells in the development of cervical diseases.Methods This study enrolled 120 HPV-positive patients with cervical diseases (18 cases of CE, 50 cases of CIN, 52 cases of CC), 20 HPV-negative patients with cervical diseases (10 cases of CE and 10 cases of CIN) and 20 healthy subjects from March 2011 to December 2016.Changes in MDSC, Treg and Th17 cells in peripheral blood of all patients were detected by flow cytometry.Results Percentages of MDSC, Treg and Th17 cells in peripheral blood of patients who were positive for HPV were significantly higher than those in HPV-negative patients (P<0.05).Besides, percentages of these cells gradually increased with the exacerbation of cervical disease (CE-CINⅠ-CINⅡ-CINⅢ-CC) (P<0.05).Patients who were infected with high-risk HPV had significantly higher percentages of MDSC, Treg and Th17 cells in peripheral blood than those infected with low-risk HPV (P<0.05).Changes of MDSC, Treg and Th17 cells in peripheral blood of HPV-positive patients with cervical cancer were related to International Federation of Gynecology and Obstetrics (FIGO) stage, tumor differentiation, lymph node metastasis and vascular invasion (P<0.05).MDSC, Treg and Th17 cells in peripheral blood of all patients were significantly reduced after treatment (P<0.05).Patients whose status changed from HPV-positive to HPV-negative following treatment also showed significantly decreased MDSC, Treg and Th17 cells in peripheral blood as compared with those who remained HPV-positive (P<0.05).MDSC, Treg and Th17 cells in peripheral blood of HPV-positive patients with cervical diseases were positively correlated (r=0.599, 0.677, 0.648;P<0.05).Conclusion MDSC, Treg and Th17 cells in peripheral blood of patients with HPV infection are associated with the pathological process, severity and clinical pathological features of cervical diseases as well as therapeutic effects.Therefore, they can be used as biomarkers to detect the occurrence and development of HPV-positive cervical diseases.

Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA.Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers.A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments.Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively.HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor.Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups.Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression.ResultsRestriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully.The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+mmu-miR-30b, pmirGLO+mmu-miR-30b inhibitor, pmirGLO-mFoxO3+mmu-miR-30b, pmirGLO-mFoxO3+mmu-miR-30b+mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively.Compared with pmirGLO+mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+mmu-miR-30b group (P<0.05), but showed no significant change in pmirGLO-mFoxO3 mt+mmu-miR-30b group (P>0.05).In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05).Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05).Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b.There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA.

Objective To investigate the changes of phenotypes and function of CD56+T cells during primary HIV-1 infection and their relationship with disease progression.Methods Peripheral blood mononuclear cells (PBMCs) were collected from 53 subjects with primary HIV-1 infection and 31 HIV-1-negative healthy subjects.The percentages of CD56+T cells and the expression of several phenotypic markers on CD56+T cells including CD16, CD161, NKB1, NKG2A, NKp46, NKG2D, NKG2C and CD158a were analyzed by flow cytometry.IFN-γand TNF-αreleased by CD56+T cells with and without K562 stimulation and the levels of cytotoxic molecular CD107a were measured.Results The percentages of CD56+T cells in patients with primary HIV-1 infection were significantly lower than those of healthy subjects (P=0.025). The levels of CD56+T cells were negatively related to the viral loads in plasma samples ( P=0.021, r=-0.316).Compared with healthy subjects, the expression of CD16 (P=0.003), CD161 (P=0.023), NKB1 (P=0.023) and NKp46 (P=0.021) on CD56+T cells were decreased in patients with primary HIV-1 infection.The levels of NKB1 were positively related to the CD4+T cell counts ( P=0.007, r=0.364), but were negatively related to the viral loads in plasma samples (P=0.030, r=-0.299).Sponta-neous secretion of IFN-γand TNF-αby CD56+T cells and the expression of CD107a were dramatically in-hibited in patients with primary HIV-1 infection as compared with healthy subjects ( all P<0.001 ) . Moreover, the killing ability of CD56+T cells against K562 target cells was weakened in patients with prima-ry HIV-1 infection as the levels of IFN-γ-, TNF-α-and CD107a-producting CD56+T cells were significantly decreased (P<0.001 for IFN-γand TNF-α, P=0.016 for CD107a).Conclusion Inhibited expression and altered phenotypes of CD56+T cells were identified during primary HIV-1 infection.Lower levels of cy-tokines and cytotoxic molecular were also detected, indicating the dysfunction of CD56+T cells appeared dur-ing early stage of HIV-1 infection and was associated with disease progression.

Objective To establish a TaqMan real-time PCR assay for the detection of encephalo-myocarditis virus ( EMCV) .Methods Based on the conservative region of 3D gene of EMCV published in GenBank , a pair of primers and one TaqMan probe were designed and synthesized .Then a TaqMan real-time PCR assay was set up and the reactive system was optimized .The sensitivity and specificity of the assay was evaluated respectively .The TaqMan real-time PCR assay was then carried out to detect 98 randomly selected swine serum samples and the results were compared with those by using ELISA .Results The Ct value of the templates had a good linear relationship with the log starting quantity , with a correlation coefficient of 0.995.The TaqMan real-time PCR assay was only specific for EMCV and its sensitivity was 100 times higher than that of the ordinary PCR .The coincidence rate between the established assay and the ELISA assay was 98.0%in the detection of 98 blood samples.Conclusion The TaqMan real-time PCR assay for the detec-tion of EMCV was successfully established with advantages of high sensitivity and good specificity .It could be used for detection of EMCV and quantitative analysis .

Objective To analyze the structures and molecular weight distributions of the capsular polysaccharides from 6 serotypes of pneumococcus .Methods The structures of pneumococcal capsular pol-ysaccharides of 6 serotypes were analyzed by 1 H nuclear magnetic resonance ( NMR) .Chemical shifts of all characteristic protons were investigated to analyze polysaccharide integrity and inter -assay consistency .High performance size exclusion chromatography-multi angle laser light scattering ( HPSEC-MALLS) was used to measure the molecular weights .Results The chemical shifts of all characteristic protons of the pneumococ-cal capsular polysaccharides of 6 serotypes were consistent with the standard chemical shift .The weight-aver-age molecular mass of the pneumococcal capsular polysaccharides ranged from 7.182×104 g/mol(for serotype 19A) to 1.273×106 g/mol(for serotype 9V)examined by HPSEC-MALLS.Conclusion The structures and molecular weight distributions of pneumococcal capsular polysaccharides could be rapidly and effectively ana -lyzed by 1 H NMR and HPSEC-MALLS.Moreover, C-PS and acetate contained in capsular polysaccharides could also be detected .HPSEC-MALLS is an applicable method for the quantitative analysis of molar mass distributions in different serotypes of pneumococcal capsular polysaccharides . Although 1 H NMR and HPSEC-MALLS have been accepted as the quality control measurements by WHO , to use them as the re-placements of the traditional QC method still needs further investigation .

Objective To construct the recombinant protein AtlM ( rAtlM) of Staphylococcal au-reus through prokaryotic expression system and to investigate its antibacterial activity in vitro.Methods The specific primers were designed according to atlM gene sequence of Staphylococcal aureus recorded in Gen-Bank, and atlM gene was amplified by PCR from the Staphylococcal aureus strain (ATCC25923).The re-combinant plasmid pET-32а(+)/atlM was constructed and transformed into Transetta ( DE3 ) to express AtlM after induced by IPTG .The expressed protein AtlM was further analyzed by SDS-PAGE and purified by electroeluting of bag filter.The minimal inhibitory concentrations (MICs) of rAtlM to ATCC25923 and oxac-illin-resistant S.aureus strain were determined by the broth microdilution method .S.aureus ATCC25923 strain and oxacillin-resistant S.aureus strain (final concentration of 5×105 CFU/ml) were exposed to rAtlM (50 μg/ml) respectively to test its antibacterial activity in vitro.Results The recombinant protein AtlM was expressed and purified successfully with a relative molecular weight of 80 ×103 and a concentration of 1.25 mg/L.The MICs of rAtlM to ATCC25923 strain and oxacillin-resistant S.aureus strain were 8 μg/ml and 64 μg/ml, respectively.In vitro test showed that rAtlM had inhibitory effects on the growth of ATCC25923 strain and oxacillin-resistant S.aureus strain after 1 h of intervention (P=0.004 and P=0.026, respectively), which lasted to 5 h for ATCC25923 strain (P=0.012) and 3 h for oxacillin-resistant strain (P=0.001).Conclusion This study shows that rAtlM has a certain antibacterial effects on S.aureus ATCC25923 strain and oxacillin-resistant S.aureus strain, suggesting a possibility of serving as antimicrobial agent.