Objective To investigate the diagnostic value of microscopic observation drug susceptibility assay (MODS) in extrapulmonary tuberculosis. Methods MODS technology was constructed by using 24-well cell culture plate and liquid culture.Ziehl-Neelsen smear,Lowenstein-Jensen culture and MODS were used to detect Mycobacterium tuberculosis in 74 pleural fluid samples collected from patients with tuberculous pleurisy,63 cerebrospinal fluid samples collected from patients with tuberculous meningitis and 18 samples collected from non-tuberculosis suspects.The immunochromatography was used to distinguish Mycobacterium tuberculosis from nontuberculosis mycobacteria. The results of Ziehl-Neelsen smear, Lowenstein-Jensen culture and MODS were compared by x2 test.Results The positive rates of MODS,Lowenstein-Jensen culture and Ziehl-Neelsen smear were 58.1 ％ (43/74),18.9 ％ (14/74 ) and 6.8％ (5/74),respectively in tuberculous pleurisy patients; 54.0％(34/63),20.6％ (13/63) and 4.8％ (3/63),respectively in tuberculous meningitis patients.The positive rate of MODS technology was significantly higher than that of Lowenstein Jensen culture in tuberculous pleurisy patients (x2 =24.00,P＜0.01) and tuberculous meningitis patients (x2 =14.97,P ＜ 0.01). Each Mycobacterium obtained from MODS and Lowenstein-Jensen culture was identified as Mycobacterium tuberculosis by immunochromatography.All of the 18 pleural fluid and cerebrospinal fluid samples which collected from non-tuberculosis suspects were all negative detected by Ziehl-Neelsen smear,Lowenstein-Jensen culture and MODS.The median time to culture positive of MODS was 9 days in cerebrospinal fluid and 14 days in pleural fluid samples,which were both significantly shorter than that of Lowenstein-Jensen culture (31 days in both cerebrospinal fluid and pleural fluid samples). Conclusion Compared to conventional microbiological diagnosis methods,MODS is a rapid detection method of Mycobacterium tuberculosis with a higher positive detection rate,which is suitable for rapid diagnosis of extrapulmonary tuberculosis.

Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.

OBJECTIVETo detect the expression levels of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) in the peripheral blood of patients with oral squamous cell carcinoma (OSCC) and to discuss their biological and clinical significance.

METHODSPD-1/PD-L1 expression on the surface of T-lymphocytes and the counts of T-lymphocyte subpopulations of peripheral blood in 82 patients with OSCC (OSCC group) and 25 healthy controls (control group) were examined via flow cytometry. The expression levels of soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) in the serum were observed through enzyme-link immunology method. The data were tested and analyzed with SPSS 17.0 software.

RESULTSThe percentage of CD8+ T cells in the OSCC group was significantly higher than that in the control group (P<0.05), whereas the percentages of CD3+ and CD4+ T cells as well as CD4+/CD8+ ratio were significantly lower than those in the control group (P<0.05). The positive rates of PD-1 and PD-L1 in CD3+ and CD4+ T cells in OSCC peripheral blood were remarkably higher than those in the control group (P<0.01). Difference was not observed between the expression levels of sPD-1 in the serum of OSCC group and those in the control group (P>0.05), but the average of sPD-L1 was remarkably higher than that in the control group (P<0.05). sPD-L1 expression was related to clinical stage, tumor cell differentiation, and lymph node status (P<0.05) but not related to sex, age, tumor location, and tumor size.

CONCLUSIONT-lymphocyte subpopulations in the peripheral blood of patients with OSCC developed immunosuppression with different degrees. PD-1 and PD-L1 expression levels on the surface of CD3+ and CD4+ T cells significantly increased. Abnormal increase in sPD-L1 expression may be associated with OSCC development.

B7-H1 Antigen ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; Case-Control Studies ; Flow Cytometry ; Humans ; Mouth Neoplasms ; metabolism ; T-Lymphocyte Subsets

Objective To investigate the effects of lincRNA-cox2 on the polarization of murine RAW264. 7 macrophages by analyzing the expression of lincRNA-cox2 in RAW264. 7 macrophages of M1 and M2 phenotypes. Methods Murine RAW264. 7 cells were induced by IFN-γand LPS to polarize to M1 phenotype, and were induced by IL-4 to polarize to M2 phenotype. The expression of lincRNA-cox2 in M1 and M2 macrophages were analyzed by real-time quantitative PCR ( RT-PCR) . We designed and synthesized siRNA oligo for lincRNA-cox2 and unrelated sequences. Then the siRNA oligo and NC oligo were transfected into RAW264. 7 cells by LipofectmineTM 2000. The transfected RAW264. 7 cells were induced by IFN-γand LPS or by IL-4 to polarize to M1 or M2 macrophages. Enzyme linked immunosorbent assay ( ELISA) was performed to measure the secretion of IL-10 and IL-12 induced in different conditions. The expression of in-ducible nitric oxide synthase ( iNOS ) , TNF-α, arginase 1 ( Arg-1 ) and found in inflammatory zone 1 (Fizz1) at mRNA level were detected by RT-PCR. The M1 macrophages were transfected with siRNAs to knock down the expression of lincRNA-cox2 for analyzing the biological effects of lincRNA-cox2 on the polar-ization of macrophages. Results The relative expression of lincRNA-cox2 in M1 macrophages was signifi-cantly higher than that in RAW264. 7 cells and M2 macrophages. Compared with the control group, the RAW264. 7 cells transfected with lincRNA-cox2-siRNA showed decreased secretion of IL-12 and inhibited expression of iNOS and TNF-αat mRNA level after IFN-γand LPS induction, but increased secretion of IL-10 and enhanced expression of Arg1 and Fizz1 at mRNA level after IL-4 induction. Transfecting the M1 mac-rophages with lincRNA-cox2-siRNA inhibited the secretion of IL-12, but promoted the secretion of IL-10. Conclusion This study indicated that lincRNA-cox2 was involved in the regulation of macrophage pheno-types by promoting the polarization to M1 macrophages and inhibiting the polarization to M2 macrophages.

Objective To analyze the expression profile of long non-coding RNA (lncRNA) in the lipopolysaecharide (LPS)-induced inflammation of monocyte-derived macrophages.Methods Peripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. The macrophages were divided into blank control group and LPS (1 mg/L) stimulated 12 hours group. Culture supernatants and cell pellets were harvested in each group, enzyme linked immunosorbent assay (ELISA) was used to assay the production changes of interleukins (IL-1β and IL-6), and tumor necrosis factor-α (TNF-α) in the supernatant. The technique of lncRNA microarray was used to test the lncRNA expression profile in LPS-induced inflammation of macrophages and control macrophages. The raw data of lncRNA were pretreated for normalization. Five lncRNA expressions were validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Furthermore, qRT-PCR was used to detect the expression of NR_028034 in macrophages after LPS-induced inflammation.Results ① The contents of IL-1β (ng/L:562.93±61.17 vs. 59.74±15.68), IL-6 (ng/L: 702.46±92.31 vs. 71.66±18.25) and TNF-α (ng/L: 794.50±63.89 vs. 85.12±22.07) in the LPS group were significantly higher than those in the blank control group (allP < 0.01). These results indicated that the inflammatory model of human macrophages was constructed successfully. ② Compared with blank control group, and 1479 lncRNA which have more than 2 folds variation and significant difference (P < 0.05) by statistical analysis was defined as lncRNA with differential expression. Among these lncRNA, LPS group showed 953 up- regulated and 526 down- regulated genes by 2 folds and 49 up- regulated and 35 down- regulated genes by 5 folds. ③ qRT-PCR results were generally consistent with the microarray data. ④ The expression of NR_028034 was increased by (4.41±0.65), (11.56±2.04), (18.58±1.36) folds compared with blank control group at 3, 6, 12 hours after LPS stimulation (allP < 0.01).Conclusions These data show a significantly altered lncRNA expression profile in the LPS-induced inflammation of monocyte-derived macrophages, suggesting that lncRNA may be involved in regulation of macrophages inflammatory response.

Objective To construct a lentiviral vector-based short hairpin RNA (shRNA) targeting the gene encoding tryptophan-aspartate containing coat protein ( TACO) and to evaluate its inhibitory effect on the expression of TACO , and to further elucidate its effects on the phagocytosing and intracellular killing of My-cobacterium tuberculosis (M.tb) by macrophages and the possible mechanisms.Methods Three shRNA frag-ments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentivi -ral vector pSicoR .The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells.Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW 264.7 cells transfected with the concentrated lentivirus .The most effective lentivirus was screened out to transfect the RAW 264.7 cells for 48 hours, followed by infection those cells with M.tb strains.The entry and intracellular survival of M .tb strains in RAW264.7 cells were de-termined by bacterial culture at indicated time points .The colocalization of M .tb and lysosomes was detected by immunofluorescence staining .The cyto-ID autophagy kit was used to detect the cellular autophagy and the auto-phagy-associated protein LC 3 was determined by Western blot assay .Results The recombinant lentiviral vec-tors were successfully constructed and confirmed by sequencing analysis .Decreased expression of TACO in RAW264 .7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours.The most effective lentivirus , LV-pSRT1, decreased the expression of TACO by 85.24%and 69.00%at the mRNA and protein levels, respectively.The bacterial loads in LV-pSRT1 trans-fected RAW264.7 cells were significantly decreased at the time point of 0 h after M.tb infection as compared with those in the control lentivirus treated cells (5.50×104 vs 8.1 ×104, P<0.05).Compared with the RAW264 .7 cells transfected with control lentivirus , the survival rate of intracellular M .tb strains in LV-pSRT1 transfected cells was significantly decreased at the time point of 48 h (134.54% vs 213.58%, P<0.05) and 72 h (148.18%vs 262.96%, P<0.05) considering the bacterial load at the time point of 0 h as the standard. The immunofluorescence staining demonstrated that the colocalization of M .tb strains with lysosomes was signifi-cantly enhanced in LV-pSRT1 transfected cells as compared with that in control lentivirus treated cells (75.67%vs 10.66%, P<0.05).Moreover, significantly enhanced autophagy and relative expression of LC 3Ⅱ protein were observed in RAW264.7 cells with TACO gene knockdown (16.20%vs 8.50%, P<0.05;0.51 vs 0.34, P<0.05).Conclusion The lentiviral vector-based shRNA targeting TACO gene could effectively knockdown the expression of TACO protein , decrease the entry and increase the intracellular killing of M .tb strains in mac-rophages.The enhanced intracellular killing of M .tb strains by macrophages was associated with the increased fusion of M.tb-containing phagosome and lysosome .

Objective To analyze the expression profile of circular RNA (circRNA) in LPS-treated murine peritoneal macrophages (MPMs) and to investigate the effects of mmu_circ_0000790 (circ790) on the secretion of pro-inflammatory cytokines in MPMs following LPS stimulation.Methods MPMs were isolated from C57BL/6 male mice and then stimulated with (LPS treatment group) or without (blank control group) 1 mg/L of LPS for 12 hours.Supernatants of cell culture and cell pellets were collected from each group.Enzyme linked immunosorbent assay (ELISA) was used to measure the changes in the secretion of IL-1β,IL-6 and TNF-α in the supernatants.Expression profile of circRNA in macrophages from the two groups (n=3) was analyze by circRNA microarray.Some differentially expressed circRNAs were validated by real-time PCR (RT-PCR).Moreover,RT-PCR was also performed to detect the expression of circ790 in MPMs after LPS stimulation.Small interfering RNA (siRNA) oligo specific for circ790 was designed and synthesized.Then the synthesized siRNA oligo and normal control (NC) oligo were transfected into MPMs by LipofectamineTM2000.The transfected MPMs were treated with LPS.ELISA was used to detect the levels of IL-1β,IL-6 and TNF-α in the supernatants.Arraystar's home-made microRNA target prediction software was used to predict circ790/microRNA.Results The concentrations of IL-1β,IL-6 and TNF-α in the LPS treatment group were significantly higher than those in the blank control group (P<0.01).These results indicated that the inflammatory model of MPMs was successfully constructed.Statistical differences in 34 differentially expressed circRNAs were found between the two groups (P<0.05).Among them,12 circRNAs were up-regulated and the other 22 circRNAs were down-regulated in the LPS treatment group.RT-PCR results were generally consistent with the microarray data.The expression of circ790 was increased by (1.94±0.15),(3.18±0.13) and (4.21±0.22) folds as compared with that of the blank control group at 3,6 and 12 h after LPS stimulation (P<0.05).After transfecting the MPMs with circ790-siRNA,the secretion of IL-6 was significantly decreased (P<0.05) without notable influence on the secretion of IL-1β and TNF-α.circ790 could function as a microRNA sponge to regulate the gene expression.Conclusion These data show a significantly altered circRNA expression profile in the LPS-treated MPMs.circ790 may be involved in the regulation of IL-6 secreted by macrophages.

Objective To investigate the expression of T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT) on peripheral CD4+ T and CD8+ T cells from patients with rheumatoid arthritis (RA) and its significance in order to clarify its role in the development of RA.Methods Peripheral blood samples were collected from 81 patients with RA and 33 healthy controls (HC).Expression of TIGIT on the surface of peripheral blood leukocytes was detected by flow cytometry.Differences in TIGIT expression between RA and HC groups were comparatively analyzed.Correlations of TIGIT expression on CD4+ T and CD8+ T cells with several laboratory indexes were analyzed.All data were statistically analyzed.Results (1) The percentage of TIGIT-expressing CD3+ T cells in patients with RA was significantly higher than that in HC (P<0.01).Moreover, the median fluorescence intensity (MFI) of TIGIT on CD3+ T cells was significantly elevated in patients with RA as compared with that in HC (P<0.01).No significant difference in the expression of TIGIT on B cells, monocytes or neutrophils was observed between RA and HC groups.(2) The percentages of TIGIT-expressing CD4+ T and CD8+ T cells were significantly elevated in patients with RA as compared with those in HC (P<0.01).Moreover, the MFI of TIGIT on CD4+ T and CD8+ T cells were significantly elevated in patients with RA as compared with those in HC (P<0.01).(3) The percentages of both TIGIT-expressing CD4+ T cells and TIGIT-expressing CD8+ T cells in patients with RA were positively correlated with erythrocyte sedimentation rate (ESR) (rs=0.355, P<0.01;rs=0.277, P=0.013).(4) The percentages of both TIGIT-expressing CD4+ T cells and TIGIT-expressing CD8+ T cells in patients with RA were positively correlated with rheumatoid factor (RF) (rs=0.265, P=0.017;rs=0.366, P<0.01).The MFI of TIGIT on CD4+ T cells was positively correlated with RF in RA group (rs=0.226, P=0.043).The percentage of TIGIT-expressing CD4+ T cells was positively correlated with anti-cyclic citrullinated peptide (anti-CCP) antibody in patients with RA who were positive for anti-CCP antibody (rs=0.324, P=0.012).(5) The percentage of TIGIT-expressing CD4+ T cells as well as the MFI of TIGIT on CD4+ T cells in patients with RA was positively correlated with Disease Activity Score-28 (DAS28) (r=0.232, P=0.038;r=0.343, P<0.010).Conclusion The expression of TIGIT on T cells is elevated in patients with RA and correlated with inflammatory markers, antibody production and disease activity.

Objective To detect the serum level of long non-coding RNA (lncRNA ) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in pulmonary tuberculosis (TB) patients ,and to evaluate its diagnostic value .Methods The expression of serum MALAT1 in 56 hospitalized TB patients , 35 latent TB infection (LTBI) individuals and 40 healthy controls were detected by real-time quantitative PCR .Serum levels of MALAT1 before and 3 ,6 months after anti-TB therapy in 16 TB patients were determined .Receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of serum MALAT 1 .The comparison between two groups was performed by Student t-test , and the comparison among three groups was performed with one-way analysis of variance test .Results Serum level of MALAT1 in TB patients was (2 .10 ± 1 .05) ,which was significantly higher than those in LTBI individuals (1 .16 ± 0 .51) and healthy controls (1 .02 ± 0 .44 ,F= 28 .53 ,P< 0 .01) .The MALAT1 level in TB patients with positive sputum smear was significantly higher than that in patients with negative sputum smear (2 .42 ± 1 .03 vs 1 .43 ± 0 .74 ,t= 2 .66 ,P< 0 .01) .Compared with pre-treatment (2 .28 ± 0 .79) ,the serum MALAT 1 levels decreased significantly in 3 months (1 .35 ± 0 .39) and 6 months (1 .05 ± 0 .30) after anti-TB therapy (t= 4 .33 ,6 .05 ;both P< 0 .01) .The area under the curve (AUC) of serum MALAT1 was 0 .821 , with sensitivity and specificity of 0 .732 and 0 .850 , respectively .Conclusion The expression of MALAT1 is up-regulated in TB patients , and could be used as potential novel biomarkers for the diagnosis of TB .

Objective Detecting plasma level of circular RNA(circRNA)hsa_circ_0009024 in pulmonary tuberculosis(TB)patients,and evaluating its diagnostic value for TB.Methods From January 2016 to December 2016, a hosptial-based, case-control study was performed, which include 90 untreated active pulmonary tuberculosis patients(TB group),75 healthy people(healthy control)and 84 patient with other diseases(other disease group).Plasma level of circRNA hsa_circ_0009024 was detected by real-time quantitative polymerase chain reaction.Furthermore, the 90 patients with TB were divided into different subgroups according to cavity formation and the lung fields involvement: patients without lung cavity(55 cases)vs those with lung cavity(35 cases),patients with involvement of <2 lung fields(49 cases)vs≥2 lung fields(41 cases).Plasma levels of hsa _circ_0009024 of 41 TB patientswere monitored andcomparedbefore and after 3 months anti-TB therapy.The sensitivity and specificity of plasma hsa_circ_0009024 were analyzed by using the receiver operating characteristic(ROC)curve.The comparison between two groups was performed with Mann-Whitney U test and the comparison among multigroupswas conducted with Kruskal-Wallis H test.Results Plasma levels of hsa_circ_0009024 in TB patients[1.98(1.42, 2.71)]were significantly higher than healthy controls[1.03(0.78,1.33)]and other disease groups[1. 13(0.77,1.51)](H=76.58,P<0.0001).Plasma levels of hsa_circ_0009024 in cavity pulmonary TB patients were higher than pulmonary TB patients without cavity(U=392.50,P<0.0001).Plasma levels of hsa_circ_0009024 in TB patients with involvement of ≥2 lung fields were higher than <2 lung fields(U=590.50,P=0.0008).As compared to pre -treatment[2.01(1.41, 2.71)], the plasma hsa_circ_0009024 levels decreased significantly in 3 months[1.22(0.85,1.47)](U=292.50,P<0.0001)after anti-TB therapy.The area under the receiver operating characteristics curve(AUC)of plasma hsa_circ_0009024 in discriminating the patients with TB from normal controls, pneumonia patients and lung cancer patients were 0.841 and 0.811, respectively.Conclusion The hsa_circ_0009024 can be used as a potential biomarker in TB diagnosis and monitoring.