Objective To study the cell death in macrophages (THP-1) stimulated with different agonists (H37Rv-PPD or BCG-PPD) and to investigate the relationship between Toll like receptor (TLR)-2 and THP-1 apoptosis. Methods H37Rv-PPD and BCG-PPD were used to stimulate THP-1 cells for 3 h, 8 h, 15 h and 24 h, respectively with or without TLR-2 blockade. Cells were analyzed by flow cytometry to detect the TLR-2 expression. Annexin V staining and Hochest staining were performed to evaluate apoptosis. Results The apoptosis cells were increased when stimulated with BCG-PPD and the percentage was 30.2% at 24 h, which were confirmed by Hochest staining.However, the expression of TLR-2 did not increase simultaneously with percentage of 8.8% at 24 h.Nevertheless, most cells presented with necrosis form when stimulated with H37Rv-PPD and the expression of TLR-2 remained at high level with the percentage of 17.2% at 24 h, while the percent of apoptosis rate was only 7.7%. Under treatment of TLR-2 antibodies, the percentage of apoptosis decreased to 10.5% at 24 h of BCG-PPD stimulation and TLR-2 expressions were down-regulated to less than 3% at all time points; but after H37Rv-PPD stimulation, the percentage of apoptosis and TLR-2 expression did not changed obviously. Conclusions The attenuated BCG-PPD induces THP-1 apoptosis predominately, which is partially correlated with TLR-2 expression. While virulent H37Rv-PPD induces THP-1 necrosis predominately.

Objective To evaluate the number and function of peripheral Vγ2Vδ2+T lymphocytes during Mycobacterium tuberculosis(MTB)infection in human immunodeficiency virus (HIV)infected individuals.Methods Seventy-six HIV/acquired immune deficiency syndrome(AIDS) patients co-infected with MTB were divided into active tuberculosis(TB)group(HIV+TB)and latent TB group(HIV+LTB).T cell subsets of peripheral blood lymphocytes were analysed by flow cytometry.Stimulated by protein purified derivative(PPD)and hydroxymethylbutenyl diphosphate (HMBPP),specific interferon(IFN)-γ producing T cells were detected using enzyme-linked immunospot(ELISPOT)and intracellular cytokine staining(ICS).Data were analyzed by t test.Results The absolute number of CD3'T cells and DroDortion of V72VB2'T cells in CD3+T cells in HIV+TB group were both significantly lower than those in HIV+LTB group(t=-3.67,P<0.01;t=-2.06,P<0.05).PPD-specific IFN-y-producing T cells and percentage of PPD-specific CD4+Tcells in CD3+T cells in HIV+LTB group were both similar with those in HIV+TB group.While HMBPP-specific IFN-γ-producing T cells and percentage of HMBPP-specific Vγ2Vδ2+T cells in CD3+Tcells in HIV+LTB group were both higher than those in HIV+TB group(t=2.71 and t=3.003,respectively;both P<0.0 1).Conclusion The number and function of Vγ2Vδ2+T cells were impaired in HIV/AIDS patients coinfected with active MTB infection,which indicates that Vγ2Vδ2+T cells may be the key immune cells against MTB in individuals with impaired CD4+T cells.

Objective To study the different response in macrophages treated with different agoβ and IL-10 in Mycobacterium tuberculosisnists(H37Rv-PPD and BCG-PPD)related with Mycobacterium tuberculosis and the relationship with TNF-αt,IL-1β and IL-10.Methods Using H37Rv-PPD and BCG-PPD to stimulate THP-1 cell for 3h,8h,15h,24h respectively.Cells were ananlyzed by Hochest staining under fluorescence microscopy to assay cell death(apoptosis and necrosis).At each stimulating time,TNF-α,IL-1β and IL-10 were examined by ELISA.Results Under fluorescence microscopy,it could easily see oval apoptotic bodies of THP-1 stimulated by BCG-PPD.However ,the nucleus were often isolated and necrosis-like when cells were stimulated by H37Rv-PPD.In a word ,BCG-PPD tend to induce THP-1 cells to apoptosis,but H37Rv-PPD inclined to induce cells to undergo necrosis.In supernatant of cells stimulated by BCG-PPD,the expression of TNF-αand IL-10 were lower than the cells stimulated by H37Rv-PPD,but the expression of IL-1β was higher than the latter.Conclusion It indicated that the necrosis of cells stimulated by H37Rv-PPD was asossiated with the excessive expression of TNF-α and IL-10,and the apoptosis of cells induced by BCG-PPD was IL-1β related.Perhaps the mechanism of differences in virulence exist in protein of strain,and associated with cytokines IL-1β,TNF-α and IL-10.

Objective To investigate the incidence,bacterial pathogen and risk factors of bacterial meningitis after major craniotomy.Methods Clinical data of patients who underwent at least one craniotomy in Huashan Hospital Affiliated to Fudan University in 2008 were collected.All subjects were ≥ 18 years old,and survived at least 7 days after surgery.Patients with only cerebrospinal fluid drainage,burr holes,cranioplasty,vascular interventional surgery,transsphenoidal or spinal surgery were excluded.Risk factors for bacterial meningitis after major craniotomy were analyzed by Logistic regression.Results A total of 691 patients were enrolled,in 60 of whom (8.68％) bacterial meningitis was identified.Among 44 samples,5 were positive in culture with 2 of Acinetobacter baumannii,1 of Enterococcus faecalis,1 of Streptococcus intermedius and 1 of Klebsiella pneumonia.Diabetes (OR =5.79,95％ CI:1.40-23.93,P =0.02),Glasgow Coma Scale score ＜ 12 (OR =6.56,95％ CI:1.17-36.80,P =0.03),external ventricular drainage (OR =4.31,95％ CI:1.57-11.82,P =0.01),and continuous lumbar cistern drainage (OR =22.82,95％ CI:10.31-50.52,P =0.00) were independent risk factors for bacterial meningitis after major craniotomy.Patients with external ventricular drainage ＞ 7 d were 11.82 times more likely to develop bacterial meningitis,and those with continuous lumbar cistern drainage ＞ 10 d were 25.50 times more likely to develop bacterial meningitis.Conclusions Bacterial meningitis remains a common complication after major craniotomy,and most are induced by Gram-negative bacilli.Diabetes,Glasgow Coma Scale score,external ventricular drainage and continuous lumbar cistern drainage may increase its incidence.

Objective To evaluate the performance of peripheral blood T-SPOT.TB and cerebrospinal fluid (CSF) interferon (IFN)-γ detection in the diagnosis of tuberculous meningitis (TBM).Methods Among the 182 consecutive cases with suspected TBM in Huashan Hospital from March 2011 to March 2013,30 patients were included in the case group according to the latest diagnostic criteria of TBM.Thirty-nine patients diagnosed with non-tuberculous meningitis were included in the control group.T-SPOT.TB was employed to detect tuberculosis-specific IFN-γ-secreting T cells in the peripheral blood.And IFN-γ in CSF was detected simultaneously by enzyme-linked immunosorbent assay (ELISA) without antigen stimulation.The CSF was collected from 10 patients of TBM group after anti tuberculosis treatment for 4 weeks to observe the dynamic changes.The t-test was used for analysis of continuous variables with normal distribution and Kruskal-Wallis test was used for analysis of variables with abnormal distribution.Results ()f the 30 TBM cases,6 were confirmed cases and 24 were highly suspected cases.The control group was comprised of 12 viral encephalitis,16 suppurative meningitis and 11 cryptococcal meningitis.The positive rate of T-SPOT.TB was significantly higher in TBM group compared with control group (70％ vs 13％,x2 =12.15,P＜0.01).The mean concentration of CSF IFN γ of TBM group was 244.35 pg/mL,which was significantly higher than that of control group 9.48 pg/mL (Z=-4.646,P＜0.01).The CSF IFN-γ was significantly decreased after 4 weeks of treatment (271.02 pg/mL vs 81.36 pg/mL,Z=-3.099,P=0.002).The sensitivity and specificity of peripheral blood T-SPOT.TB in the diagnosis of TBM were 70％ and 87％,respectively.The area under the receiver operating characteristic (ROC) curve of CSF IFN-γ for TBM diagnosis was 0.819; the optimal cut-off point was 81.36 pg/mL; the corresponding sensitivity and specificity were 83 ％ and 85 ％,respectively.Conclusion Both the detection of peripheral blood T-SPOT.TB and CSF IFN-γ are of great importance for the diagnosis of TBM.Dynamic observation of CSF IFN-γ is important for disease monitoring.

Objective To analyse the clinical feature of infective endocarditis (IE) in recent years.Methods Clinical profiles including age of onset,predisposing factor,clinical manifestation,blood culture and ultrasonic cardio gram (UCG) of 97 cases from Huashan Hospital in the recent 10 years were collected and analyzed retrospectively. Descriptive data were represented as mean ±standard deviation form.Positive rate was represented as percentage form.Fisher's exact test were used to determine two groups' comparison.Results The mean age of the population was (49±17)years.Seventy-three patients (75.3％) had background heart disease,the top 3 of which was rheumatic heart disease in 27 patients (27.8％),congenital heart disease in 23 patients (23.7％) and idiopathic mitral valve prolapse in 18 patients (18.6 ％).The most common clinical manifestation were fever (99.0％),murmurs (95.9％) and anemia (84.5％).Sixty-six patients (68.0％) had positive result of blood cultures. Streptococcus viridans,which was found in 28 patients with native valve endocarditis (42.4 ％),was still the most common pathogen.Staphylococcus,which was found in 18patients (27.3％),had an elevated ratio.Staphylococcus aureus was found in 10 patients (15.2％)and 3 of which were MRSA.Coagulase-negative staphylococcus was found in 8 patients (12.1 ％ ) and 2 of which were MRCNS. Drug-resistant bacteria was increased and pathogens were varied.Vegetations were found in 79 patients (81.4％) by UCG.ConclusionsClinical manifestation,predisposing factor and pathogen have changed in IE patients. Attaching importance to physical examination,multiple-time blood culture and UCG helps the diagnose of IE.

Objective To observe the role of intermediate conductance calcium?activated potassium channels (KCa3.1) in alkalinization and β?glycerophosphate induced vascular calcification. Methods Vascular smooth muscle cells (VSMCs) and aortic rings were obtained from rat thoracic aorta, and then randomly divided into control group (pH was provided into 7.4, 8.0), high phosphorus groups (pH was provided into 7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10 mmol/L β?glycerophosphate; HCl and NaHCO3 were used to adjust the pH) and TRAM?34 group (20 nmol/L was added into pH8.0 high phosphorus dulbecco's modified eagle's medium). Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, calcium content and enzyme linked immunosorbent assay after cells were simulated for 12 days. Intracellular free Ca2 + was measured by ELISA. The expression of KCa3.1, runt?related transcription factor 2 (Runx2) were detected by RT?PCR and Western blotting 4 days after cells were stimulated. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. The expressions of KCa3.1 and Runx2 were detected by immunohistochemistry after aortic rings were cultured for 4 days. Results Compared with control group, calcification in VSMCs and aortic rings were significantly increased in high phosphorus group (P<0.05) while decreased in TRAM?34 group (P<0.05). Compared with control group, the expressions of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased (P<0.05) while decreased in TRAM?34 group (P<0.05). Besides, expressions of Runx2 and KCa3.1 were augmented as the pH was higher (P<0.05). The expression of Runx2 in aortic rings was the same situation. Besides, the Ca2+ influx was blocked by TRAM?34 (P<0.05). Conclusions Alkalinization contributes to β?glycerophosphate induced VSMCs calcification through increase of Ca2 + influx, up?regulation of KCa3.1 and promotion of osteogenic/chondrogenic differentiation.

Objective:To investigate the clinical characteristics and prognosis of Epstein-Barr virus-related diseases in adults.Methods:The clinical data of 59 patients with Epstein-Barr virus-related diseases in Huashan Hospital, Fudan University, Shanghai from January 2017 to August 2019 were analyzed retrospectively. The clinical manifestations of patients with infectious mononucleosis (IM), chronic active Epstein-Barr virus infection (CAEBV) and lymphoma in patients were compared. Patients were divided into acute course group (IM) and chronic course group (CAEBV+ lymphoma), and the results of labratory indications (blood rontine, liver function, imflammatory indications, Epstein-Barr virus DNA, Epstein-Barr virus antibody and T lymphocyte) were compared between two groups. Statistical analysis was performed by Mann-Whitney U test, chi-square test or Fisher exact probability test. Results:Among the 59 patients, 23 cases (39.0%) were diagnosed with IM, 23 cases (39.0%) were lymphoma and 13 cases (22.0%) were CAEBV. The clinical manifestations of patients with Epstein-Barr virus-related diseases were fever (57/59, 96.6%), lymphadenopathy (37/59, 62.7%) and splenomegaly (36/59, 61.0%). There were 17 patients in the chronic course group experienced hemophagocytic lymphohistiocytosis (HLH). The white blood cell counts, hemoglobin levels and platelet counts of patients in the chronic course group (4.07(1.94, 8.35)×10 9/L, 89.5(74.5, 108.0) g/L and 100(37, 161)×10 9/L, respectively) were all lower than those in the acute course group (9.91(6.75, 17.38)×10 9/L, 132.5(118.2, 152.0) g/L and 197(129, 233)×10 9/L, respectively), with statistically significant differences ( U=3.69, 5.22 and 3.61, respectively, all P<0.01). The levels of procalcitonin, C-reactive protein and serum ferritin in the chronic course group (0.45(0.15, 1.13) μg/L, 47.75(17.57, 84.67) mg/L and 2 000(682, 2 002) μg/L, respectively) were all higher than those in the acute course group (0.12(0.07, 0.28) μg/L, 6.39(3.13, 11.38) mg/L and 482(159, 1 271) μg/L, respectively), with statistically significant differences ( U=-2.95, -3.77 and -4.16, respectively, all P<0.01). The counts of CD4 + T lymphocytes, CD8 + T lymphocytes, CD19 + B lymphocytes and natural killer cells in the chronic course group (259.15(101.98, 509.26), 214.69(119.31, 529.47), 46.14(4.44, 135.87) and 81.09(41.53, 118.46)/μL, respectively) were all lower than those in the acute course group (738.88(592.20, 893.94), 1 609.17(920.88, 3 952.34), 144.52(83.65, 215.14) and 309.82(123.78, 590.68)/μL, respectively), with statistically significant differences ( U=3.66, 3.80, 2.90 and 3.40, respectively, all P<0.01), while the CD4 + /CD8 + T lymphocytes ratio in the chronic course group was higher (0.90(0.60, 1.70) vs 0.45(0.10, 1.28))( U=-2.29, P=0.02). Twenty-three patients with IM were all cured, while 10 patients with lymphoma died and 13 received chemotherapy. Seven patients with CAEBV died and six improved. Conclusions:The clinical characteristics of Epstein-Barr virus-related diseases in adults are fever, lymphadenectasis, splenomegaly.Chronic Epstein-Barr virus infection may be associated with HLH. The prognosis of adults with acute Epstein-Barr virus infection is good, while that of long-term chronic Epstein-Barr virus infection is poor.

Objective:To analyze the differences of peripheral blood transcriptome between mild and severe influenza A (H1N1) patients, and to find indicators for the assessment of disease severity.Methods:A total of ten patients (five patients with mild disease and five patients with severe disease) diagnosed with H1N1 infection from January to May 2018 at Huashan Hospital, Fudan University in Shanghai were enrolled, and five healthy people were also enrolled as controls. The peripheral blood of patients was collected for transcriptome sequencing at the time when they were first diagnosed. Measurement data were compared using t test or Mann-Whitney U test. The count data were compared using Fisher exact test when appropriate. Data analysis of transcriptome predictions was performed using bioinformatics methods. Results:The platelet counts were significantly different between mild and severe groups ((163.4±21.5 )×10 9/L vs (255.6±52.5)×10 9/L, t=3.636, P=0.007). There were no differences between the two groups in gender, age, white blood cell counts, neutrophil percentage, lymphocyte percentage and hemoglobin levels (all P>0.05). However, the average expression levels of matrix metalloproteinase (MMP) 8 and MMP9 in severe group (18.41 and 174.00, respectively) were both higher than those in mild group (2.33 and 22.91, respectively) and healthy control (1.43 and 34.65, respectively; all P<0.01). Conclusion:MMP8 and MMP9 could be expected to serve as the molecular biological markers for predicting the disease severity in patients with influenza A (H1N1) infection.

Dysregulation of mTORC1/mTORC2 pathway is observed in many cancers and mTORC1 inhibitors have been used clinically in many tumor types; however, the mechanism of mTORC2 in tumorigenesis is still obscure. Here, we mainly explored the potential role of mTORC2 in esophageal squamous cell carcinoma (ESCC) and its effects on the sensitivity of cells to mTOR inhibitors. We demonstrated that RICTOR, the key factor of mTORC2, and p-AKT (Ser473) were excessively activated in ESCC and their overexpression is related to lymph node metastasis and the tumor-node-metastasis (TNM) phase of ESCC patients. Furthermore, we found that mTORC1/ mTORC2 inhibitor PP242 exhibited more efficacious anti-proliferative effect on ESCC cells than mTORC1 inhibitor RAD001 due to RAD001-triggered feedback activation of AKT signal. Another, we demonstrated that down-regulating expression of RICTOR in ECa109 and EC9706 cells inhibited proliferation and migration as well as induced cell cycle arrest and apoptosis. Noteworthy, knocking-down stably RICTOR significantly suppresses RAD001-induced feedback activation of AKT/PRAS40 signaling, and enhances inhibition efficacy of PP242 on the phosphorylation of AKT and PRAS40, thus potentiates the antitumor effect of RAD001 and PP242 both and . Our findings highlight that selective targeting mTORC2 could be a promising therapeutic strategy for future treatment of ESCC.