The role and treatment progress of ferroptosis in osteoarthritis / 中国医师杂志

Osteoarthritis is a chronic inflammatory disease characterized by non inflammatory degeneration of articular cartilage and the formation of osteophytes at the edge of the joint, caused by complex causes. Its pathology is complex, and its pathogenesis is not yet clear, ultimately leading to joint stiffness and functional activity disorders. At present, the treatment for osteoarthritis is limited to alleviating symptoms and improving function, with varying degrees of side effects. Ferroptosis is a new type of programmed cell death discovered in recent years, which is related to the pathological and physiological processes of osteoarthritis and plays an important regulatory role in the occurrence and development of osteoarthritis. Its main characteristics include iron metabolism imbalance and accumulation of reactive oxygen species. Therefore, ferroptosis inhibitors targeting ferroptosis have shown great application prospects in the treatment of osteoarthritis. In this review, the author summarizes the relevant mechanisms of ferroptosis in the occurrence and development of osteoarthritis, outlines a large number of specific therapeutic drugs and their corresponding targets, with the aim of delaying and reversing the progression of osteoarthritis by regulating chondrocyte ferroptosis, which has certain clinical guiding significance.

Activation-induced markers for evaluation of specific CD4 + T cell immune response in HIV-1-infected patients / 中华微生物学和免疫学杂志

Objective:To establish a method using activation-induced markers (AIM) to detect the function of HIV-1-specific CD4 + T cell subsets for evaluating the immune response of HIV-1-specific CD4 + T cells more effectively. Methods:Twelve chronically HIV-1-infected patients without antiviral therapy and six healthy people without HIV-1 infection were enrolled in this study. The function of HIV-1-specific T lymphocytes was detected by AIM and ICS based on polychromatic flow cytometry. The performance of the two methods in assessing HIV-1-specific CD4 + T cell immune response in HIV-1-infected patients was evaluated. Results:The positive rates of HIV-1-specific PD-1 + CD25 + CD4 + T, CD69 + CD200 + CD4 + T, CD69 + ICOS + CD4 + T, CD69 + ICOS + CD8 + T、CD137 + CD69 + CD8 + T、PD-1 + CD25 + CD8 + T and OX40 + PD-1 + CD8 + T cells in all of the HIV-1 patients were 11/12, 8/12, 7/12, 8/12, 8/12, 7/12 and 7/12 using AIM method. ICS results showed that the positive rates of HIV-1-specific IL-2 + CD4 + T, IFN-γ + CD4 + T, TNF-α + CD4 + T, IFN-γ + CD8 + T, TNF-α + CD8 + T and IL-2 + CD8 + T cells were 2/12, 2/12, 0, 12/12, 10/12 and 5/12, respectively. Conclusions:AIM method was more sensitive in antigen-specific CD4 + T cell detection, and could be used as a complementary method to ICS in assessing antigen-specific T cell response.

SARS-CoV-2 vaccine research and immunization strategies for improved control of the COVID-19 pandemic / 医学前沿

The record speed at which Chinese scientists identified severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) and shared its genomic sequence with the world, has greatly facilitated the development of coronavirus disease (COVID-19) diagnostics, drugs, and vaccines. It is unprecedented in pandemic control history to develop a dozen successful vaccines in the first year and to immunize over half of the global population in the second year, due to the efforts of the scientific community, biopharmaceutical industry, and regulatory agencies worldwide. The challenges are both great and multidimensional due to the rapid emergence of virus variants and waning of vaccine immunity. Vaccination strategies need to adapt to these challenges to keep population immunity above the herd immunity threshold, by increasing vaccine coverage, especially for older adults and young people, and providing timely booster doses with homologous or heterologous vaccine boosts. Further research should be undertaken to develop more effective vaccines against SARS-CoV-2 variants and to understand the best prime-boost vaccine combinations and immunization strategies to provide sufficient and sustainable immune protection against COVID-19.

Neutrophils mediate T lymphocyte function in septic mice via the CD80/cytotoxic T lymphocyte antigen-4 signaling pathway / 中华危重病急救医学

Objective:To investigate the effect of neutrophils on T lymphocyte function in septic mice and the role of CD80/cytotoxic T lymphocyte antigen-4 (CTLA-4) signaling pathway in this modulated effects.Methods:① In vivo experiment: 6-8 weeks old male C57BL/6 mice were divided into sham operation group (Sham group, n = 20), Sham+CTLA-4 antibody treatment group (Sham+aCTLA-4 group, n = 20), cecal ligation and perforation (CLP) induced sepsis model group (CLP group, n = 30) and CLP+CTLA-4 antibody treatment group (CLP+aCTLA-4 group, n = 30) according to the random number table. CLP was used to reproduce mouse sepsis model. The mice in the Sham group were treated identically but their cecums were neither punctured nor ligated. In CTLA-4 antibody treatment groups, 50 μg CTLA-4 antibody was injected intraperitoneally 6 hours and 24 hours after the operation. Forty-eight hours after operation, 6 mice in Sham group and Sham+aCTLA-4 group, 14 mice in CLP group and CLP+aCTLA-4 group were randomly selected to detect the expression of CD69 in spleen. At the same time, spleen, bone marrow and peripheral blood were collected, and the expression of CD80 on neutrophils was detected by flow cytometry. The expression of CTLA-4 on the surface of T lymphocytes in spleen was detected by immunofluorescence and flow cytometry. The remaining mice in each group were used to observe the 96-hour survival after operation.② In vitro experiment 1: neutrophils were extracted from bone marrow of healthy mice and stimulated with LPS (1 mg/L) for 4, 8 and 12 hours respectively. The control group was added with the same amount of phosphate buffered saline (PBS) at each time point, and the expression of CD80 was detected at each time point.③ In vitro experiment 2: splenic T lymphocytes of healthy mice were extracted and divided into PBS control group, LPS group (final concentration of LPS 1 mg/L), neutrophil group and neutrophil+LPS group. In the latter two groups, the co-culture model of neutrophils and T lymphocytes was established, and then the corresponding treatment was given to detect the expression of CTLA-4 on the surface of T lymphocytes. With the above four groups as controls, CTLA-4 antibody treatment groups (final concentration of CTLA-4 antibody 50 mg/L) were set up respectively. After 48 hours, the level of interleukin-2 (IL-2) in the cell supernatant was detected by enzyme linked immunosorbent assay (ELISA). Results:① Results of in vivo experiment: compared with Sham group, the expression of CD80 on neutrophils in spleen, bone marrow and peripheral blood was significantly up-regulated, while the expression of CTLA-4 on the surface of T lymphocytes was significantly increased [(9.98±0.84)% vs. (3.48±0.64)%, P < 0.05]. It suggested that neutrophils may affect T lymphocytes function through CD80/CTLA-4 pathway in sepsis. Compared with CLP group, CTLA-4 antibody could significantly improve the 96-hour cumulative survival rate of CLP mice (56.25% vs. 18.75%, P < 0.05), and increase the expression of CD69 on the surface of T lymphocytes. It suggested that CTLA-4 antibodies might increase T lymphocytes activation in sepsis and improve survival. ② Results of in vitro experiment: with the prolongation of LPS stimulation, the expression of CD80 on neutrophils gradually increased in time-dependent manner as compared with PBS control group [4 hours: (6.35±0.40)% vs. (3.41±0.40)%, 8 hours: (8.57±0.64)% vs. (3.09±0.27)%, 12 hours: (19.83±1.06)% vs. (5.16±0.36)%, all P < 0.05]. Compared with PBS control group, the expression of CTLA-4 on CD4 +/CD8 + T lymphocytes was not significantly affected by LPS stimulation alone, but CTLA-4 was increased after co-culture with neutrophils [CD4 +: (4.92±0.30)% vs. (3.33±0.25)%, CD8 +: (4.26±0.21)% vs. (2.53±0.66)%, both P < 0.05], and the increased trend of CTLA-4 was more obvious after co-culture with LPS-stimulated neutrophils [CD4 +: (6.34±0.50)% vs. (3.33±0.25)%, CD8 +: (6.21±0.41)% vs. (2.53±0.66)%, both P < 0.05]. In the PBS control group and LPS group, CTLA-4 antibody had no significant effect on IL-2 secretion of T lymphocytes. Compared with PBS control group, co-culture with neutrophils could inhibit the secretion of IL-2 by T lymphocytes (ng/L: 1 938.00±68.45 vs. 2 547.00±218.00, P < 0.05), and the inhibitory effect of neutrophils stimulated by LPS was more obvious (ng/L: 1 073.00±34.39 vs. 2 547.00±218.00, P < 0.05). CTLA-4 antibodies could partially restore IL-2 secretion. In conclusion, after promoting the expression of CTLA-4 on the surface of T lymphocytes, neutrophils might mediate the inhibition of T lymphocytes function by reducing the production of IL-2. Conclusions:Neutrophils mediate T lymphocytes dysfunction in sepsis, and the CD80/CTLA-4 pathway plays an important role. The CTLA-4 antibody improves survival and T lymphocytes function in sepsis mice, which may be a new method of immunotherapy for sepsis.

Characteristics of amino acid and codon usage of Env genes in HIV-1 infected individuals with highly broad cross-neutralizing activity / 中华微生物学和免疫学杂志

Objective:To study the amino acid and codon usage profile of HIV-1 Env gene in donors whose serum exhibit highly broad cross-neutralizing activity. Methods:The samples were divided into highly broad cross-neutralizing activity group (hBCN + group) and non-highly broad cross-neutralizing activity group (hBCN - group) based on whether the neutralization breadth was higher than 90% or not. Full-length Env genes were amplified by single genome amplification (SGA) method from patients′ plasma samples, and the characteristics of Env sequences in hBCN + group were compared with hBCN - group. The correspondence analysis (COA) on relative amino acid usage (RAAU), adaptability to host based on similarity index D( A, B) and relative synonymous codon usage (RSCU) values of Env genes (hBCN + and hBCN -) with respect to human host RSCU were analyzed. Results:Correspondence analysis showed that the RAAU data of hBCN + group and hBCN - group were distributed along the two main axes to form two relatively separated clusters, indicating that the Env genes of the two groups had relatively unique amino acid usage patterns; the similarity index calculation results showed that hBCN + group (0.097) was lower than the hBCN - group (0.102), in addition, the Env gene of the hBCN + group had less frequency of similarly selected codons with human host system compared to hBCN - group. Conclusions:Env genes in hBCN + group and hBCN - group may have relatively unique amino acid usage patterns, and virus strains in hBCN + group are less adaptable to the host than those in hBCN - group.

Review on effects of heavy metals on sleep / 环境与职业医学

　　Heavy metals including lead, cadmium, manganese, and mercury are important raw materials and auxiliary materials in industrial production, but they are also seriously polluting the environment. While most of the early toxicological data on heavy metals are derived from studying occupational exposure populations, the general adult population and the infants and adolescents are now increasingly being studied for the health hazards of heavy metals. Epidemiological and laboratory studies have clearly demonstrated the neurotoxicity of heavy metals and sleeping is heavily regulated and coordinated by nervous system. Based on available epidemiological studies, the paper reviewed the effects of heavy metals on sleep status of occupational and non-occupational (general adults as well as infants and adolescents) populations. In addition, it presented the associated mechanisms in terms of sleep-wake cycle and sleep-related neurochemicals.

Advances on molecular mechanism of hepatitis B virus-induced hepatocellular carcinoma / 浙江大学学报·医学版

The pathogenesis of hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC） is complicated with the crosstalk of multiple factors and the multi-step processes. The main mechanisms underlying the HBV-induced HCC include:①integration of HBV DNA into the host hepatocyte genome to alter gene function at the insertion site，resulting in host genome instability and expression of carcinogenic truncated proteins;②HBV gene mutations at S，C，and X coding regions in the genome;③HBV X gene-encoded HBx protein activates proto-oncogenes and inhibits tumor suppressor genes，leading to the HCC occurrence. In this article，the recent research progress on the molecular mechanism of HBV-induced HCC is comprehensively reviewed，so as to provide insights into the prevention，early prediction and postoperative adjuvant therapy of HCC.

HIV genetic subtypes and comparison of the first CD4+T cell counts in newly diagnosed HIV infected patients in Liuzhou, 1998-2012 / 中华流行病学杂志

Objective@#To analyze the change trend of HIV genetic subtypes and compare the first CD4+T cell counts of newly diagnosed HIV infected patients in Liuzhou from 1998 to 2012, and provide a reference for AIDS prevention and control.@*Methods@#Newly diagnosed HIV-infected patients from 1998 to 2012 in Liuzhou were selected through national HIV/ADIS comprehensive response information management system. Their plasma samples were used for RNA gene extraction, amplification, sequencing and genotyping. Coharan-Armitage trend test was used to analyze the ratio trend of genetic subtypes and phylogenetic clusters of HIV and Wilcoxon Rank Sum Test was used to compare the first CD4+T cell counts (CD4) of the different subtype HIV infected patients.@*Results@#A total of 1 877 newly diagnosed HIV infected patients were included in the study. From 1998 to 2012, the proportions of CRF01_AE and CRF01_AE (Cluster 1) increased from 78.4% (76/97) to 91.5% (1 441/1 574), from 63.9% (62/97) to 74.0% (1 164/1 574), and the proportion of CRF07_BC decreased from 17.5% (17/97) to 4.6% (72/1 574), respectively (Z=4.632, P<0.001; Z=2.455, P=0.014; Z=-5.943, P<0.001). The median and interquartile range of the first CD4 of the patients infected with subtype CRF01_AE (Cluster 1), CRF01_AE (Cluster 2), CRF07_BC and CRF08_BC were 230 (83-375), 215 (48-351), 365 (254-503) and 334 (206-479) cell/μl, respectively. The first CD4 levels of the patients infected with subtype CRF01_AE (Cluster 1) or CRF01_AE (Cluster 2) were significantly lower than those of CRF07_BC (Z=-4.795, P<0.001; Z=-4.238, P<0.001).@*Conclusion@#The genetic subtypes of HIV were mainly CRF01_AE in newly diagnosed HIV-infected patients and this subtype proportion was in increase and the first CD4 levels of the patients were low in Liuzhou during 1998 to 2012.

Eukaryotic expression and serodiagnostic potential of HIV-1 p24 antigen / 中华微生物学和免疫学杂志

Objective To express HIV-1 capsid p24 antigen in an eukaryotic expression system and to evaluate its antigenicity and potential in the early diagnosis of HIV. Methods The full-length gene of HIV-1 p24 and the signal peptide DRVI gene were amplified by PCR. The signal peptide DRVI preceding the p24 gene was introduced using fusion PCR, and cloned into vector pDRVI1. 0. Two recombinant plas-mids pDRVI-p24 and pDRVI-p24s were constructed and transfected into 293F cells. Expression and secre-tion of p24 protein were detected by SDS-PAGE, Ni-NTA column chromatography and molecular sieve were used to purify p24s protein. The purified protein was identified by Western blot and indirect ELISA using hu-man/mouse HIV-1-positive serum samples. Results The eukaryotic expression system for HIV-1 p24 anti-gen was successfully established with high efficiency. The target protein of interest with the signal peptide DRVI was obviously detected in the supernatants of cell culture. The recombinant protein had good specifici-ty and sensitivity based on the results of serological tests using serum samples of five HIV-1-positive and five HIV-negative mice , 30 HIV-1-positive patients and 50 HIV-1-negative healthy individuals . Conclusions The eukaryotic expression system for HIV-1 p24 antigen was successfully established. The purified HIV-1 p24s antigen had good antigenicity. An indirect ELISA assay with good specificity and sensitivity for the de-tection of HIV-1 was preliminarily constructed and showed great potential for application.

Effect of baseline CD4+ T cell count on drop-out of antiretroviral therapy in HIV infected persons in Guangxi Zhuang Autonomous Region, 2008-2015 / 中华流行病学杂志

Objective To investigate the effect of baseline CD4+T cell count (CD4) on drop-out of antiretroviral therapy (ART) in HIV infected persons.Methods Retrospective cohort was conducted in this study.HIV infected persons aged≥ 18 years and receiving free ART for the first time in Guangxi Zhuang Autonomous Region (Guangxi) from 2008 to 2015 were selected from the antiretroviral treatment database of National Comprehensive HIV/AIDS Information System,with follow-up conducted till May 30,2016.Cause-specific Cox proportional hazard models were used to evaluate effect of different CD4 on the drop-out of ART in the HIV infected persons.Results A total of 58 502 eligible study participants were included in this retrospective cohort study.The average drop-out ratio was 4.8/100 person-years.After controlling the following baseline covariates:age,sex,marital status,route of HIV infection,WHO clinical stage before ART,initial/current ART regiment,ART regiment adjustment,and year of initiating ART for potential confounding,the adjusted HR of drop-out for HIV infected persons with 200-cells/μl,351-cells/μl and ≥500 cells/μl were 1.110 (95％CI:1.053-1.171,P＜0.001),1.391 (95％CI:1.278-1.514,P＜0.001) and 1.695 (95％CI:1.497-1.918,P＜ 0.001),respectively,in risk for drop-out compared with those with baseline CD4 ＜200 cells/μ 1.Among the HIV infected persons,56.0％ (1 601/2 861) of drug withdrawal was due to poor compliance with medication.Conclusions With the increase of baseline CD4 when initiating ART,the risk for the drop-out in HIV infected persons increased significantly.To further reduce the drop-out of ART,it is important to take CD4 into account in initiating ART and to strengthen the health education on treatment compliancy and training for healthcare providers.

Effect of baseline CD4+ T cell count on drop-out of antiretroviral therapy in HIV infected persons in Guangxi Zhuang Autonomous Region, 2008-2015 / 中华流行病学杂志

Objective To investigate the effect of baseline CD4+T cell count (CD4) on drop-out of antiretroviral therapy (ART) in HIV infected persons.Methods Retrospective cohort was conducted in this study.HIV infected persons aged≥ 18 years and receiving free ART for the first time in Guangxi Zhuang Autonomous Region (Guangxi) from 2008 to 2015 were selected from the antiretroviral treatment database of National Comprehensive HIV/AIDS Information System,with follow-up conducted till May 30,2016.Cause-specific Cox proportional hazard models were used to evaluate effect of different CD4 on the drop-out of ART in the HIV infected persons.Results A total of 58 502 eligible study participants were included in this retrospective cohort study.The average drop-out ratio was 4.8/100 person-years.After controlling the following baseline covariates:age,sex,marital status,route of HIV infection,WHO clinical stage before ART,initial/current ART regiment,ART regiment adjustment,and year of initiating ART for potential confounding,the adjusted HR of drop-out for HIV infected persons with 200-cells/μl,351-cells/μl and ≥500 cells/μl were 1.110 (95％CI:1.053-1.171,P＜0.001),1.391 (95％CI:1.278-1.514,P＜0.001) and 1.695 (95％CI:1.497-1.918,P＜ 0.001),respectively,in risk for drop-out compared with those with baseline CD4 ＜200 cells/μ 1.Among the HIV infected persons,56.0％ (1 601/2 861) of drug withdrawal was due to poor compliance with medication.Conclusions With the increase of baseline CD4 when initiating ART,the risk for the drop-out in HIV infected persons increased significantly.To further reduce the drop-out of ART,it is important to take CD4 into account in initiating ART and to strengthen the health education on treatment compliancy and training for healthcare providers.

Involvement of PML proteins in treatment of acute promyelocytic leukemia with arsenic trioxide / 浙江大学学报·医学版

Promyelocytic leukemia (PML) protein, a tumor suppressor, plays an important role in patients with acute promyelocytic leukemia (APL) receiving arsenic trioxide (AsO) therapy. APL is a M3 subtype of acute myeloid leukemia (AML), which is characterized by expression of PML-RARα (P/R) fusion protein, leading to the oncogenesis. AsO is currently used as the first-line drug for patients with APL, and the mechanism may be:AsO directly binds to PML part of P/R protein and induces multimerization of related proteins, which further recruits different functional proteins to reform PML nuclear bodies (PML-NBs), and finally it degraded by SUMOylation and ubiquitination proteasomal pathway. Gene mutations may lead to relapse and drug resistance after AsO treatment. In this review, we discuss the structure and function of PML proteins; the pathogenesis of APL induced by P/R fusion protein; the involvement of PML protein in treatment of APL patient with AsO; and explain how PML protein mutations could cause resistance to AsO therapy.

Single B cell sorting-based amplification and functional identification of HIV-1 monoclonal antibody genes / 中华微生物学和免疫学杂志

Objective@#To amplify and identify monoclonal antibody genes from HIV-1-infected patients.@*Methods@#Single cell sorting was used to isolate antigen-specific single B cells. Sequence Identity Matrix and the international ImMunoGeneTics information system were used to analyze antibody variable region genes. Binding abilities were detected by enzyme linked immunosorbent assay. Neutralizing activities were tested by TZM-bl/pseudovirus assay.@*Results@#The heavy and light chain genes of four, seven, and eleven antibodies were amplified and sequenced from three HIV-1-infected patients, respectively. They were derived from various germline genes with flexible CDR3 lengths and somatic mutations. A1 and B3 antibodies bound to HIV-1 clade B, CRF01_AE, and CRF07_BC antigens. The half maximal inhibitory concentration values of A1 and B3 against MW965 virus were 0.04 μg/ml and 37.34 μg/ml.@*Conclusion@#In this study, we acquired a lot of monoclonal antibody genes and two HIV-1 monoclonal binding and neutralizing antibodies, which would provide basic data for further research on monoclonal antibody identification.

Study on the relationship between HIV drug resistance and CD4+T cell counts among antiretroviral therapy patients with low viral load / 中华预防医学杂志

Objective@#To explore drug resistance of different viral loads, and investigate the relationship between drug resistance and CD4+T cell counts in patients with HIV antiretroviral therapy (ART) in China from 2003 to 2015.@*Methods@#Data were extracted from the Chinese National HIVDR Surveillance database from 2003 to 2015. For this study, the data collected were as follows: having received ART for ≥12 months; 18 years or older; demographic characteristics, information of ART, CD4+T cell counts, viral load (VL) and HIV drug resistance of a total of 8 362 patients were collected. Multi-variables non-conditional logistic regression model was used to study the relationship between viral load, HIV drug resistance and CD4+T cell counts.@*Results@#Participants with age of (41.8±10.5) years were enrolled in this study. Among them, 59.9% (5 009 cases) were men. The percentage of CD4+T cell counts <200 cells/μl in the total population was 17.9% (1 496 cases), the highest was in VL ≥1 000 copies/ml with drug resistance, which was 43.0% (397/923) , followed by VL 50-999 copies/ml with drug resistance, which was 31.1% (69/222), and the lowest was in VL 50-999 copies/ml without drug resistance 13.2% (273/2 068). Compared to VL 50-999 copies/ml without drug resistance, VL<50 copies/ml, VL 50-999 with drug resistance, VL≥1 000 copies/ml without drug resistance, and VL ≥1 000 copies/ml with drug resistance, the OR (95%CI) of CD4 <200 cells/μl were 0.9 (0.7-1.0), 3.2 (2.3-4.4), 2.6 (2.1-3.2), and 4.9 (4.0-5.9), respectively. Among 222 patients with VL 50-999 and HIVDR, the most frequent antiretroviral drugs were EFV and NVP, both of which were NNRTI, and whose percentage both were 94.1% (209 cases). The most frequent mutations were M184V/I (NNRTI), and the percentage was 26.1% (58 cases). The second one was K103N (NNRTI), and the percentage was 22.5% (50 cases). The percentage of V32L/E (PI) and V82A (PI) were lower, they were 0.9% (2 cases) and 0.5% (1 case) respectively.@*Conclusion@#Decreased CD4+T cell counts were associated with HIV drug resistance at low viraemia. In the case of low viral load, the most vulnerable were the NNRTI antiviral drugs such as EFV and NVP.

Changes in follicular helper T ( Tfh) cells and Tfh-related molecules during HIV-1 infection / 中华微生物学和免疫学杂志

Objective To analyze the changes in follicular helper T (Tfh) cells during HIV-1 in-fection, to investigate the influences of Tfh cells and Tfh-related molecules on HIV-1 progression and to pro-vide references for further research on using Tfh cells in highly active antiretroviral therapy ( HAART) and vaccines. Methods This study enrolled 33 patients with HIV-1 infection, including 11 long-term nonpro-gressors (LTNP), 10 rapid progressors (RP) and 12 typical progressors (TP), and 11 healthy subjects (normal controls, NC). Peripheral blood mononuclear cells were isolated from each subject. Multicolor flow cytometry was performed to detect CD4+CD45RA-CXCR5+Tfh and CD4+CD45RA-CXCR3-CXCR5+PD-1+Tfh subsets and the levels of inducible costimulatory molecule (ICOS), IFN-γ and IL-21. Moreover, the levels of IL-10 and the percentages of CD19+B cells in plasma samples of each group were also analyzed. Relationships among Tfh, CD4 and B cells were analyzed. Results The percentages of both Tfh subsets were higher in patients with HIV-1 infection than in NC. Compared with NC, LTNP had the highest percent-age of CD4+CD45RA-CXCR3-CXCR5+PD-1+Tfh cells (P<0. 05). Expression of Tfh-related molecules ICOS, IFN-γ and IL-21 were enhanced significantly upon Staphylococcus enterotoxin B ( SEB) stimulation, ICOS+Tfh cells were negatively related with HIV-1 progression, but had a positive correlation with CD19+B cells (r=-0. 49, P<0. 01; r=0. 60, P<0. 05). IL-10 level in plasma increased significantly in patients withHIV-1 infection , especially in TP and RP ( TP vs NC : P＜0. 01 ; RP vs NC : P<0. 05 ) . Conclusion HIV-1 patients and NC had significant differences in the expression of Tfh cells and Tfh-related molecules in peripheral blood. ICOS+Tfh cells were closely related to the progression of HIV-1 infection and the function of B cells.

Isolation and identification of neutralizing monoclonal antibodies to two Tier 2 viruses in a patient with HIV-1 infection / 中华微生物学和免疫学杂志

Objective To isolate neutralizing monoclonal antibodies to Tier 2 viruses in a Chinese patient with HIV-1 infection. Methods Monoclonal antibodies were isolated using single B cell sorting and monoclonal antibody expression technique. The international ImMunoGeneTics database (IMGT) was used to analyze antibody variable region genes. Antibody binding ability and neutralizing activity were tested by enzyme linked immunosorbent assay and TZM-bl/pseudovirus assay,respectively. Results Two monoclonal antibodies (E11 and H2) that could neutralize two Tier 2 viruses were isolated from the patient with clade B HIV-1 infection. Heavy chains of E11 and H2 were derived from IGHV4-4*08 with a somatic mutation rate of 15.79% and 14.74%,respectively. Light chains were both derived from IGKV3-15*01 with a somatic mutation rate of 8.33% and 7.95%, respectively. E11 and H2 could bind to HIV-1 clade B, CRF01_AE and CRF07_BC viruses. The half maximal inhibitory concentration(IC50) values of E11 and H2 were 18.78 μg/ml and 22.43 μg/ml against 398-F1 virus and 43.35 μg/ml and 39.45 μg/ml against 25710 virus. Conclusion In this study, two neutralizing monoclonal antibodies to two Tier 2 viruses were identified in the patient with HIV-1 infection,which might provide reference for the development of AIDS vaccines.

Establishment of a real-time fluorescent quantitative PCR for the rapid detection of viral reservoir in the peripheral blood of SIV-infected rhesus monkeys / 中华微生物学和免疫学杂志

Objective To establish a TaqMan probe-based real-time fluorescent quantitative PCR ( real-time PCR) for the quantitative and rapid detection of viral reservoir in peripheral blood mononuclear cells (PBMCs) isolated from rhesus monkeys with simian immunodeficiency virus (SIV) infection and to evaluate its preliminary application. Methods A pair of primers and one TaqMan probe were designed ac-cording to the conserved sequence of SIVmac239 strain for real-time PCR amplification. A length of 2 090 bp of nucleotide fragment was digested from the plasmid p239SpSp5 containing 5′-end long segments of SIV-mac239 strain by restriction enzymes EcoRⅠand SpeⅠ. The standards used for quantitative detection of SIV DNA in peripheral blood samples were prepared by a 10-fold serial dilution and used for graphing the stand-ard curve. The numbers of SIV DNA ( copies per 106 PBMCs) in rhesus monkeys during acute and chronic phases of SIVmac239 infection were determined and the virological characteristics of SIV DNA at different phages of infection were analyzed. Results A linear positive correlation between cycle threshold ( Ct) val-ues and concentrations (10 copies/μl to 109 copies/μl) of the standards was found. High levels of SIV DNA were monitored in SIV-infected monkeys 14 to 22 days after acute infection. The levels of SIV DNA in the acute phase of infection were about 1 to 2 logs higher than those in the chronic phase of infection. The num-bers of SIV DNA ( copies per 106 PBMCs) were 1 log lower than the SIV viral load in peripheral blood of the same monkey. The ratios of SIV DNA load to SIV RNA load ( DNA/RNA) in chronic phase of infection were higher than those in the acute phase. Conclusion The established TaqMan probe-based real-time fluorescent quantitative PCR was a highly sensitive and specific assay for the detection of SIV DNA with an advantage of wide linear range. It could be used for the quantitative evaluation of latent reservoirs of SIV.

CD137 molecule permits single cell sorting and expansion of HIV-specific T cells in vitro / 中华微生物学和免疫学杂志

Objective To isolate HIV-specific T cell clone and to expand them in vitro through the activation-induced expression of CD137 molecule.Methods The peripheral blood mononuclear cells were isolated from HIV-infected patients and HIV Gag specific CD3+CD8+CD137+T cell subset were sorted to 96-well plate in 1 cell/well by multicolor flowcytometry and single cell sorting.After 14 days in vitro culture with feeder cells and cytokines, the numbers and phenotypes of the cultured HIV-specific T cells were calcu-lated and identified.Results The CD137 expression was low on rested T cells but up regulated by the stim-ulation with Gag peptide pool.The CD8+CD137+T cells could secret IFN-γ.The number of CD8 T cells reached to 106 after 14 days in culture and expanded to 107-108 cells after 28 days of culture in vitro 100%of the cells remained activated upon Gag stimulation.Conclusion In stead of using IFN-γ, CD137 could be utilized as a novel molecule to isolate and expand HIV specific T cells in vitro.The expanded antigen spe-cific T cell clones could maintain good activation status.

The humoral immune response in guinea pigs primed with recombinant vaccinia virus strains ex-pressing Transmitted/Founder virus HIV-1 membrane proteins and boosted with gp140 protein / 中华微生物学和免疫学杂志

Objective To analyze the antibody responses in guinea pigs vaccinated with recombi-nant vaccinia virus( rTT) strains expressing transmitted/founder ( T/F) HIV-1 membrane proteins in combi-nation with gp140 protein.Methods Guinea pigs were primed with rTT strains and boosted twice with gp140 protein in every four weeks.Serum samples were collected from guinea pigs before immunization and in 2, 6 and 10 weeks after the last immunization for the detection of HIV-1-specific binding antibodies, neu-tralizingantibodiesandtherelativeavidityofantibodies.Results (1)Thebindingantibodiesspecificto HIV-1 B′/C, B, AE subtypes were efficiently induced by the immunization of rTT-B, rTT-C and rTT-CON vaccinia strains in combination with gp140 protein.The antibody titers ranged from 111 430 to 1 024 000. More antibodies against HIV-1 B′/C and AE subtypes were induced in guinea pigs by the immunization of rTT-C and rTT-CON strains in combination with gp140 protein than those by using rTT-B strain prime-protein boost strategy (P<0.05).No significant differences with the titers of HIV-1 B subtype specific antibody were observed among the guinea pigs immunized with the three strategies.( 2 ) High titers of SF162 and ZM109 neutralizing antibodies were induced in guinea pigs immunized with rTT-B, rTT-C and rTT-CON vac-cinia strains in combination with gp140 protein, ranging from 83.76 to 649.30.No significant differences were found among the three groups.(3) The HIV-1 V1V2-gp70 specific antibodies associated with protec-tive immunity were induced by immunization of the three virus prime-protein boost strategies.No significant differences were observed among them.(4) Antibodies induced in guinea pigs by immunization of the three strategies showed strong affinity to membrane proteins of HIV-1 B′/C, B, AE subtype strains.No significant differences were found among the three immunization strategies.Conclusion A strong humoral immune re-sponse was induced in guinea pigs primed with recombinant vaccinia virus strains expressing T/F virus HIV-1 membrane proteins and boosted with gp140 protein.

Analysis of the phenotypes and the function of CD56+T cells during primary HIV-1 infection / 中华微生物学和免疫学杂志

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.