Background::Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods::The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 +) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. Results::The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10-unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10-unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. Conclusions::This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Background::T-cell-mediated immunity is crucial for the effective clearance of viral infection, but the T-cell-mediated immune responses that are induced by booster doses of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in people living with human immunodeficiency virus (PLWH) remain unclear.Methods::Forty-five PLWH who had received antiretroviral therapy (ART) for more than two years and 29 healthy controls (HCs) at Beijing Youan Hospital were enrolled to assess the dynamic changes in T-cell responses between the day before the third vaccine dose (week 0) and 4 or 12 weeks (week 4 or week 12) after receiving the third dose of inactivated SARS-CoV-2 vaccine. Flow cytometry, enzyme-linked immunospot (ELISpot), and multiplex cytokines profiling were used to assess T-cell responses at the three timepoints in this study.Results::The results of the ELISpot and activation-induced marker (AIM) assays showed that SARS-CoV-2-specific T-cell responses were increased in both PLWH and HCs after the third dose of the inactivated SARS-CoV-2 vaccine, and a similar magnitude of immune response was induced against the Omicron (B.1.1.529) variant compared to the wild-type strain. In detail, spike-specific T-cell responses (measured by the ELISpot assay for interferon γ [IFN-γ] release) in both PLWH and HCs significantly increased in week 4, and the spike-specific T-cell responses in HCs were significantly stronger than those in PLWH 4 weeks after the third vaccination. In the AIM assay, spike-specific CD4 + T-cell responses peaked in both PLWH and HCs in week 12. Additionally, significantly higher spike-specific CD8 + T-cell responses were induced in PLWH than in HCs in week 12. In PLWH, the release of the cytokines interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-α), and IL-22 by peripheral blood mononuclear cells (PBMCs) that were stimulated with spike peptides increased in week 12. In addition, the levels of IL-4 and IL-5 were higher in PLWH than in HCs in week 12. Interestingly, the magnitude of SARS-CoV-2-specific T-cell responses in PLWH was negatively associated with the extent of CD8 + T-cell activation and exhaustion. In addition, positive correlations were observed between the magnitude of spike-specific T-cell responses (determined by measuring IFN-γ release by ELISpot) and the amounts of IL-4, IL-5, IL-2 and IL-17F. Conclusions::Our findings suggested that SARS-CoV-2-specific T-cell responses could be enhanced by the booster dose of inactivated COVID-19 vaccines and further illustrate the importance of additional vaccination for PLWH.

Background::Cluster of differentiation 8 (CD8 T) cells play critical roles in eradicating human immunodeficiency virus (HIV)-1 infection, but little is known about the effects of T cells expressing CD8 at low levels (CD8 low) or high levels (CD8 high) on HIV-1 replication inhibition after HIV-1 invasion into individual. Methods::Nineteen patients who had been acutely infected with HIV-1 (AHI) and 20 patients with chronic infection (CHI) for ≥2 years were enrolled in this study to investigate the dynamics of the quantity, activation, and immune responses of CD3 +CD8 low T cells and their counterpart CD3 +CD8 high T cells at different stages of HIV-1 infection. Results::Compared with healthy donors, CD3 +CD8 low T cells expanded in HIV-1-infected individuals at different stages of infection. As HIV-1 infection progressed, CD3 +CD8 low T cells gradually decreased. Simultaneously, CD3 +CD8 high T cells was significantly reduced in the first month of AHI and then increased gradually as HIV-1 infection progressed. The classical activation of CD3 +CD8 low T cells was highest in the first month of AHI and then reduced as HIV-1 infection progressed and entered the chronic stage. Meanwhile, activated CD38 -HLA-DR +CD8 low T cells did not increase in the first month of AHI, and the number of these cells was inversely associated with viral load ( r = -0.664, P = 0.004) but positively associated with the CD4 T-cell count ( r = 0.586, P = 0.014). Increased programmed cell death protein 1 (PD-1) abundance on CD3 +CD8 low T cells was observed from the 1st month of AHI but did not continue to be enhanced, while a significant T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) abundance increase was observed in the 12th month of infection. Furthermore, increased PD-1 and TIGIT abundance on CD3 +CD8 low T cells was associated with a low CD4 T-cell count (PD-1: r = -0.456, P = 0.043; TIGIT: r = -0.488, P = 0.029) in CHI. Nonetheless, the nonincrease in PD-1 expression on classically activated CD3 +CD8 low T cells was inversely associated with HIV-1 viremia in the first month of AHI ( r = -0.578, P = 0.015). Notably, in the first month of AHI, few CD3 +CD8 low T cells, but comparable amounts of CD3 +CD8 high T cells, responded to Gag peptides. Then, weaker HIV-1-specific T-cell responses were induced in CD3 +CD8 low T cells than CD3 +CD8 high T cells at the 3rd and 12th months of AHI and in CHI. Conclusions::Our findings suggest that CD3 +CD8 low T cells play an anti-HIV role in the first month of infection due to their abundance but induce a weak HIV-1-specific immune response. Subsequently, CD3 +CD8 low T-cell number decreased gradually as infection persisted, and their anti-HIV functions were inferior to those of CD3 +CD8 high T cells.

BACKGROUND: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), an inhibitory receptor expressed on T cells, plays a dysfunctional role in antiviral infection and antitumor activity. However, it is unknown whether TIGIT expression on T cells influences the immunological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccines. METHODS: Forty-five people living with HIV (PLWH) on antiretroviral therapy (ART) for more than two years and 31 healthy controls (HCs), all received a third dose of a SARS-CoV-2 inactivated vaccine, were enrolled in this study. The amounts, activation, proportion of cell subsets, and magnitude of the SARS-CoV-2-specific immune response of TIGIT + CD4 + and TIGIT + CD8 + T cells were investigated before the third dose but 6 months after the second vaccine dose (0W), 4 weeks (4W) and 12 weeks (12W) after the third dose. RESULTS: Compared to that in HCs, the frequency of TIGIT + CD8 + T cells in the peripheral blood of PLWH increased at 12W after the third dose of the inactivated vaccine, and the immune activation of TIGIT + CD8 + T cells also increased. A decrease in the ratio of both T naïve (T N ) and central memory (T CM ) cells among TIGIT + CD8 + T cells and an increase in the ratio of the effector memory (T EM ) subpopulation were observed at 12W in PLWH. Interestingly, particularly at 12W, a higher proportion of TIGIT + CD8 + T cells expressing CD137 and CD69 simultaneously was observed in HCs than in PLWH based on the activation-induced marker assay. Compared with 0W, SARS-CoV-2-specific TIGIT + CD8 + T-cell responses in PLWH were not enhanced at 12W but were enhanced in HCs. Additionally, at all time points, the SARS-CoV-2-specific responses of TIGIT + CD8 + T cells in PLWH were significantly weaker than those of TIGIT - CD8 + T cells. However, in HCs, the difference in the SARS-CoV-2-specific responses induced between TIGIT + CD8 + T cells and TIGIT - CD8 + T cells was insignificant at 4W and 12W, except at 0W. CONCLUSIONS TIGIT expression on CD8 + T cells may hinder the T-cell immune response to a booster dose of an inactivated SARS-CoV-2 vaccine, suggesting weakened resistance to SARS-CoV-2 infection, especially in PLWH. Furthermore, TIGIT may be used as a potential target to increase the production of SARS-CoV-2-specific CD8 + T cells, thereby enhancing the effectiveness of vaccination.
Humans ; Antibodies, Viral ; CD8-Positive T-Lymphocytes ; COVID-19/complications* ; COVID-19 Vaccines/immunology* ; HIV Infections/complications* ; Receptors, Immunologic ; SARS-CoV-2

Objective To investigate the expression of autophagy marker in peripheral blood T and B lymphocytes of patients with autoimmune hepatitis (AIH) and its clinical significance. Methods Peripheral blood samples were collected from 62 AIH patients who were treated in Beijing YouAn Hospital affiliated to Capital Medical University from October 2019 to October 2020 who were treated in Beijing YouAn Hospital affiliated to Capital Medical University from October 2019 to October 2020 and 8 healthy controls to detect autophagy of T and B lymphocyte subsets, and then subgroup analyses were performed based on treatment, diagnostic type, and presence or absence of liver cirrhosis and liver failure. The t -test was used for comparison of normally distributed continuous data between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Mann-Whitney U test was used for comparison between two groups; the chi-square test or the Fisher's exact test was used for comparison of categorical data between two groups. Results Compared with the healthy control group, the AIH group had a significantly higher mean fluorescence intensity (MFI) of the autophagy marker LC3B in CD4 + T, CD8 + T, CD19 + B, and CD4 + CD25 + T lymphocytes (all P < 0.05), especially in CD19 + B lymphocytes. The non-treatment group and the partial remission group had a significantly higher MFI of autophagy marker in CD19 + B lymphocytes than the complete remission group ( P =0.037 and 0.040); the idiopathic AIH (I-AIH) group and the drug-induced AIH(DI-AIH) group had a significantly higher MFI than the primary biliary cholangitis (PBC)-AIH overlap syndrome group ( P =0.037 and 0.031); the non-cirrhosis group and the decompensated cirrhosis group had a significantly higher MFI than the compensated cirrhosis group ( P =0.009 and 0.003); the liver failure group had a significantly higher MFI than the non-liver failure group ( P =0.042). The PBC-AIH group had a significantly higher MFI of autophagy marker in CD4 + CD25 + T lymphocytes than the I-AIH group and the DI-AIH group ( P =0.042 and 0.044), the compensated cirrhosis group had a significantly lower MFI than the non-cirrhosis group ( P =0.037), and the non-liver failure group had a significantly higher MFI than the liver failure group ( P =0.043). Conclusion AIH patients have a significant increase in the expression of autophagy marker in peripheral blood T and B lymphocyte subsets compared with healthy individuals, and the level of autophagy is associated with treatment, diagnostic type, and disease severity.

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.
Animals ; DNA, Mitochondrial ; genetics ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; MELAS Syndrome ; genetics ; Male ; Mice ; Microsatellite Repeats ; genetics ; Mitochondria ; genetics ; metabolism ; Mutation ; genetics

Objective To evaluate whether an intervention with a health promotion program can reduce prevalence of the health risk factors of non-communicable chronic diseases (NCD) and improve the knowledge awareness of NCD prevention of employees from a security corporation in Beijing selected by the study team. Methods A cross-sectional study was conducted by selecting 157 employees of a security corporation in Beijing, and a quasi-experiment method and a self-developed Questionnaire of Intervention for Health Risk Factors were used. Based on the results of the distribution of risk factors for NCD and the focus-group discussion, one year health promotion program intervention was performed. According to the comparison of prevalence of risk factors between baselines and after intervention of the studied employees, the intervention effectiveness was evaluated. Results After the health intervention, rate of reported unhealthy diet reduced from 75.0%to 56.8%, which showed statistically significant (?2=8.53, P<0.05);rate of reported less physical activity reduced from 35.5%to 17.4%, with statistical significance (?2=19.60, P<0.05);rate of reported cigarette smoking reduced from 16.8%to 11.6%with statistically significance. There was no statistically significant reduction in the rate of the other health risk factors (P>0.05). The median value of health knowledge awareness of the studied employees was significantly improved from 10.20 to 11.33(S=-2 545, P<0.05) and awareness of recommended daily intake of dietary salt increased significantly from 46.20%to 67.09%after intervention (χ2=17.85, P<0.05). Conclusion The non-communicable chronic diseases intervention program can reduce the health risk factors and improve health knowledge awareness of the studied population.

Objective To understand the affecting factors of health and health promotion needs of one company staff so as to provide evidence for effective health intervention.Methods A convenience sampling method was used to enroll subjects in this cross-sectional study.All the participants were required to fill in a questionnaire to learn their demographics,health related factors and health promotion needs in the 2 work days of June 2012 had been studied.Chi-square test and unconditional logistic regression were used for data analysis.Results More males showed higher risk of disease than females (x2 =5.394,P ＜ 0.05).In conditional logistic regression,age was the main factor affecting health (odds ratio (OR) =3.127,95％ (confidence interval) CI 1.020-9.591,P ＜ 0.05) when gender,marital status,educational level and occupation were adjusted.Dietary nutritional knowledge (69.6％) and physical check-up (64.6％) were urgent needs among those surveyed.Conclusion Staff with varied demographic characteristics have different health related factors and physical check-up may provide a tool to meet their urgent needs of health promotion.

Objectives To evaluate the association between serum cystatin C and carotid atherosclerosis.Methods Medical data,including physical examinations,serum levels of cystatin C,bilirubin,uric acid and other biochemical markers and duplex ultrasonographic scanning for the carotid artery,of 1741 healthy adults (1193 men and 548 women) who sought physical check-up at Peking Union Medical College Hospital during January 2010 and December 2011 were collected,and those with hypertension,diabetes and liver or renal function failure were excluded.Based on the results of carotid duplex uhrasonography,all the adults checked were assigned to two groups,one with thickened intima-media and atherosclerotic plaque (group A),the other with non-thickened intima-media and non-atherosclerotic plaque (group B).Difference in serum cystatin C,bilirubin,uric acid and other biochemical markers between the two groups was analyzed by using Student's t test.The correlation between the markers and carotid plaque was analyzed by using Spearman correlation analysis.The correlated factors of the carotid thickness and plaque appearance were analyzed by using unconditional Logistic regression analysis.Results Serum level of cystatin C was significantly increased in group A (t =-8.85,P ＜ 0.05).Thickness of carotid intima was positively correlated with serum cystatin C (r =0.21,P ＜ 0.05).In multivariate Logistic regression analysis,high level of serum cystatin C was not an independent risk factor of carotid atherosclerosis.Conclusion Relationship between serum cyatatin C and carotid atherosclerosis was not confirmed in our populations,which should be explored further in cohort studies.

ObjectiveTo explore the treatment and economic burden of urine incontinence (UI) in the rural elderly,and provide evidences for preventive intervention.MethodsA cluster sampling was carried out in 743 people (aged 60 years and over) from 5 villages dominated by Yanliu and Dafengshang health centers,Jixian County,Tianjin from 2007 to 2008.The information about general state of health,treatment and economic burden of disease were analyzed. Results The prevalence of UI was 33.4％ (248 cases) among the 743 elderly people.Only 12 patients (4.8％) with UI ever visited community health-care service centers or hospitals for diagnosis and treatment,among whom only 1 case visited hospital within 1 year,8 cases during 1 to 5 years,and 3 cases over 5 years.And the method of treatment was all drug therapy.Among 248 cases with UI,there were 227 cases (91.5％) without any treatment,9 cases (3.6％) buying medicines by themselves.The direct cost for treatment of UI was 80-12000 yuan RMB,including 2 cases below 100 yuan RMB,2 cases 100-1000 yuan RMB,7 cases 1001-5000 yuan RMB and 1 case over 10000 yuan RMB. Conclusions Prevalence of UI is high while hospital-visiting rate is very low,and the financial burden of disease is serious,thus intervention measures for UI should be taken for the elderly people in rural areas of Jixian County,Tianjin.