Objective:To explore the dynamic changes of inflammatory cytokines and T lymphocyte activation in the peripheral blood of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients during anti-retroviral therapy (ART).Methods:Two hundred and six HIV/AIDS patients with ART at clinic of the Department of Infectious Diseases in Second Xiangya Hospital, Central-South University between January 2017 and December 2019 were selected as HIV infection group. They were followed up regularly and the blood samples before treatment and at month 6, month 12, month 24 of treatment were collected. Meanwhile, 52 healthy cases were enrolled in the healthy control group and their blood samples were collected. Enzyme-linked immunosorbent assay was used to detect the plasma concentrations of interleukin (IL)-6, hypersensitive C-reactive protein (hsCRP) and tumor necrosis factor (TNF)-α. Flow cytometry was used to detect the CD3 + CD4 + T lymphocytes count and the percentage of CD4 + CD38 + T lymphocytes and CD8 + CD38 + T lymphocytes in the peripheral blood mononuclear cells. Plasma HIV RNA viral load was determined using a quantitative real-time polymerase chain reaction technique. Statistical methods used paired t test and Pearson correlation analysis. Results:The concentrations of IL-6, hsCRP and TNF-α in HIV infection group were (13.42±2.35) pg/mL, (4 012.46±1 012.35) μg/L and (51.78±11.32) μg/L, respectively, which were higher than those in healthy control group ((6.14±0.78) pg/mL, (707.21±305.76) μg/L and (19.01±6.48) μg/L, respectively). The differences were all statistically significant ( t=12.56, 16.79 and 13.45, respectively, all P<0.001). They decreased gradually after initiation of ART in HIV infection group, and returned to normal levels at month 24 of ART. CD3 + CD4 + T cells count was (256.00±65.32)/μL and HIV RNA viral load was (4.467±4.244) lg copies/mL before ART in HIV infection group, which were negatively correlated ( r=-0.625, P=0.041). The percentages of CD8 + CD38 + T lymphocytes before treatment and at month 12 or month 24 of treatment in HIV infection group were higher than those in healthy control group. The differences were all statistically significant ( t=3.85, 6.84 and 2.57, respectively, all P<0.050). The percentage of CD8 + CD38 + T lymphocytes was positively correlated with HIV RNA viral load before ART ( r=0.768, P=0.026). The percentages of CD4 + CD38 + T lymphocytes before treatment and at month 12 or month 24 of treatment in the HIV infection group were lower than those in the healthy control group, and the differences were all statistically significant ( t=6.80, 1.10, and 2.11, respectively, all P<0.050). Conclusions:HIV infection could not only cause insufficiency in immune system, but also abnormal activation of immune system, which could get better gradually with ART.

Objective To observe the expression of apoptosis related protein c-inhibitor of apoptosis protein (IAP)2 by RNA interference of human immunodeficiency virus-1 (HIV-1)vpr gene and analyze the apoptosis of Jurkat cells.Methods Vector (NC),HIV-1vpr (vpr),vpr+ pRNAT-U6.1/Neo-vpr-56 (Si56) and vpr+ pRNAT-U6.1/Neo-vpr-160 (Si160) were transfected to Jurkat cells and cultured for 48 hours.The total RNA and protein were extracted.Expression of vpr gene was detected by reverse transcription (RT)-polymerase chain reaction (PCR) to confirm the success of transfection.Expression of c IAP2 was detected by RT-PCR and Western Blot.The apoptosis of Jurkat cells was observed by flow cytometry.Results Expression of vpr gene was detected in vpr,Si56 and Si160 groups.The mRNA expression levels in Si56 and Si160 groups were significantly lower than that in vpr group,which declined 87.2％ and 82.2％,respectively (P＜0.05).The levels of c IAP2 mRNA expression in vpr,Si56 and Si160 groups were increased by 3.75,2.49 and 2.65 folds,respectively,compared to that in NC group.However,the c-IAP2 mRNA expressions in Si56 and Si160 groups were lower than that in vpr group,which declined 33.7％ and 29.5％,respectively (P ＜ 0.05).The c-IAP2 protein expression was consistent with mRNA by immunoblotting,and those in Si56 and Si160 groups were declined 42.2％ and 46.8％,respectively,compared to that in vpr group (P＜0.05).The apoptosis of J urkat cells was detected in all groups.Compared to NC group,the apoptotic rate in vpr group was increased by 1.76 folds.However,the differences of apoptosis rate among NC,Si56 and Si160 groups were not statistically significant (all P＞0.05).Compared to vpr group,the apoptotic rates in Si56 and Si160 were significantly decreased by 19.26％ and 18.05％,respectively (P＜0.05).Conclusions The expression of c-IAP2 could be downregulated by knockdown of HIV-1 vpr gene in transcription and protein levels,and the apoptosis of Jurkat cells is inhibited.

Objective The plasma levels of interleukin 6 (IL-6),high sensitive C reactive protein (hs-CRP) and the level of T cell activation were detected in the peripheral blood inflammatory factors of acquired immunodeficiency syndrome (AIDS) patients,and the relationship with opportunistic infection and prognosis was analyzed.Methods 79 human immunodeficiency virus (HIV)-positive/aids cases from May 2014 to January 2015 in first hospital of Changsha were enrolled in the study.They were divided into three groups:HIV-infected without opportunistic infections group (n =20),HIV-infected with infections group (n =43,including HIV-infected with tuberculosis group,HIV-infected with merge fungus group.HIV combined hepatitis C),death group (n =16).Serum IL-6 and the concentration of hs-CRP were detected by enzyme-linked immunosorbent assay (ELISA).Flow cytometry was used to test the CD3 + CD4 + cell count,the percentage of CD4 + CD38 + cell and CD8 + CD38 + cell in peripheral blood mononuclear cell (PBMC).Compare the differences among the three groups.Results The results showed that:the concentration of hsCRP and IL-6 in peripheral blood of HIV death group was significantly higher than that in other three groups (P ＜ 0.05).The concentration of hs-CRP and IL-6 in the peripheral blood of the HIV-infected with tuberculosis group and HIV-infected with merge fungus group were significantly higher than that in the HIV-infected without opportunistic infections group (P ＜ 0.05),and the hs-CRP in the peripheral blood of the HIV combined with the hepatitis C group was higher than that in the HIV-infected without opportunistic infections group (P ＜ 0.05).The number of CD3,CD4T lymph nodes in the death group and the combined opportunistic infection group was significantly lower than that in the HIV-infected without opportunistic infections group (P ＜ 0.05).The HIV-RNA expression in peripheral blood of the death group and the combined opportunistic infection group was significantly lower than that in the HIV-infected without opportunistic infections group (P ＜ 0.05).The expression of CD8+CD38+ on PBMC in the HIV-infected without opportunistic infections group was significantly lower than that in the death group,the tuberculosis group,the fungus group and the hepatitis C group (P ＜ 0.05).The expression of CD4+ CD38 + on PBMC in the HIV-infected without opportunistic infections group was higher than that in the death group (P ＜ 0,05).Conclusions The concentration of inflammatory cytokines (IL-6,hs-CRP) and the expression level of T cell surface CD8 + CD38 + related to immune activation were associated with opportunistic infection and prognosis of AIDS.

Objective: To investigate the relationship between M2-polarized macrophages and early response in multiple myeloma and its molecular mechanism. Methods: Two hundred and forty bone marrow biopsy tissue were collected and M2-polarized macrophages were stained by anti-CD163 monoclonal antibody. In vitro M2-polarized macrophages were derived from human peripheral blood mononuclear cell or THP-1 cells and identified by flow cytometry. Two myeloma cell lines RPMI 8226 and U266 were co-cultured with M2 macrophages using a transwell system. We measured myeloma cells proliferation through CCK-8 method and the pro-inflammatory cytokines expression (TNF-α and IL-6) by ELISA. Real time PCR was applied to measure chemokines (CCL2 and CCL3) , chemokine receptors (CCR2, CCR5) , VEGF and their receptors. In addition, flow cytometry was used to analyze the apoptosis of myeloma cells induced by dexamethasone. Results: ①Patients with high percentage of M2 macrophage involvement in bone marrow showed poorer response (23.9% versus 73.0%, χ²=60.31, P<0.001). ② In vitro the proliferation of RPMI 8226 cells (P=0.005 at 24 h, P=0.020 at 36 h) or U266 myeloma cells (P= 0.030 at 24h, P=0.020 at 36h) co-cultured with M2-polarized macrophages was higher than control group. ③In vitro the apoptotic rate of RPMI 8226 cells (29.0% versus 71.0%, t=4.97, P=0.008) or U266 myeloma cells (24.9% versus 67.7%, t=6.99, P=0.002) co-cultured with M2-polarized macrophages was lower than control group. ④ In vitro M2-polarized macrophages promoted myeloma cells secreting higher level of IL-6, TNF-α and higher expression of CCL2, CCL3, CCR2, CCR5, VEGFA, VEGFR-1,-2 compared with the non-macrophage co-culture system. Conclusion M2-polarized macrophages promote myeloma cells proliferation and inhibit apoptosis through a very complex mechanism involving pro-inflammatory cytokines IL-6 and TNF-α, chemokines and related receptors such as CCL2, CCL3, CCR2, CCR3, and VEGF as well as related VEGFR.

Objective To investigate the regulatory effects of IFN-γon Treg cells from HIV/AIDS patients receiving highly active antiretroviral therapy (HAART) for one year.Methods Thirty HIV/A1DS patients whose CD4+T cells were below 350/μ1 were recruited for HAART therapy.Blood samples were collected at the time points of 0,24,48 weeks after HAART.PBMCs were isolated and randomly divided into two culture groups.One group was cultured directly in medium and another group was co-cultured with IFN-γ (40 pg/ml).The supernatants and cells were separated after 5 days of culture for analysis.The concentrations of IL-12 and CD4+CD25+Foxp3 Treg cells were measured by ELISA and flow cytometry,respectively.Results The levels of IL-12 in the supernatants from the culture without IFN-γ at time points of 0,24,48 weeks after HAART were lower than those from the co-cultured group [(37.02±12.76) vs (41.79± 15.02),t=2.336,P=0.03; (41.76±17.01) vs (47.2±14.26),t=2.702,P=0.014; (48.01± 11.84) vs (53.44± 11.30),t =3.14,P =0.003].The percentages of CD4+ CD25 + Foxp3 Treg cells in CD4+ T cells from the direct-cultured group were higher than those from the co-cultured group at the three time points [(10.41±1.10)％ vs (2.40±1.11)％,t=13.89,P=0.000; (8.33±2.03)％ vs (1.99± 0.86)％,t=12.93,P=0.000; (5.65±1.55)％ vs (1.32±0.73)％,t=10.61,P=0.000].Moreover,the results within the same group at the time points of 0,24,48 weeks upon HAART were also significantly different.Conclusion With the interference of HAART,IL-12 levels were increased,while CD4+CD25+ Foxp3 Treg cells were decreased in patients with HIV/AIDS.IFN-γ plays an important role in this process.

OBJECTIVE: To observe the dynamic changes of 3 types of viral reservoir cells (NK cells, T lymphocytes and monocytes), and its relationship with treatment effect in Chinese HIV-1 infected patients receiving highly active antiretroviral treatment (HAART) for 2 years. METHODS: A total of 40 chronic HIV-1-infected adults who initiated HAART were enrolled in this study and followed up for 2 years. Peripheral whole blood was obtained from each patient at baseline (0 month), 6, 12, 18 and 24 months. Real-time fluorescent quantitative PCR was used to detect the HIV-1 RNA in the plasma and HIV-1 DNA in NK cells, T lymphocytes and monocytes. All the data were statistically analyzed. RESULTS: CD4 count increased with the decrease of the viral load during HAART. After HAART initiation, HIV-1 DNA showed a significant decrease in NK cells, T lymphocytes and monocytes. The HIV-1 DNA from T lymphocytes, NK cells and monocytes correlated positively with the HIV- 1 RNA (P<0.05) while NK cells and T lymphocytes correlated negatively with CD4+ T cell count. However we did not find significant correlation between CD4+ T cell count and HIV-1 DNA in monocytes at the baseline of HAART. CONCLUSION This study found that NK cell was an important HIV cellular reservoir besides T lymphocytes and monocytes. T lymphocytes may be the main long lasting HIV reservoir. HIV-1 proviral DNA may play an important role in the efficacy of treatment and monitoring the disease progression.
Adult ; Antiretroviral Therapy, Highly Active ; DNA, Viral ; analysis ; Female ; HIV Infections ; drug therapy ; virology ; HIV-1 ; genetics ; Humans ; Killer Cells, Natural ; virology ; Male ; Middle Aged ; Monocytes ; virology ; RNA, Viral ; blood ; T-Lymphocytes ; virology ; Viral Load

Objective To observe the Th17 and regulatory T cells(Tr) equilibrium state as well as their changes of tuberculosis patients in six-month's anti-Tuberculosis treatment.Methods Select thirty-two tuberculosis patients received anti-Tuberculosis treatment while thirty-two healthy volunteers as controls.Flow cytometry was used to analyze Th17 and Tr cells in venous blood at the time of pre-therapy,3th,6th month.Results The ratio of Th17 cells in CD4 cells in tuberculosis patients and volunteers were (1.10±0.39)％,(2.50±1.03) ％,(3.90±1.34) ％,(4.50±1.52)％,respectively; the ratio of Tr cells were (9.17±3.26)％,(6.85±2.73)％,(5.46±1.69)％,(4.35±0.86)％,respectively.Conclusion Tuberculosis could make Th17 cells and Tr cells lost their balance,but the immune equilibrium state may gradually recover after anti-tuberculosis.The change of the amount of immune cells was likely to be the reference indexes to observe the progress of tuberculosis and the treatment effect of anti-tuberculosis.

Objective The objective of this study was to investigate the course of certain common gamma cytokines ( IL-2 and IL-7 ) and their role on the control of the viral infection in a short term antiviral therapy.Methods A total of 35 adults with chronic HIV infection,responding to combined antiretroviral therapy (cART) guideline criteria were enrolled in this one year follow-up study.After signing an informed consent,20 ml blood were collected from each patient at base line,week 0,week 24 and week 48.1 ml serum collected from each patient was kept at -80 * C until use.Serum concentration of IL-2 and IL-7 was determined using ELISA kit from ebioscience Beijing.CD4 and CD8 cells were counted and quantified using flux cytometry,and serum HIV RNA was quantified using real time PCR.Results All patients had a mean baseline IL-2 level [ (9.67 ± 2.6 ) pg/ml ]lower than the controls [ ( 27.36 ± 5.05 ) pg/ml ].After treatment for 48 weeks,IL-2 increased[ ( 19.8 ± 3.3 ) pg/ml ].However,the mean baseline 1L-7 [ ( 81.74± 20.47 ) pg/ml ]in patients was higher than controls [ ( 2.06 ± 1.52 ) pg/ml ].After treatment for 48 weeks,IL-7 decreased [ (8.36 ± 2.16)pg/ml ].IL-2 showed a significant increase and positive correlation with CD4 cells after HAART initiation (0week:R =0.21,P =0.063,24week:R =0.24,P =0.033,48week:R =0.19,P =0.103; IL-7 showed a significant decrease after HAART initiation but it did not show correlation with CD4 cells.We noted there was a negative correlation between IL-2 and CD4 count in HAART baseline (R =0.28,P =0.012 ),but no correlation between IL-7 and CD4 count from 6 month after HAART.IL-2 showed negative correlation with HIV RNA ( R =- 0.17,P =0.032),but IL-7 showed a relationship with the HIV RNA Conclusions The increase of IL-2 coupled with the decrease of IL-7 revealed a partial restoration of immune response during HAART.However,the absence of relationship with HIV RNA suggested that these cytokines might not be directly involved in the reduction of viral load.

Objective To observe that antiretroviral efficacy, immune reconstitution of four-year highly active antiretroviral therapy (HAART), and evaluate its side effect in Chinese HIV-1-infected patients. Methods A total of 258 HIV-1 infected patients, given HAART regimens composed of two nucleoside reverse transcriptase inhibitor (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) for mean 51.5 months, measured HIV RNA viral load(VL) and the counts of CD4+ T cell,CD8+ T cell at the baseline and 6, 12, 24, 36 and 48 months after HAART initiation, respectively,monitoring side effect, blood routine, main biochemical parameters, and other disadvantageous accidents during the 51.5-month treatment. Results Plasma HIV-1 RNA level was determined by fluorescent quantitative polymerase chain reactions (FQ-PCR) at the baseline and 6, 12, 24, 36 and 48 months after starting HAART, and showed 5.27, 2.97, 2. 74, 2. 62, 2. 67 and 2.75 lg (copies/ml), respectively. The counts of CD4+ T cell from (127±63) cells/μl at the baseline increased to (190±115), (248±93),(269±127), (296 ± 156) and (317 ± 195) cells/μl at 6, 12, 24, 36 and 48 months after starting HAART. A total of 149 treated patients (57.8%)had gastrointestinal side effects, peripheral polyneuropathy, various rashes, central nervous system disorders, fever or baldness. Twenty-two patients changed one of three medicines to another because toxicity. Sixteen changed the regimen to the second line HAART for lactic acidosis or other serious toxicities. Conclusions A total of 258 HIV-1 infected Chinese patients treated with two NRTI and one NNRTI as first line HAART regimen during mean 51.5 months,showed a good antiretroviral efficacy and immune reconstitution, but a few site-effects at the parts of patients. It is necessary to treat adverse effect and change HAART regimen for severe toxicity in time.

Objective To observe the Th17, IL-17 and Tr cells equilibrium state as well as their changes of HIV infected or AIDS suffered patients in one-year HAART treatment. Methods Select 33 HIV/AIDS patients received HAART treatment while 33 healthy volunteers as controls. Flow cytometry was used to analyze Th17 and Tr cells in venous blood at the time of pre-therapy, 6th, 12th month when IL-17 levels in serum are tested by ELISA. Results The ratio of Th17 cells in CD4 cells in HIV/AIDS patients and volunteers were (1.20±0.37)%, (2.50±1.03)%, (3.70±1.56)%, (4.70±1.43)%, respectively; The ratio of Tr cells were (9.16±3.33)%, (7.19±2.91)%, (5.53±1.88)%, (4.43±0.97)%, respectively; The levels of IL-17 in serum were (5.3±2.5) pg/ml, (7.7±2.4) pg/ml, (10.4±3.1) pg/ml, (17.7±6.6) pg/ml respectively. The Th17 cells' level was positively correlative with the amount of CD4 cells, negatively correlate with the count of viral load. However, the Tr cells level is positively correlative with the count of viral load, negatively relate to the quantity of CD4 cells. Conclusion HIV could make IL-17, Th17 cells and Tr cells lost their balance, but the immune equilibrium state may gradually recover after HAART treatment. Which indicates the IL-17, Th17 cells and Treg cells may play an important role in the pathogenesis of AIDS, and they are likely to be the effective indexes to observe the progress of AIDS and the treatment effect of highly active antiretroviral therapy(HAART).