Objective To clarify the mechanism that HIV infection mediates mitochondrial damage of CD4⁺ T lymphocytes (CD4⁺ T cells) through mitogen-activated protein kinase (MAPK) pathway. Methods From October 1st, 2022 to March 31st, 2023, 47 HIV-infected people who received antiretroviral therapy (ART) for 4 years were recruited, including 22 immune non-responders (INR) and 25 responders (IR); and 26 sex and age-matched control participants (HC) who were negative for HCV, HBV, and HIV infections. The immune parameters were analyzed by flow cytometry. Finally, peripheral blood mononuclear cells (PBMCs) from HC or HIV patients were treated with MAPK pathway inhibitor SB203580, and the changes of mitochondrial function of CD4⁺ T cells were observed. Results Compared with HC group, the proportion of CD4⁺ T cells in PBMCs in INR group and IR group was significantly lower, and the proportion of CD4⁺ T cells in PBMCs in INR group was significantly lower than that in IR group. In addition, the proportion of naive (CD45RA⁺CD27⁺)T cells in PBMCs in INR group was significantly lower than that in HC group and IR group. Compared with HC group and IR group, the proportions of CD4⁺PD-1⁺, CD4⁺Av⁺ and CD4⁺MO⁺ in PBMCs in INR group and the proportions of CD45RA⁺CD27⁺PD-1⁺, CD45RA⁺CD27⁺Av⁺, CD45RA⁺CD27⁺MO⁺ in CD4⁺ T cell subsets increased significant. Compared with HC-con group, the basal respiration, maximal respiration and adenosine triphosphate(ATP) production of CD4⁺ T cells in HIV-con group decreased significantly, and JC-1 (green/red) in CD4⁺ T cells increased significantly. Compared with HIV-con group, the basal respiration, maximal respiration, ATP production and respiratory potential of CD4⁺ T cells in HIV-SB203580 group increased significantly, and the JC-1 (green/red) in CD4⁺ T cells decreased significantly. Conclusion Abnormal activation of the MAPK signaling pathway is observed in HIV patients receiving ART treatment, especially in CD4⁺ T cells of INR patients, which may lead to impaired mitochondrial function and abnormal CD4⁺ T cell homeostasis.
Humans ; HIV Infections/immunology* ; Male ; Mitochondria/drug effects* ; Female ; CD4-Positive T-Lymphocytes/metabolism* ; Adult ; Middle Aged ; MAP Kinase Signaling System/drug effects* ; Pyridines/pharmacology* ; Imidazoles/pharmacology* ; Leukocytes, Mononuclear/immunology*

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects

OBJECTIVETo explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol.

METHODSThe value of 50% inhibition concentration (IC₅₀) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry.

RESULTSResveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target.

CONCLUSIONResveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; metabolism ; Stilbenes ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.
Apoptosis ; B7-2 Antigen ; immunology ; Cell Survival ; Flow Cytometry ; HL-60 Cells ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; drug effects ; Leupeptins ; pharmacology ; Lymphocyte Culture Test, Mixed ; Proteasome Inhibitors ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

OBJECTIVETo observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.

METHODSLeukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).

RESULTSThe optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).

CONCLUSIONSThe RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Coculture Techniques ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; virology ; Diterpenes ; pharmacology ; Fetal Blood ; cytology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; immunology ; Influenza, Human ; drug therapy ; immunology ; virology ; Interferon-beta ; genetics ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; immunology ; virology ; Macrophages ; drug effects ; virology ; NF-kappa B ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; immunology ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; genetics ; immunology

This study was aimed to investigate the effect of dexamethasone (Dex) on immunosuppressive ability of mesenchymal stem cells (MSC) during expansion and differentiation of MSC. MSC were cultured in 96-well flat-bottom plates. Proliferation assays were performed by using the BrdU colorimetric ELISA Kit. To explore the effect of Dex on MSC immunosuppressive ability, MSC were firstly cultured in complete culture medium for 14 d with Dex (10 nmol/L), and then, peripheral blood mononuclear cells (PBMNC) were co-cultured with MSC in 96-well flat-bottom plates for 3 d. Phytohemagglutinin A (PHA, 10 µg/ml) was used to stimulate activation of PBMNC. The concentrations of IFN-γ in culture supernatants was detected by ELISA. The results indicated that there was no obvious difference in representative phenotypes of MSC between experimental and control groups after MSC were treated with low concentration of Dex (10 nmol/L) for 14 d, but the suppression of Dex-treated MSC on lymphocyte activation in same concentration of cells was significantly reduced as compared with control group. After the Dex-treated MSC were co-cultured with IFN-γ for 12 h, the immunoregulatory ability of MSC was recovered in a certain degree. It is concluded that the Dex impairs the immunosuppressive ability of MSC, the IFN-γ can protect and reverse the immunosuppressive ability of MSC impaired by Dex, so that, when the immunoregulatory activity of MSC is investigated, it is necessary to avoid adding Dex in the culture medium.
Cells, Cultured ; Dexamethasone ; adverse effects ; Humans ; Immune Tolerance ; drug effects ; Interferon-gamma ; immunology ; Leukocytes, Mononuclear ; Lymphocyte Activation ; immunology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology

OBJECTIVETo study the effects of TNF-α on ICAM-1 and LFA-1 expression in peripheral blood mononuclear cells (PBMC) of children with febrile seizures (FS).

METHODSSixteen children with FS and 16 age- and gender-matched healthy children were enrolled. The samples of PBMC from FS children were randomized into two groups with or without TNF-α treatment (TNF-α concentration 1.0 ng/mL). PBMC were purified and cultured with a conventional method in vitro. The expression of ICAM-1 and LFA-1 in PBMC was determined by flow cytometry (FCM).

RESULTSICAM-1［(20±9)% vs (14±7)%)］and LFA-1［(43±16)% vs (30±16)%］expression in PBMC in the untreated FS group was significantly higher than that in the normal control group (P<0.05). Compared with the untreated FS group, the treatment with TNF-α remarkably increased the ICAM-1 expression［(27±11)%］(P<0.05). PBMC LFA-1 expression［(52±21)%］in the TNF-α-treated group was higher than that in the untreated FS group, although there were no statistical differences between the two groups.

CONCLUSIONSTNF-α treatment may increase LFA-1 and ICAM-1 expression in PBMC of children with FS.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Intercellular Adhesion Molecule-1 ; blood ; Leukocytes, Mononuclear ; chemistry ; drug effects ; Lymphocyte Function-Associated Antigen-1 ; blood ; Male ; Seizures, Febrile ; immunology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo study the effects of Radix Astragali on serum cytokines IL-1beta, TNFalpha and antigen expression of peripheral blood mononuclear cells (PBMCs) in patients with Graves disease (GD).

METHODSEighty GD patients at their first visit were randomly assigned to the methimazole (MMI) group (Group A) and the MMI combined Radix Astragali group (Group B), 40 in each. The improvement of clinical symptoms and thyroid functions were observed after one-month treatment. The serum IL-1beta and TNF-alpha levels in the peripheral blood were determined using radioimmunoassay. The expression levels of surface antigen CD80, CD54, and HLA-DR of PBMCs were detected using flow cytometry.

RESULTSThe improvement of the thyroid gland function was similar in the two groups. There was no obvious change in the levels of autoantibody TGAb or TPOAb of the two groups. Symptoms such as fear of heat, hidrosis, palpitation, and so on were more obviously improved in Group B than in Group A (P < 0.05). The serum IL-betaP, TNFalphaa, CD00 levels in the peripheral blood were all improved in the two groups after treatment when compared with before treatment ( P < 0.05 or P < 0.01). But the serum levels of IL-beta and TNFalpha decreased more obviously in Group B than in Group A ( P < 0.05). The expression of CD54 decreased more obviously in Group B (P < 0.01), showing statistical difference when compared with Group A at the same time point (P < 0.05).

CONCLUSIONRadix Astragali could significantly relieve the clinical symptoms such as hidrosis and palpitation, regulate the immune function of GD patients, playing an important role in the adjuvant therapy for GD.

Adult ; Astragalus Plant ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Graves Disease ; blood ; drug therapy ; immunology ; HLA Antigens ; metabolism ; Humans ; Interleukin-1beta ; blood ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Methimazole ; therapeutic use ; Middle Aged ; Tumor Necrosis Factor-alpha ; blood

BACKGROUNDIt is well known that dehydration can impair bodily functions. To evaluate the impact of hydration status under ambient environmental temperature on the immune system, 25 male collegiate wrestlers were recruited to undergo an experimental dehydration program.

METHODSThirteen subjects had controlled diets with individual energy requirements to prevent body mass loss and restricted water intake to cause 4.52% dehydration; they formed the dehydrated group (DE). These subjects developed a urine specific gravity of about 1.030 in 84 hours. Twelve other subjects had no water restriction and maintained their total body weight comprised the euhydrated group (EU). Peripheral blood monocytes (PBMNC) were isolated after dehydration to perform immune response testing by being incubated with a polysaccharide fraction from fu-ling, Poria cocos (polysaccharide fraction from Poria cocos, PCPS, 1 - 30 £g/L), to prepare a conditioned medium termed conditioned medium of PBMNC stimulated by PCPS (PCPS-MNC-CM). More PCPS (25 µg/L) was needed in the DE group to prepare the PCPS-MNC-CM, which was assayed with a growth inhibitory curve for treated U973 cells.

RESULTSThe treated U937 cells, incubated together with PCPS-MNC-CM from the DE group, exhibited a much lower nitroblue tetrazolium (NBT) positive value of (63.7 ± 4.7)%. The concentration of interferon-gamma (IFN-γ), interleukin (IL)-1β and tumor necrosis factor (TNF)-α in PCPS-MNC-CM from subjects after dehydration was much lower than in the CM from the EU group.

CONCLUSIONThe immune response to PCPS in the DE group was lower than in normally hydrated subjects.

Adolescent ; Adult ; Dehydration ; physiopathology ; Drugs, Chinese Herbal ; chemistry ; Fever ; Humans ; Immunologic Factors ; Interferon-gamma ; metabolism ; Interleukin-1beta ; metabolism ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Male ; Polysaccharides ; chemistry ; immunology ; Tumor Necrosis Factor-alpha ; metabolism ; U937 Cells ; Universities ; Wolfiporia ; Wrestling ; Young Adult