Mitogen-induced Proliferation Assay Research Articles

Immunosuppressive effect of voacamine from Voacanga africana Stapf based on SPRi experiment

Purpose: To investigate the affinity of a bis-indole alkaloid - voacamine from Voacanga Africana Stapf for IL-2Rα - and its immunosuppressive effect on concanavalin A-induced T cell proliferation and lipopolysaccharide -induced B cell proliferation in vitro.
 Methods: Surface plasmon resonance imaging (SPRi) was used to screen the target protein of voacamine, while CCK-8 kit was used to evaluate cytotoxicity. Mitogen-induced proliferation assay was carried out to assess the inhibitory effect of voacamine on Con A-induced T cell proliferation and LPSinduced B cell proliferation. The binding characteristics of voacamine were investigated using a binding model with IL-2Rα constructed based on molecular docking simulation.
 Results: Voacamine had a high-affinity for IL-2Rα with an equilibrium dissociation constant (KD) of 1.85×10-8 M. Cytotoxicity data showed that voacamine did not exhibit cytotoxicity at concentrations lower than 0.32 µM. However, it exerted significant immunosuppressive effect on B cells at a lower concentration, but had no influence on proliferation of T cells. Autodock results indicate that voacamine has a good interaction with the enzyme active site.
 Conclusion: Voacamine and its analogues exert influence on the immune system.

Extracellular Vesicles Released from Human Umbilical Cord-Derived Mesenchymal Stromal Cells Prevent Life-Threatening Acute Graft-Versus-Host Disease in a Mouse Model of Allogeneic Hematopoietic Stem Cell Transplantation.

Mesenchymal stromal cells (MSCs) are attractive agents for the prophylaxis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, safety concerns remain about their clinical application. In this study, we explored whether extracellular vesicles released from human umbilical cord-derived MSCs (hUC-MSC-EVs) could prevent aGVHD in a mouse model of allo-HSCT. hUC-MSC-EVs were intravenously administered to recipient mice on days 0 and 7 after allo-HSCT, and the prophylactic effects of hUC-MSC-EVs were assessed by observing the in vivo manifestations of aGVHD, histologic changes in target organs, and recipient mouse survival. We evaluated the effects of hUC-MSC-EVs on immune cells and inflammatory cytokines by flow cytometry and ProcartaPlex™ Multiplex Immunoassays, respectively. The in vitro effects of hUC-MSC-EVs were determined by mitogen-induced proliferation assays. hUC-MSC-EVs alleviated the in vivo manifestations of aGVHD and the associated histologic changes and significantly reduced the mortality of the recipient mice. Recipients treated with hUC-MSC-EVs had significantly lower frequencies and absolute numbers of CD3+CD8+ T cells; reduced serum levels of IL-2, TNF-α, and IFN-γ; a higher ratio of CD3+CD4+ and CD3+CD8+ T cells; and higher serum levels of IL-10. An in vitro experiment demonstrated that hUC-MSC-EVs inhibited the mitogen-induced proliferation of splenocytes in a dose-dependent manner, and the cytokine changes were similar to those observed in vivo. This study indicated that hUC-MSC-EVs can prevent life-threatening aGVHD by modulating immune responses. These data provide the first evidence that hUC-MSC-EVs represent an ideal alternative in the prophylaxis of aGVHD after allo-HSCT.

Immunosuppressive effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) in the rat

THI is a component of ammonia caramel, a widely used food colouring. The effect of THI on the immune system has been determined in the male F344 rat. THI was given in the drinking water at doses of 1, 10 and 50 mg/l (equivalent to 0.1, 1 and 5 mg/kg per day) to animals on a vitamin B6-deficient diet. After 1 week, the immune competence of the animals was assessed under continued THI treatment. No marked changes in thymus or spleen weight were observed after THI treatment, although there was an increased number of pyknotic cells in the thymic cortex, mainly engulfed by macrophages and there appeared to be a slight thinning of the cortex area. THI produced a significant loss in T and B lymphocytes in peripheral blood but not in the spleen. No change in natural killer (NK) cell activity against YAC-1 target cells was observed in the spleen. The observed increase in NK cell activity in peripheral blood was due to an increase in circulating large granular lymphocytes (LGL). Although the serum antibody titre against keyhole limpet haemocyanin (KLH) was not affected by THI treatment, B cells showed less proliferation when cultured with lipopolysaccharide. T cell function was impeded as measured in mitogen-induced proliferation assay, delayed-type hypersensitivity assay and host versus graft (popliteal lymph node) assay. The results indicate that THI is an immunosuppressor in the rat, in whom it can produce profound lymphopenia and suppression of cell-mediated immunity.

CD40 ligation induced phenotypic and functional expression of CD80 by human cardiac microvascular endothelial cells.

CD40/CD40L (gp39) interactions are known to play a central role in the function of the immune system (1). CD40 is constitutively expressed on professional antigen presenting cells, such as macrophages and dendritic cells, as well as at low levels on other cell lineages, including human umbilical vein endothelial cells (HUVECs). On antigen-presenting cells, ligation of CD40 causes expression of the costimulatory molecule CD80 (B7-1). Similar ligation of CD40 on HUVECs, however, leads to up-regulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin, but not CD80. In efforts to provide evidence that microvascular endothelial cells (MECs) are distinct from HUVECs and that the distinguishing features play a role in allograft rejection, MEC cultures were prepared from the explanted hearts of human heart transplant recipients and primary cell lines were established. These MECs were induced to express higher levels of CD40 with interferon-gamma pretreatment, co-cultured with CD40L-transfected HeLa cells, and fluorescence-activated cell sorter-assisted phenotypic studies, in addition to functional allogeneic mixed lymphocyte reaction and accessory-cell dependent mitogen induced proliferation assays were performed. CD40-CD40L interactions induced the expression of the adhesion molecules VCAM-1 and E-selectin and the costimulatory molecule CD80 but not CD86 (B7-2) on the MECs. The expressed CD80 proved functional in both allo-MLR assays and accessory-cell dependent mitogen proliferation assays. MECs are distinct from HUVECs by their potential to express VCAM-1 after interferon-gamma pretreatment and CD80 after CD40 ligation, properties which enable this cell lineage to play a central role in initiating and maintaining allograft rejection in human cardiac transplants.

Purified porcine seminal plasma protein enhances in vitro immune activities of porcine peripheral lymphocytes.

The porcine seminal plasma protein (PSP) accounts for much more than 50% of the total proteins in seminal plasma. PSP has been previously purified and its biochemical properties characterized. However, the biological functions of PSP remain to be elucidated. We hypothesize that PSP is involved in the regulation of uterine immune activity. In the current study, effects of PSP on in vitro lymphocyte activities and the presence of PSP binding sites on lymphocytes were examined. In mitogen-induced proliferation assay, lymphocytes from peripheral blood of gilts were cultured with pokeweed mitogen (PWM), phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of PSP. PSP at 50, 125, and 250 ng/well augmented PWM-induced [3H]thymidine uptake in a dose-responsive manner by 152.8 +/- 8.1%, 225.9 +/- 35.2%, and 274.8 +/- 53.6%, respectively, compared with that of control. PSP did not alter lymphocyte proliferation in the absence of PWM. Similarly, PSP had little or no effect on PHA- or Con A-induced lymphocyte proliferation. In one-way mixed lymphocyte reactions, PSP at 50, 125, and 250 ng/well enhanced [3H]thymidine uptake in a dose-responsive manner by 181.5 +/- 16.5%, 339.9 +/- 48.2%, and 600.1 +/- 84.8% of control, respectively. Using biotinylated PSP-I, PSP binding sites were localized on approximately 3-5% of the lymphocyte population. In summary, we have demonstrated that PSP itself is not a mitogen/antigen to porcine lymphocytes but that it has a stimulatory effect on lymphocyte activities initiated by PWM or surface antigens of lymphocytes. PSP may exert its functions by interacting with PSP binding sites on a subpopulation of porcine lymphocytes. The high potency of PSP on lymphocyte activities and the abundance of PSP in seminal plasma have suggested that PSP may play an important role in regulating immune responses in the porcine uterine environment.

The photodynamic effect of Victoria blue BO on peripheral blood mononuclear and leukemic cells.

The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cytometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12% vs 80% of PI-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells.

Variability in the growth sustaining capacity of medium batches

Medium batches, analysed in various spontaneous and mitogen induced proliferation assays, revealed heterogeneity in their growth promoting activity. This can critically affect test results and suggests that culture media can be a source of variability and problems in cell culture work. The manufacturers of media should broaden their quality control of medium batches.

In vitro inhibition of cellular immune responses by benzodiazepines and PK 11195. Effects on mitogen- and alloantigen-driven lymphocyte proliferation and on IL-1, IL-2 synthesis and IL-2 receptor expression.

In vitro mitogen-driven lymphocyte proliferation tests (Con A, LPS) on murine lymph node and spleen cells revealed inhibition of T and B cell stimulation by different benzodiazepines and by PK 11195, with IC50 values in the low micromolar range. T cell responses as a consequence of recognition of alloantigens, as measured in mixed lymphocyte cultures (MLC), were affected in an analogous way. In all systems, agonists at peripheral type benzodiazepine receptors (Ro 5-4864 and the non-benzodiazepine compound PK 11195) and diazepam which acts on both, central and peripheral type benzodiazepine receptors, were most potent; clonazepam, a central type agonist, proved about half as active. The central type antagonist Ro 15-1788 failed to antagonize the action of diazepam and clonazepam. Variations among cells from several congenic strains of mice were modest. Cytotoxicity could not be made responsible for drug effects. The most susceptible stage of mitogen-triggered T and B lymphocyte proliferation was found to be at incipience. Radioresistant, adherent spleen cells, upon LPS-stimulation formed only small amounts of the cytokine IL-1. Its release was affected only at very high drug concentrations. Similar small amounts of IL-1 were generated during MLC; in this case, the drugs were about 10 times less potent than in mitogen-induced proliferation assays. Peripheral agonists were more active on IL-1 synthesis. Spleen cells stimulated with Con A and cultivated with the highest concentration of diazepam and clonazepam formed markedly greater amounts of IL-2 than those cultivated in medium, while at this concentration PK 11195 allowed no formation of the lymphokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Abrogation of glucocorticoid-mediated inhibition of T cell proliferation by the synergistic action of IL-1, IL-6, and IFN-gamma.

Glucocorticoids (GCS) inhibit the transcription of multiple activation-associated cytokine genes. By Northern blot analysis now we demonstrate that antiproliferative concentrations of dexamethasone and 6 alpha-methylprednisolone block mitogen-induced IL-2 gene expression in human peripheral blood mononuclear leukocytes in a concentration-dependent fashion. In addition, using a mitogen-induced proliferation assay of human peripheral blood mononuclear leukocytes, we show that GCS-mediated anti-proliferative effects are not blocked by rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, rTNF-alpha, rTNF-beta, and rIFN-gamma, individually at 1 to 1000 U/ml, but are totally abrogated, in a concentration-dependent fashion, by the combination of rIL-1, rIL-6, and rIFN-gamma (25 to 50 U/ml for each cytokine). Thus, blockade of cytokine expression is the primary mechanism by which GCS inhibit mitogen-driven and alloantigen-induced T cell proliferation. The immunosuppressive effects of GCS are almost certainly exacted at the level of cytokine gene transcription.

Accessory function of human leukemic cell lines: properties of B and B-K562 hybrid cell lines.

The accessory function (AF) of various B cell lines has been investigated by studying their ability to replace monocytes inducing the proliferation of highly purified T lymphocytes in the presence of phytohemagglutinin. AF could be exerted by B cells from different origin, including Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL), Burkitt's lymphoma (BL)-derived lines, either EBV-positive or not, pre-B and B leukemias and lymphomas. The EBV-converted BJAB-B95 BL cells could exert an AF as efficient as BJAB cells, their EBV-negative counterparts, which demonstrates that EBV itself does not play any role in this function. The HLA-DR antigen-negative BL-K562 hybrids PUTKO and DUTKO, T51.4.1.6 cells which derived from an HLA-DR-negative variant of the T51 LCL and 721.84.5 cells from a mutagenically dissected HLA-DR-negative clone of the 721 LCL, also exerted very efficient AF. 721 LCL, PUTKO and DUTKO hybrids could produce interleukin 1 activity when triggered with 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Furthermore, addition of anti-DR monoclonal antibodies which blocked completely the AF of the B cell lines in mixed lymphocyte-tumor cell reaction did not affect the mitogen-triggered AF exerted by the same B cell lines. The induction of cellular differentiation with TPA markedly reduced the AF exerted by all B cell lines studied as well as by the hybrid cells DUTKO. By contrast, the PUTKO hybrid was unsensitive to sodium butyrate and TPA treatments, which were unable to abrogate its AF. Taken together, these data indicate that the AF in mitogen-induced T lymphocyte responses is dependent upon some precise maturational stages of accessory cells and HLA-DR antigen expression is not required for AF in mitogen-induced proliferation assays.