Determination of human T cell activity in response to allogeneic cells and mitogens. An immunochemical assay for gamma-interferon is more sensitive and specific than a proliferation assay

T lymphocytes proliferate and secrete lymphokines in response to allogeneic cells, mitogens and other stimuli. Cell proliferation as measured by [3H]thymidine ([3H]Tdr) incorporation into DNA has been routinely used to determine T cell responses in research and clinical laboratories. We have compared the sensitivity of an immunoradiometric assay (IRMA) for human gamma-interferon (IFN-gamma) (Chang et al., 1984), with that of the conventional [3H]Tdr incorporation assay in the measurement of T cell responses to antigens and mitogens in culture. Peripheral blood mononuclear cells (PBMs) were incubated in the presence and absence of phytohemagglutinin (PHA) or mononuclear cells from another individual for various periods of time. The culture fluids were collected for determining IFN-gamma and the cells were assayed for [3H]Tdr incorporation. Results of measurements were expressed in terms of stimulation indices. Both IFN-gamma secretion and thymidine incorporation were measurable in mixed lymphocyte cultures after incubation for 3 days, and in PHA stimulated culture after 24 h of incubation. The stimulation indices reflecting increased gamma-interferon were found to be more pronounced and more consistent than those of [3H]Tdr incorporation.     

Effects of different protein supplements on mitogen responses of human peripheral blood lymphocytes

We evaluated the effects of 6 supplements often used in human lymphocyte cultures, including fetal calf serum, autologous human serum, pooled human AB serum, hypogammaglobulinemic human serum, bovine serum albumin and human serum albumin. Lymphocyte proliferation of unstimulated and mitogen activated peripheral blood mononuclear cells was measured by [³H]thymidine incorporation. The responses of cells stimulated with the T-cell mitogen phytohemagglutinin-P were significantly lower when cultured in bovine serum albumin supplemented media, but were otherwise not supplement dependent. In contrast, responses of cells stimulated with the B-cell mitogens Cowan I strain of S. aureus and antisera against the μ or δ chain of human immunoglobulin were significantly effected by supplement. Cultures containing fetal calf serum and bovine serum albumin had high background responses without a proportional rise in cellular proliferation when B-lymphocyte-specific mitogens were utilized. Autologous human serum and pooled human AB serum contained immunoglobulin which interacted with each of the B cell mitogens, thus limiting their usefulness as in vitro supplements. Cells grown in human serum albumin supplemented media had minimal background and high stimulated responses to B-cell mitogens. These results indicate that human serum albumin is an optimal supplement for in vitro human lymphocyte proliferative assays since it supports high stimulated cell responses with low background activity, is devoid of immunoglobulin and had minimal variability among lots.     

New Lymphocyte Stimulating Monocot Lectins from Family Araceae

Three monocot lectins from underground tubers of plants belonging to the family Araceae were investigated for their mitogenic potential towards human peripheral blood lymphocytes. All the three lectins turned out to be potent mitogens in the [³H]-thymidine uptake assay. Gonatanthus pumilus lectin was mitogenic at an optimum concentration of 25 μg/ml while Alocasia indica and Sauromatum guttatum lectins were most effective at a concentration of 50 μg/ml. [³H]-thymidine incorporation studies further revealed that the lectins were T-cell mitogens and did not induce any appreciable DNA synthesis in B-enriched lymphocytes. The proliferation kinetic studies detected maximum incorporation on day 3 and the mitogenic response was shown to be inhibited by asialofetuin in a concentration-dependent manner.     

Immunotoxic effects of gold and silver nanoparticles: Inhibition of mitogen-induced proliferative responses and viability of human and murine lymphocytes in vitro

Understanding the effects of nanoparticles (NP) on immune cell functions is essential in designing safe and effective NP-based in vivo drug delivery systems. The immunomodulatory potential of gold nanoparticles (GNP) and silver nanoparticles (SNP) was investigated in vitro using murine splenic and human peripheral blood lymphocytes (PBL) in terms of effects on viability and mitogen-induced proliferation. Hydrodynamic size and number of NP were characterized using NP tracking analysis (NTA); modal diameters of GNP and SNP were 28 (±1.5) and 66 (±?2.7) nm, respectively, with a unimodal distribution. Lymphocytes were incubated with GNP or SNP in the presence/absence of B- or T-cell mitogens and proliferative responses then determined using [³H]-thymidine incorporation. Concanavalin A (T-cell-specific) and lipopolysaccharide- (B-cell-specific) stimulated responses of murine splenic lymphocytes, as well as phytohemagglutinin (T-cell-specific) and pokeweed mitogen- (B-and T-cell specific) induced responses of human lymphocytes, were significantly inhibited by GNP (25–200?μg/ml) and SNP (12.5–50?μg/ml). However, [³H]-thymidine incorporation by unstimulated lymphocytes was unaffected in the presence of GNP or SNP. Viability of lymphocytes was determined using trypan blue dye exclusion and was significantly inhibited only at 200 μg GNP/ml and 25 or 50?μg SNP/ml. As mitogen responses are most useful to provide supportive mechanistic information on primary immunotoxicologic functional observations, and so far more comprehensive data (in vivo and in vitro) is still needed, the results nevertheless suggest to us that GNP and SNP might potentially be able to modulate immune responses by impacting on lymphocyte activation.     

Kinetics of inhibition of mitogen-induced proliferation of human lymphocytes by alpha 2-macroglobulin in serum-free medium

The effect of alpha 2-macroglobulin (alpha 2M) on the ability of human lymphocytes to proliferate in response to stimuli by 3 mitogens, concanavalin A, phytohaemagglutinin and pokeweed mitogen was investigated in in vitro lymphocyte cultures in serum-free medium. The following experiments were performed: 1. lymphocytes were treated with alpha 2M prior to stimulation with the mitogens; 2. alpha 2M and mitogens were added simultaneously to the lymphocyte cultures; and 3. alpha 2M was added to the lymphocyte cultures after they were stimulated with the mitogens. In all cases, alpha 2M was found to inhibit mitogen induced proliferation of the lymphocytes as evaluated by (6-3H)-thymidine uptake of the cells. The mechanisms involved in the inhibition of lymphocyte proliferation by alpha 2M are discussed.     

Lymphocyte proliferation in response to exercise

Lymphocyte proliferative responses are often used to evaluate the functional capacity of the immune system in response to exercise. Blood mononuclear cells (BMNC) are stimulated in vitro with polyclonal mitogens and the incorporation of ³H-thymidine into the DNA reflects cell proliferation. The BMNC are most often stimulated with either phytohaemagglutinin (PHA), poke weed mitogen (PWM), concanavalin A (Con-A), interleukin-2 (IL-2), or purified derivative of tuberculin (PPD). The literature concerning lymphocyte proliferation and exercise is reviewed with respect to the type and intensity of exercise, and also the effect of training status. The proliferative responses to exercise are highly heterogeneous, the most consistent finding being that PHA-stimulated cell responses decrease during exercise which may reflect a decreased fraction of CD3+ cells. In contrast, reduced, elevated or even unchanged lymphocyte proliferative response to PHA, PWM, Con-A, IL-2 and PPD have been demonstrated in the recovery period following exercise. Also variable responses are present in trained athletes compared to less fit subjects. Even though this may reflect that the time of ³H-thymidine incorporation into lymphocytes varies, we conclude that a functional evaluation of the immune system in response to exercise cannot be based solely upon measurements of lymphocyte proliferation.     

Cellular immunity to bacteria: impairment of in vitro lymphocyte responses to Pseudomonas aeruginosa in cystic fibrosis patients.

Lymphocyte responses to the mitogens phytohemagglutinin and concanavalin A and to Streptococcus pyogenes, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa were evaluated in patients with cystic fibrosis and in normal individuals. Lymphocyte proliferation in vitro was stimulated by gentamicin-killed whole bacteria, and the proliferative response was measured by [3H]thymidine incorporation. The in vitro lymphocyte responses to antibiotic-killed bacterial reached maximum thymidine incorporation after 5 days in culture and followed a unimodal dose-response curve for each of the bacteria studied. A significant specific incapacity to respond to P. aeruginosa was detected in cystic fibrosis patients with advanced clinical disease.     

Suppression of murine lymphocyte proliferation induced by a small RNA purified from theTaenia solium metacestode

A substance fromTaenia solium metacestodes that decreases lymphocyte proliferation induced by concanavalin A was isolated. The molecular weight of this substance was estimated to be slightly more than 1,450 Da. Crude metacestode factor was fractionated through a Bio-gel P-6 column. Peak 1 showed suppressive activity. After incubation with RNase the substance lost its activity. Incubation of this material with trypsin or papain increased its suppressive activity. It was stable at boiling temperature for 10 min. The incubation of this substance with murine macrophages had no effect on [³H]-thymidine uptake by cocultured fresh splenic lymphocytes stimulated with concanavalin A. Conversely, cocultures of lymphocytes pretreated with the substance and fresh splenic lymphocytes showed a decreased incorporation of [³H]-thymidine. These results suggest that this substance is a RNA-peptide molecule whose RNA moiety accounts for its suppressive activity. The findings also suggest that in vivo the factor may be a modulator of the immune response.     

Ambient pressure stimulates immortalized human aortic endothelial cells to increase DNA synthesis and matrix metalloproteinase 1 (tissue collagenase) production

In the present study, we investigated the effect of ambient pressure on [³H]-thymidine incorporation and on the production of matrix metalloproteinase 1 (tissue collagenase/proMMP-1) using human aortic endothelial cells immortalized with simian virus 40 (SE-1). Incubation of cells at ambient pressures of 50 and 100 mmHg for 24 h slightly increased [³H]-thymidine incorporation when directly compared with normal culture conditions. The amount of [³H]-thymidine incorporated in SE-1 reached a maximum at 150 mmHg, while a further increase in pressure to 200 mmHg decreased incorporation. The same ambient pressure slightly stimulated human aortic intimal smooth muscle cells (SMC) to increase [³H]-thymidine incorporation but not medial SMC. Immunoblot analysis also showed that ambient pressure, ranging from 50 to 200 mmHg, like 12-O-tetradecanoyl-phorbol-13-acetate stimulated SE-1 to produce proMMP-1, an effect not seen with either intimal or medial SMC. The amount of proMMP-1 produced also reached a maximum level at 150 mmHg. We postulate that human endothelial cells are ambient pressure sensitive and that relatively lower ambient pressures play an important role in the growth of endothelial cells, while higher pressures injure endothelial cells, resulting in the initiation of atherosclerosis. This cell line may prove useful in the investigation of both the physiological and pathological roles of blood pressure on endothelial cell function.     

New Lymphocyte Stimulating Monocot Lectins from Family Araceae

Three monocot lectins from underground tubers of plants belonging to the family Araceae were investigated for their mitogenic potential towards human peripheral blood lymphocytes. All the three lectins turned out to be potent mitogens in the [³H]-thymidine uptake assay. Gonatanthus pumilus lectin was mitogenic at an optimum concentration of 25 μg/ml while Alocasia indica and Sauromatum guttatum lectins were most effective at a concentration of 50 μg/ml. [³H]-thymidine incorporation studies further revealed that the lectins were T-cell mitogens and did not induce any appreciable DNA synthesis in B-enriched lymphocytes. The proliferation kinetic studies detected maximum incorporation on day 3 and the mitogenic response was shown to be inhibited by asialofetuin in a concentration-dependent manner.     

Lymphocyte subpopulation profiles and mitogen kinetics in immunological deficiency testing

The correlation between lymphocyte proliferative responses to mitogens and T4/T8 ratios was analyzed in a cross section of patients who either were in a high-risk group for HTLV-III infection or fulfilled clinical criteria for acquired immune deficiency syndrome (AIDS). The patient results showed that, correlated with decreased T4/T8 ratios, there was a decrease in mitogen responsiveness first to pokeweed mitogen (PWM), followed by concanavalin A (Con A) and then phytohemagglutinin (PHA). Parallel to this decrease there was a shift toward higher concentrations of mitogens needed for optimal proliferation. In comparison, depletion of T4⁺ lymphocytes from normal healthy controls also decreased lymphocyte proliferative responses to all three mitogens but shifted the amount of mitogen needed for optimal proliferation toward lower concentrations. The differences in mitogen-induced proliferation between patients and healthy controls suggest a model whereby there is a functional defect(s) in mitogen responsiveness of the remaining T4^– lymphocyte population that can be overcome when higher concentrations of mitogen are used.     

Measurement of T-lymphocyte responses in whole-blood cultures using newly synthesized DNA and ATP

The proliferative response is most frequently determined by estimating the amount of [(3)H]thymidine incorporated into newly synthesized DNA. The [(3)H]thymidine procedure requires the use of radioisotopes as well as lengthy periods of incubation (>72 h). An alternative method of assessing T-lymphocyte activation in whole-blood cultures involves the measurement of the nucleotide ATP instead of [(3)H]thymidine incorporation. In addition, the Luminetics assay of T-cell activation measures specific T-lymphocyte subset responses through the use of paramagnetic particles coated with monoclonal antibodies against CD antigens. This assay permits rapid (24 h) analysis of lymphocyte subset activation responses to mitogens and recall antigens in small amounts of blood.     

牛磺酸减轻β-甘油磷酸诱导的大鼠血管平滑肌细胞钙化氧化应激在反流物所致食管粘膜损伤中的作用

目的：观察牛磺酸对钙化的形成及逆转作用的影响，探讨牛磺酸在血管钙化发生中的作用。方法：利用β-甘油磷酸制备钙化血管平滑肌细胞（VSMCs），测定细胞钙含量、碱性磷酸酶活性、[⁴⁵Ca]沉积及[³H]-胸腺嘧啶。结果：与对照组相比，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均明显升高（P<0.05），而牛磺酸与β-甘油磷酸同时孵育呈剂量依赖性地抑制钙化发生。在牛磺酸逆转实验中，钙化细胞的钙含量、ALP活性和[⁴⁵Ca]沉积均较换液前明显降低（P<0.01）；且牛磺酸呈剂量依赖性地逆转已钙化细胞。与对照组相比，钙化细胞的细胞数量和[³H]-TdR掺入量增加（P<0.01），牛磺酸早期干预组显著抑制钙化细胞的增殖，但牛磺酸对已钙化的细胞,增殖抑制效应不明显。结论：牛磺酸可抑制细胞钙化形成且能逆转已形成钙化。     

The effect of organic acids on phytohaemagglutinin-activated proliferation of human lymphocytes in vitro

The present study assesses the effects of 25 organic acids on in vitro proliferation of human peripheral lymphocyte stimulated with phytohaemagglutinin (PHA). Lymphocytes were cultured in flat-bottomed 96-well microplates at 37°C for 96 h in the presence of the mitogen and of one acid. The concentrations of organic acids tested in the cultures were from 1 to 5 mM, corresponding to those usually found in the blood of patients with organic acidaemias. Cellular growth was measured by the incorporation of 6[³H]-thymidine into cellular DNA. We observed that tiglic (2-methylcrotonic), α-keto-β-methylvaleric, aminoadipic, sebacic and alpha-ketoisocaproic acids strongly inhibited lymphocyte DNA synthesis, whereas α-ketoisovaleric, propionic, α-hydroxy-β-methylvaleric, α-methylbutyric and isobutyric acids moderately suppressed DNA synthesis. Lactic and ethylmalonic acids, however, stimulated DNA synthesis. The most inhibitory acids were added to cultures at different times after the beginning of the incubation period. Except for tiglic acid, whose action persisted even after 48 h from the onset of cultures, the others acted only when added during the first 24 h. The present study demonstrated that organic acids modulate DNA synthesis in mitogen-stimulated human lymphocytes.     

Enhancement of T-cell response to PWM by neuraminidase-treated non-T cells.

The effect of neuraminidase (from Cl. perfringens, CPN) on the human lymphocyte response to pokeweed mitogen (PWM) was studied. CPN treatment greatly increased [3H]-thymidine incorporation by human lymphocytes at lower concentrations of PWM. This enzyme acted specifically on T-cell proliferation and had no direct effect on non-T-cell proliferation. When CPN-treated non-T cells were added to autologous T cells, [3H]-thymidine incorporation was markedly enhanced at lower concentrations of PWM. However, the addition of T cells pre-treated with mitomycin C (MMC) to CPN-treated non-T cells failed to increase [3H]-thymidine incorporation in contrast to the controls. MMC-blocked, CPN-treated non-T cells significantly enhanced T-cell proliferation, whereas the effect of MMC-blocked, CPN-treated adherent cells was equal to that of the controls. These data indicate that cell-surface properties of non-T cells other than monocytes affect T-cell proliferation in some situations.     

Protease inhibitors reduce mitogen induced lymphocyte stimulation.

Proteolytic events might play a role during lymphocyte activation. Mouse spleen cells were therefore stimulated in serum-free cultures by PHA, ConA, LPS and dextran sulphate and the effect of various added protease inhibitors on [3H]-thymidine incorporation investigated. Both soybean inhibitor and Trasylol inhibited the response of the cells to all mitogens. The other inhibitors (antipain, leupeptin, ovomucoid, alpha-1 trypsin, alpha-2 macroglobulin) had little or no effect. The marked inhibitory effect of tosyl-lysine chloromethylketone could be neutralized by reduced glutathione, indicating an effect on intracellular glutathione rather than on proteases.     

氟伐他汀抑制高血压大鼠血管平滑肌细胞增殖

目的:探讨氟伐他汀对自发性高血压大鼠血管平滑肌细胞增殖的抑制作用。方法:培养自发性高血压大鼠主动脉血管平滑肌细胞,不同浓度血管紧张素Ⅱ(AngⅡ)、血小板源生长因子(PDGF)、氟伐他汀及甲羟戊酸干预后,行细胞计数和[³H]-TdR掺入率测定。结果:①氟伐他汀呈浓度依赖性抑制10^-6mol/LAngⅡ和10μg/LPDGF刺激诱导的血管平滑肌细胞数和[³H]-TdR掺入率增加;②10^-3mol/L甲羟戊酸几乎完全逆转氟伐他汀对血管平滑肌细胞增殖的抑制作用。结论:氟伐他汀抑制AngⅡ和PDGF诱导的高血压大鼠血管平滑肌细胞增殖;甲羟戊酸代谢途径可能参与血管平滑肌细胞增殖过程。     

Determination of peripheral blood mononuclear cell responses to mitogens and woodchuck hepatitis virus core antigen in woodchucks by 5-bromo-2′-deoxyuridine or 2[3H]adenine incorporation

Summary.  Characterization of cellular immune response to antigens of woodchuck hepatitis virus (WHV) can contribute to the understanding of acute resolving and chronic outcome of hepadnavirus infection. Studies were limited because peripheral blood mononuclear cells (PBMCs) of woodchucks failed to incorporate [³H]thymidine sufficiently. Therefore, we established a non-radioactive proliferation assay for woodchuck PBMCs using 5-bromo-2′deoxyuridine (BrdU) as thymidine analogue. Mitogen- and WHV core protein(WHcAg) induced PBMC proliferation was detected by BrdU incorporation and compared to an assay using 2[³H]adenine. After stimulation with concanavalin A (ConA) and phytohaemagglutinin (PHA) we observed significant PBMC proliferation with both assays. Mitogen-induced nucleoside uptake of PBMCs into cellular DNA was confirmed by detection of 1′,2′[³H]BrdU and 2[³H]adenine in extracted DNA. PBMCs obtained during the acute phase of WHV infection could be stimulated by WHcAg, whereas no WHcAg-induced proliferation of PBMCs was found in WHV-negative animals. PBMCs of chronic WHV carriers showed only a weak response to WHcAg. The established assays will be useful in determining the kinetics of cellular immune responses to different WHV antigens in the course of WHV infection and may provide an insight into mechanisms responsible for chronic outcome of hepadnavirus infection.     

Comparison of a Simplified Whole Blood and Isolated Lymphocyte Stimulation Technique

Whole blood and isolated lymphocyte stimulation in healthy men were compared utilizing PHA, ConA, and PWM. Dose response curves with each of the 3 mitogens were found to differ with the two techniques of lymphocyte stimulation. When incubation time was lengthened PWM responses increased markedly in both the whole blood and isolated lymphocyte assays. Increased incubation time had no effect on PHA cultures and increased the responses with ConA in the whole blood method. In each study, responses with ³H-thymidine were comparable to responses utilizing ¹²⁵IUdR.     

The immunological properties of haptens coupled to thymus-independent carrier molecules. III. The role of the immunogenicity and mitogenicity of the carrier in the induction of primary IgM anti-hapten responses

Hapten (DNP-lys) conjugates of two putatively nonimmunogenic polymers, hyaluronic acid and poly-γ-D-glutamic acid, induce significant primary IgM anti-DNP responses in C3H mice. Preparations of various immunogenic (Type 3 pneumococcal polysaccharide (SIII), levan, E. coli lipopolysaccharide) and nonimmunogenic (hyaluronic acid and poly-glutamic acid) polymers were tested for their ability to act as polyclonal mitogens in vitro. In serum-containing spleen cell cultures, only lipopolysaccharide stimulated substantial cell proliferation. In serum-free medium, and using high specific activity [³H]thymidine, lipopolysaccharide, levan, SIII and to a lesser degree hyaluronic acid induced significant thymidine incorporation. However, under the latter conditions cell survival and proliferation were much less impressive. There was no apparent correlation between the capacity of various polymers to induce lymphocyte proliferation and their ?potency”? as carriers for the generation of a primary IgM anti-DNP response. Furthermore while low doses of lipopolysaccharide elicited ?polyclonal”? antibody formation in vivo, high doses of SIII, levan and hyaluronic acid did not. These results indicate that T cell-independent B cell triggering is dependent on the polymeric nature of the antigen, and that polymers need not be immunogenic or mitogenic to act as carriers for the induction of primary IgM anti-hapten antibody responses.