T-cell proliferation assays

In order to evaluate the in vitro antitumor activity of the prodrugs, compounds 20 and 21 were incubated at varied concentrations with human T-lineage acute lymphoblastic leukemia MOLT-3 cells in the presence or absence of ImM of PGA. The data from the cell proliferation assays are presented in Fig. 5.15. 

Disparate effects on T-cell proliferative responses have been reported following exposure to JP-8. Significant suppression of T-cell proliferation is reported following either inhalation or dermal exposure to JP-8 [ 18,20,36], while the response is unaffected following either the oral or dermal exposure routes in other studies [66,71,72], These differences may be explained by variations in exposure routes and in assay methodology, as agents used to evaluate T-lymphocyte activation and proliferation were diverse and included Con A plus IL-2 [18,20], anti-CD3 [36], or Con A only [66,71,72],... 

Cell-mediated immunity Mitogen-induced T-cell proliferation, NK cell activity Spontaneous cytotoxicity assays... 

Several cytokines have been characterized at the molecular level in different species of marine mammals (Table 23.2). In addition, limited evidence exists for conserved functionality of cytokines in marine mammals, such as the ability of human recombinant IL-2 to stimulate T cell proliferation [32, 33] and natural killer cell activity [39,40] in beluga whales and harbor seals. Assays were developed to quantify circulating levels of cytokines [41,42], as well as C-reactive protein, a marker of acute inflammation [43],... 

Proliferation assays performed with PBMC cultures from a number of healthy donors demonstrated that IRIV indeed elicited cell proliferation in all tested cultures to an extent variable from donor to donor (Fig. 1 A). Further proliferation assays with CD4+ T-cells or CD8-I- T-cells in coculture with CD 14+ cells demonstrated that IRIV induce CD4+ T-cell proliferation, whereas no proliferation of CD8+ T-cells could be observed (Fig. 1C). Dissection of the CD4+ T-cell population into CD4+CD45RO+ and CD4+CD45RA+ cell subsets pointed to CD4+CD45RO+ cells as proliferative responders (Fig. ID). Importantly, culture of cord blood mononuclear cells in presence of IRIV did not result in major cell proliferation (Fig. IB), underlining antigen experience as prerequisite for IRIV-induced cell proliferation. 

Okubo T, Suzuki T, Yokoyama Y, Kano K, Kano I (2003) Estimation of estrogenic and antiestrogenic activities of some phthalate diesters and monoesters by MCF-7 cell proliferation assay in vitro. Biol Pharm Bull 26 1219-1224... 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

Calcineurin is a calcium/calmodulin-dependent protein phosphatase that may be involved in neurotransmission and T-cell proliferation. This assay uses nonlabeled peptides and is comparable in sensitivity to a radioactive assay. 

Desaulniers D, Leingartner K, Zacharewski T, Foster WG (1998) Optimization of an MCF7-E3 cell proliferation assay and effects of environmental pollutants and industrial chemicals. Toxicol In Vitro 12(4) 409 422... 

Peripheral blood mononuclear cells were obtained by density gradient separation through Ficol-Hypaque technology 17). Thymidine incorporation proliferation assays were set up as previously described using human serum albumin (HSA) or STn-HSA as stimulant (77). A stimulation index of > 2 SD compared to normal donors mononuclear cells was used as evidence of positive response. Fifty seven percent of tested vaccinated patients had evidence of specific T cell proliferation against STn. 

Maghni K, Nicolescu OM, Martin JG. Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA. J Immunol Methods 1999 223 185-194. 

To cite a few examples, anticoagulants must nearly always be added to the blood sample. There are various anticoagulants, for example, heparin, citrates, or EDTA. Best quality of RNA and DNA samples may be obtained from citrate-stabilized blood, but it may lead to a higher yield of lymphocytes for culture. On the contrary, heparin-stabilized blood could influence T-cell proliferation, and moreover heparin binds to many proteins and may therefore compromise proteomic studies. EDTA is suitable for both DNA assays and proteomics, but it affects Mg" " concentration causing problems for cytogenetic analyses (e.g., decreases mitotic index). 

As well as being suppressed by VA deficiency, immune responses may be suppressed by excessive dietary VA. Sklan et al. [36] assayed a TD antibody response and antigen-specific T cell proliferation in young chicks fed diets supplemented with 0 to 13 200 pg VA/kg diet. Antibody production and T cell proliferation increased progressively with dietary VA up to 6600 pg/kg and then declined slightly. Thus, an inverted U-shaped curve may best describe the relationship of antibody production to dietary VA intake. 

Despite the complexity of the experiments and the enormous data manipulation necessary, complex biological pathways, as well as new drug targets are being identified by this method. Examples include screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway [50], or assays with primary T cells from PLP TCR transgenic mice for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells [51], and identification of small-molecule inhibitors of histone acetyltransferase activity [52]. 

Ex vivo studies have revealed that trichothecenes can both inhibit and stimulate leukocyte function.12 For example, trichothecenes are toxic to alveolar macrophages,13 but drive differentiation of human myeloid leukemic cells.14 Dose-dependent decreases or increases in B- and T-cell mitogen responses are observable in lymphocytes from animals exposed to T-2 toxin, DON, or various macrocyclic trichothecenes these toxins similarly impair or enhance mitogen-induced lymphocyte proliferation in vitro.12 Rank order of inhibitor potency in rodent and human lymphocyte proliferation assays is Type D > Type A group > Type B group and is dependent on degree of acylation as well as of uptake and metabolism. 

Nickel-specific human T-lymphocyte clones have been isolated from blood [373] and inflammatory infiltrates after nickel application [379, 381]. 7-15% of the CD4 1 8 T-lymphocyte clones appeared to be specific for nickel in a proliferation assay [379] this proliferation response required the presence of antigen-presenting cells and was restricted by HLA class II molecules. Nickel-specific T cells from each nickel-allergic patient were extremely heterogenous with respect to their genetic restriction [381], Nickel-specific T lymphocytes sometimes show unusual genetic restrictions and might even respond to nickel without the participation of HLA-II molecules. 

As described previously, the humoral immune response results in the proliferation, activation, and subsequent production of antibodies by B cells following antigenic exposure and stimulation. The functionality and interplay between the three primary types of immune cells (macrophage, B cells, and T cells) required to elicit a humoral response can be assessed through various in vitro assays using cells from the peripheral blood or lymphoid tissues. 

As some of the toxicity observed with DPP-8/9 inhibitors 4 and 7 in predinical species suggested a potential immune system role, we hypothesized that the immunological effects observed with historical DPP-4 inhibitors [14] might be due to inhibition of DPP-8/9 instead of DPP-4 as initially reported. Sure enough, when these historical compounds were assayed at DPP-8/9 and DPP-4, they possessed more potent intrinsic inhibition at DPP-8/9 than at DPP-4 [20]. Furthermore, we demonstrated that DPP-8/9 inhibitor 7 is able to attenuate proliferation and IL-2 release in human in vitro models of T-cell activation, while a selective DPP-4 inhibitor does not. Recent tissue distribution studies also suggest a role for DPP-8 in the immune system [25]. 

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