Immune cell-proliferation assay

In recent years, the mouse local lymph node assay (LLNA) has been approved as an alternative to the guinea pig test. It is based on the proliferation of lymphocytes (a type of immune cell) in lymph nodes draining the site of contact with the chemical. In the LLNA, the substance is applied to the mouse s ear on days 1-3 of the test but withheld on days 4 and 5 to give the immune system a chance to respond. On day 6, the mouse is injected in its tail vein with a small amount of a radioactive DNA base such as H-thymidine (tritiated thymidine) to label newly formed immune cells. The mouse is sacrificed, its auricular lymph nodes (located near the ears) are cut out, and their radioactivity is measured. Increased radioactivity in the lymph nodes of treated animals compared with controls indicates that the test chemical sensitizes the immune system and can cause contact dermatitis. The LLNA is more objective than the traditional guinea pig test because the amount of immune cell proliferation can be determined as a function of dose. 

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

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. 

Adjuvants enhancing HLA class I-restricted CTL responses are especially needed for treatment or prevention of chronic viral diseases and infections linked to intracellular pathogens, and for cancer immunotherapy. Among the very few adjuvants licensed for human use, we evaluated the capacity of IRIV to enhance HLA class I-restricted CTL responses in vitro. We addressed IRIV-elicited immune responses and the induction of CTL specific to IM58 66 and Melan-A/Mart-127-35 epitopes. Proliferation assays, cytokine expression studies, and phenotypes of CD4+ T-cells demonstrated that IRIV... 

Numerous reviews have documented immune modulation in response to metal, pesticide and organic contaminants in fish4,19,37,135. Assays traditionally used to assess perturbations of immune function in fish fall into three broad categories pathogen challenge models, assays that monitor immune suppression/activation or immuno-pathology. Immune suppression/activation is usually measured with assays of phagocytosis, respiratory burst, cell proliferation, as well as quantification of soluble factors such as lysozyme, serum antibody, CRP or complement. 

Characteristic of immune function evaluations, there is considerable diversity in the approaches to proliferation assays, from the experimental design to the analytical method. Experimental procedures necessitate sterile technique and cell culture expertise to ensure accurate assessment of the cellular response to a particular stimulant. Culture conditions vary depending on the cell source and type of stimulant, but generally are conducted at 5% CO2,37 °C for 48 to 96 hours. The source of lymphocytes may be from peripheral blood, spleen. 

Preclinical studies have shown that impaired immune function determined using a variety of assays, e.g., the plaque-forming cell (PFC) assay, lymphocyte proliferation, delayed-type hypersensitivity, and NK cell activity, is associated with decreased resistance toward experimental infections (Luster et al., 1994). When a drug candidate has been shown to impair immune function in animal studies, the question arises whether similarly negative effects can also be seen in treated human subjects. 

A variety of techniques have been employed to assess the immune response to beryllium. Ho vever, the ease, sensitivity, and specificity have made the lymphocyte proliferation assay using H-thymidine the standard technique (Rossman et al. 1988, Mroz et al. 1991). In the past, this technique has been referred to as the lymphocyte transformation test (LTT), but transformation is an older term that is no longer applicable. The cells respond by proliferating (a normal process) and are not transformed, which today implies a malignant process. Hence, the test is currently known as the lymphocyte proliferation test (LPT). 

Interleukin-2 Human recombinant lL-2 aldesleukin, proleukin des-alanyl-1, serine-125 human lL-2) differs from native lL-2 in that it is not glycosylated, has no amino terminal Ala, and has an Ser substituted for the Cys at amino acid 125. The potency of the preparation is represented in International Units in a lymphocyte proliferation assay such that 1.1 mg of recombinant lL-2 protein equals 18 million International Units. Aldesleukin has the following in vitro biologic activities of native lL-2 enhancement of lymphocyte proliferation and growth of lL-2-dependent cell lines enhancement of lymphocyte-mediated cytotoxicity and killer cell activity and induction of interferon-7 activity. In vivo administration of aldesleukin in animals produces multiple immunologic effects in a dose-dependent manner. Cellular immunity is profoundly activated with lymphocytosis, eosinophilia, thrombocytopenia, and release of multiple cytokines e.g., TNF-a, lL-1, interferon-7). Aldesleukin is indicated for the treatment of adults with metastatic renal cell carcinoma and melanoma. Administration of aldesleukin has been associated with serious cardiovascular toxicity resulting from capillary leak syndrome, which involves loss of vascular tone and leak of plasma proteins and fluid into the extravascular space. Hypotension, reduced organ perfusion, and death may occur. An increased risk of disseminated infection due to impaired neutrophil function also has been associated with aldesleukin treatment. 

Different formulations of saponin-adjuvanted vaccines were tested for humoral and cell-mediated responses in mice. Many antigens, often including hen egg albumin (ovalbumin, OVA) as the antigen to be tested, have been used for immunizations, enzyme immunoassays, antigen-specific CTL and cellular proliferation assays [31]. 

Mice were immunized by s.c. injection of 0.2 ml of test formulation (25 p,g of the antigen OVA and varying doses of the test adjuvants) on days 0 and 14. Mice were bled and sera collected for antibody titer determination one week after the second immunization. Several other schedules of immunization have been reported in the literature. Splenic mononuclear cells were collected two weeks after the last immunization for use as effector cells in the cytotoxic T lymphocyte assay and for the proliferation assay [32]. 

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. 

Figure 2. Bone marrow-derived DCs demonstrate a potent APC function. DCs pretreated with CHP-HER2 complex, control CHP-CAB complex, or untreated DCs were used as stimulator cells. Responder CD4 T cells and CD8 T cells were obtained from nylon fiber-purified spleen cells of mice immunized with CHP-HER2 complex. H-TdR proliferation assay was performed. Both CD4 T cells and CD8 T cells showed significantly responded only to CHP-HER2 complex pretreated DCs.

Proliferation assays were done with naive BALB/c lymphocytes (6 x 10 cells/ml) incubated with a 100 ng/ml concentration of TSST-1 plus a 1 32 dilution ofpooled sera from mice immunized with either TSST-1, HI35A, or adjuvant alone. Cells were incubated with H-thymidine, harvested, and radioactivity measured in a liquid scintillation counter. 

In addition to being able to recognize the different cells involved in the acquired immune system of marine mammals, it is important to assure that the cells perform their functions appropriately. The ability of lymphocytes to proliferate upon stimulation (usually with mitogens) has been studied for several decades [1,12,14,15, 32-35], Recent advances include the demonstration of a conserved specificity for standard mitogens used in beluga whales [32] and harbor seals [33], An assay to assess the expression of the receptor for interleukin-2 (IL-2), an early event in lymphocyte activation, was adapted in harbor seals [35], bottlenose dolphins [36], and sea otters [37], Molecular and biochemical mechanisms of activation of beluga T lymphocytes do not vary substantially from those in other mammals [38],... 

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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