Lymphocyt reaction assay

Hyrtiomanzamine (493), a compound consisting of a 6-hydroxy-(J-carboline associated with a betaine unit, was isolated from Hyrtios erecta from the Red Sea and the structure elucidated by spectral data examination. Hyrtiomanzamine exhibited immunosuppressive activity in the B lymphocytes reaction assay [419],... 

The thiol group can also be found in heterocyclic compounds such as 2-mercaptobenzothiazole (9) and echinoclathrine C (10). Compound 9 was isolated from the symbiont bacterium Micrococcus sp., which was obtained from the sponge Tedania ignis [13]. The pyridine alkaloid echinoclathrine C (10) and its S-acetylated derivative, echinoclathrine B (11), were isolated from the Okinawan sponge Echinoclathria sp. [14]. The position of the hydroxyl and acylamino group on the phenyl ring in echinoclathrines (11 and 10) was recently corrected [15]. Compound 11 showed weak immunosuppressive activity in the mixed lymphocyte reaction assay with an IC50 of 9.7 pg/ml [14]. 

The New Zealand bryozoan Cribicellina cribraria was the source of several P-carboline alkaloids. One of these examples, compound 34, has a unique methylsulfone substituent. Spectroscopic studies identified 34 as 1-ethyl-4-methylsulfone-P-carboline [34]. Hyrtiomanzamine (35) represents the first example of a 6-OH-P-carboline ring associated with a betaine unit to be obtained from a natural source. This compound was isolated from the Red Sea sponge Hyrtios erecta and its structure was determined on the basis of its spectral data [35]. Compound 35 displayed immunosuppressive activity with an ED50 of 2 mg/ml in the B lymphocytes reaction assay. The lack of cytotoxicity against KB cells indicated that the immunosuppressive activity of 35 is specific and not due to a general cytotoxic effect. 

Table 1 Selected thieno[2,3-d]pyrimidine-2,4( 1 H,3H)-dione derivatives and their corresponding physical data. When evaluated for their effectiveness as IL-2 inhibitors using the human mixed lymphocyte reaction assay, all experimental agents had IC50 values less than 1 x 10 6M...

Manzamine A was able to block the cytosolic calcium increase induced by KCl depolarization on human neuroblastoma SH-SY5Y cells. The maximum effect was observed at 1.0 jxM, inhibiting by 31.5%. 8-Hydroxymanzamine A also showed a similar effect whereas manzamines E, F, and Y, ircinal A and weo-kauluamine did not show any significant effect [44]. In the B lymphocytes reaction assay hyrtiomanza-mine showed immunosuppressive activity with an EC50 of 2.0 xg/mL, but no activity was displayed against the KB human epidermoid carcinoma cell line [53]. 

In 2013, Stella and coworkers made some pyrimidine compounds to evaluate the potential immunosuppressive activity in the mixed lymphocyte reaction assay. The chemistry is based on the direct amination of the tau-tomerizable heterocycles using BOP in the presence of DBU in MeCN (13BMC1209). 

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. 

Locai iymph node assay Cytokines (iL-1, iL-1ra, TNF-a, IL-6, mixed lymphocyte reaction TGF-(5, IL-4, IL-13)... 

The delayed type hypersensitivity response (DTH) is an assay frequently used to assess the T cell response to commonly encountered microbial antigens. It involves intradermal injection of antigens to which the majority of individuals are immune (known as recall antigens) such as vaccinia, herpes simplex, and mumps viruses, Candida, and tetanus toxoid. In normal individuals, after 24-48 hours, an inflammatory filtrate results in local edema and induration, the diameter of which can be measured. A negative reaction to all the antigens (anergy) is usually reflected by decreased lymphocyte function as measured in vitro and is frequently seen in AIDS and ARC patients. 

Fig. 9. Flow analysis of apoptotic human peripheral blood lymphocytes using direct TUNEL assay. Human peripheral blood lymphocytes (1 x 10 ) treated (A) without or (B) with dexamethasone (0.1 ijlM) for 16 h were transferred to a 15-ml tube. Paraformaldehyde (2%) was added to cells with shaking and incubated for 10-15 min in ice with occasional shaking. Cells were washed with PBS with 1% BSA and 3-4 ml cold acetone was then added to cells. After 2-3 min incubation on ice with occasional shaking, cells were washed twice and TUNEL reaction mixture including enzyme TdT and fluorescein-labeled anti-dUTP antibody was added to cells (H). For the negative control group, only label solution without TdT was added to cells ( ). Cells without any addition of reaction or label solution were used for assessment of the autofluorescence ( ). The cell mixture was incubated 1 h at 37°C in the dark. The result of the apoptosis after flow analysis was expressed as a histogram using software CellQuest (Becton Dickinson). In Fig. 9A Ml (nonapoptotic cells), 86% M2 (apoptotic cells), 14%. In Fig. 9B Ml (nonapoptotic cells), 78% M2 (apoptotic cells), 22% (our unpublished data).

Homogenates of lymphocytes were preincubated at either 15 or 30°C for 10 minutes before the reaction was started, by addition of 3-hydroxykynure-nine to a final concentration of 1 mM. When the assay was performed at 30°C, the presence of 4 pM pyridoxal-5 -phosphate was needed to obtain a linear reaction. The reaction was terminated after 5 minutes by the addition of 150 piL of 10% trichloroacetic acid. HPLC analysis was carried out on 30 / L of the supernate obtained after centrifugation. 

Modifications are made also in order to use this reaction to conjugate nucleosides to erythrocyte surfaces, allowing use of the coated cells as targets for assays of antibody-forming splenic lymphocytes. Nucleoside, 10-20 mg, is oxidized in 1.5 ml of 0.1 M sodium periodate in 0.15M NaHCOa for 20 min at room temperature the reaction is stopped by the addition of 15 /zl of ethylene glycol. Sheep erythrocytes are washed twice with 0.15 M NaHCOs, and 0.5 ml of packed cells is then suspended in 2.0 ml of the bicarbonate solution in a40-ml centrifuge tube. The oxidized nucleoside is added dropwise to the cell suspension and the mixture is kept at room temperature for 15 min. fert-Butylamine borane (Aldrich Chemical Co.), 100 mg in 5 ml of 0.15 M NaHCOs, is added. The suspension is kept at room temperature for 3 min, and the tube is then quickly filled with bicarbonate solution and centrifuged at 1500 rpm for 10 min. 

It is stiU uncertain whether cefaclor-associated serum sickness-like reactions correspond to true hypersensitivity. It has indeed been suggested that they may result from inherited defects in the metabolism of reactive intermediates and may be a unique adverse reaction requiring biotransformation of the parent drug (187). A lymphocyte-based cytotoxicity assay has shown that cefaclor metabolites, as generated by murine hepatic microsomes, mediated cytotoxicity among patients with... 

Products of what is now known as the HLA class II region were originally detected through the use of the mixed lymphocyte culture (MLC) assay (Bl, B3). This test uses the principle that when lymphocytes from genetically dissimilar individuals are mixed under appropriate cell culture conditions, the lymphocytes of one individual will stimulate those of the other to reproduce and form blast cells. The finding that lymphocytes from HLA-A and HLA-B identical siblings did not stimulate each other in the MLC, whereas lymphocytes from HLA-A and HLA-B identical Individuals did, suggested that the MLC reaction is related to the HLA system but is controlled by a locus separate from HLA-A and HLA-B (Y3). This... 

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