3). antigen and the major histocompatibility complex (MHC) present on the cell surface of APC cells, and the T-cell receptor (TCR) of T-cells. Signal 1 only induces a partial T-cell response. In addition, there is a second signal, signal 2, part of a co-stimulatory pathway for T-cells that is required for their full activation, which is induced by the binding between CD80 (also known as B7-1) of APC cells and CD28 (4) of T-cells binding between CD80 and CD28. To maintain T-cells in a healthy condition requires a balance of both positive and negative immune responses. Imbalanced bindings between CD28 and CD80 and between CTLA4 and CD86 responses, both over-active and under-active immune responses are pathological. There are many autoimmune disease examples of over-active immune responses. Compounds such as FK506 (tacrolimus), which penetrates the cell membrane and intracellularly shuts down particular pathways including signal 1, are currently being investigated or used clinically as immunosuppressive agents (5,6). While FK506 is a useful immuno-suppressive agent with significant effects on T-cells for example, in inhibition of transplantation rejection, it has severe side effects since it shuts down many pathways in the cell. This indiscriminate behavior also affects cells in other organs. For example, the calcium ion pathway in cells is altered by FK506 due to its effects on calcineurin, which may subsequently lead to the disease of osteoporosis (lack of calcium in bone). Therefore, recent efforts to find new immuno-suppressive drugs have focused on cell membrane receptors antibodies (for example anti-CD28 antibody, MMV390048 a non-mitogenic antibody being considered as outside T-cell blocker), which can efficiently block receptors but not affect other pathways inside cells. However, using antibodies as drugs has many drawbacks including their fragile nature (protease and peptidase digestion limits oral availability and half-life) and poor stability (loss of 3-D structure). Also, unlike small molecules, which mimic metabolites in the body, the large antibodies might induce other immune system problems and therefore reduce their therapeutic utility. In this study, the small organic compound, cynarin (see Fig. 1), previously isolated from the north American herb with our recently established screening method: After Flowing Through Immobilized Receptor (AFTIR; see Fig. 2) (7) was further investigated. A detailed understanding of the mode and types of binding of cynarin to T-cell receptors is essential for further development of this potentially new mild immuno-suppressive agent. Open in a separate window Fig. 1 Structure of cynarin. Cynarin (1,3-dicaffeoylquinic acid) structure was confirmed by mass spectroscopy and H1 NMR and C13 NMR5. Open in a separate window Fig. 2 AFTIR scheme. The compound cynarin was discovered in extract by the AFTIR method. This schematic diagram shows the immobilization of CD28 (extra-cellular part) in the chip. Many components from the extract of extracts on a silica gel open column with serial elution buffers of 1 1 L of 100% crude extract. A typical HPLC MMV390048 profile (absorbance at 254 nm) for cynarin fraction and for crude extract are shown in Fig. 8A, (a) and (b), respectively. Composition of collected fractions was also monitored by TLC using Silica gel 60 F254 plate in an EA: isopropanol: H2O (8:5:3, extracts by using silica gel open column as shown in A curve (was shown in A curve (addition of anti-CD3 and anti-CD28, respectively. Flat-bottom 96-well plates were coated with 10 g/mL of anti-CD3 for 24 h at 4C. Wells including anti-CD3 were then washed twice with phosphate-buffered saline (PBS) to remove unbound PCDH8 anti-CD3. Jurkat T-cells (200 L, 2106 cells/mL) with or without cynarin (PBS buffer only; control group) were then MMV390048 added to the wells. Cells were activated by anti-CD3 in wells for 15 min (signal 1 stimulation). Consequently, anti-CD28 (1 g/mL) was then added into the wells (for signal 2 stimulation) for 24 h at 37C. IL-2 release from stimulated T-cells (100 L) was then measured by enzyme-linked immunosorbent assay (ELISA; see section below for detail). (2) R2: similar procedures as shown for R1 above, 100 L Jurkat T-cells (2106 cells/mL) with or without cynarin in PBS were pre-incubated with anti-CD3 (50 ng/mL) for.