Tolerance in a rat cardiac allograft model after short-term treatment with LF 08-0299. Absence of clonal deletion and evidence of CD4+ suppressor cells.

LF 08-0299 is a new immunosuppressive compound. In a fully mismatched rat cardiac allograft model (Dark Agouti [DA]-->Lewis [LEW]), long-term unresponsiveness was observed after LF 08-0299 short-term treatment (20 days). Survival of additional cardiac and skin DA allografts, and rejection of third-party (Brown Norway [BN]) skin allografts demonstrated induction of a donor-specific tolerance state. The aim of this study was to investigate mechanisms of cardiac acceptance in this model. LEW rats with long-term surviving heart grafts (LTS LEW) were examined for their immune proliferative and cytotoxic responses toward donors (DA) and third-party (BN) antigens. Normal proliferative responses were observed and limiting dilution analysis did not reveal a reduction of T cytotoxic cell precursors. In our model, tolerance exists despite the presence of cells reactive with donor alloantigens. In vivo adoptive transfer of serum from LTS LEW failed to transfer unresponsiveness, indicating that serum factors do not seem to be involved in tolerance maintenance. Transfer of spleen cells, obtained from LTS LEW, showed specific prolongation of DA cardiac allografts in syngeneic hosts. Moreover, these cells were able to induce the rejection of third-party BN grafts. These results suggest that although LTS LEW possessed suppressor cells, they remained immunocompetent in recognizing and responding to third-party alloantigens. Purified CD4+ cells transferred unresponsiveness to secondary hosts, but CD8+ cells did not. Taken together, these results suggest that tolerance to donor alloantigens after treatment with LF 08-0299 in the rat cardiac allograft model is most likely due to induction of specific CD4+ suppressor cell activity, rather than induction of suppressive serum factor and selective elimination of antidonor helper or cytotoxic cell precursors (clonal deletion).

Structural and stereoelectronic requirements for the inhibition of mammalian 2,3-oxidosqualene cyclase by substituted isoquinoline derivatives.

2,3-Oxidosqualene lanosterol-cyclase (OSC; EC 5.4.99.7) is an attractive target for the design of compounds that block hepatic cholesterol biosynthesis. (4a alpha, 5 alpha, 6 beta, 8a beta)-Decahydro-5,8a-dimethyl-2-(1,5,9-trimethyldecyl)-6- isoquinolinol (1) and simplified analogs have been devised to inhibit this enzyme by mimicking the postulated pro-C-8 high-energy intermediary carbocation occurring during the cyclization-rearrangement pathway. In order to gain an understanding into the mechanism by which these types of molecules inhibit OSC, we have synthesized a series of substituted isoquinoline derivatives 3 and investigated the structural and stereoelectronic requirements, and their stringency, that make 3 potential high-energy intermediate analogs of OSC. Determination of the IC50 values of the different compounds with rat liver microsomal cyclase, allowed the study of the relative importance of (i) the nature and the stereochemistry of the nitrogen side chain, (ii) the presence of methyl groups at C-5 and C-8a (ring junction), (iii) the presence and stereochemistry of the C-6 hydroxyl group, (iv) the nature of the ring junction, and (v) the absolute configuration of the bicyclic system. The resulting structure-activity relationships seem to validate the mechanism of action of these inhibitors as analogs of a pro-C-8 high-energy intermediate and delineate the minimal requirements for the design of efficient isoquinoline-based, or simplified, OSC inhibitors.

Use of [35S]methionine-labelled rat lymphoblasts in micro-cytotoxic and limiting dilution assays.

When only limited numbers of effector cells are available for in vitro T cytotoxic determinations, standard assays cannot be performed. 51Cr is still the most commonly used marker of target cells in cytotoxicity assays but since the incorporation of this marker is low, especially in non-tumor cells such as lymphoblasts, larger numbers of both target and effector cells are required. Here we report the use of [35S]methionine-labelled rat ConA blasts in cytotoxic, micro-cytotoxic and limiting dilution assays. Regardless of whether [35S]methionine or 51Cr targets were employed, cytotoxic activities were identical when large numbers of target cells (10(4)) were used. The high uptake of [35S]methionine by ConA blasts (9 cpm/cell) permitted the use of a small number of target cells without any loss of sensitivity. Therefore, the number of effectors and targets required was dramatically reduced, especially with high E : T ratios such as 100 : 1. The use of low number of [35S]methionine-labelled rat ConA blasts as targets was also suitable for the measurement of alloreactive T cell precursor frequencies. This technique illustrates the possibility of studying T cytotoxicity in animal species lacking tumor target cell lines under experimental conditions where the availability of effector cells is limited and the optimal use of such cells becomes critical.