Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase.

BACKGROUND AND PURPOSE: Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. EXPERIMENTAL APPROACH: In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. KEY RESULTS: Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test. CONCLUSIONS AND IMPLICATIONS: It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

Murine APC activation in the xenogeneic MLC.

Purified human T cells proliferate in response to direct and indirect presentation of human alloantigens. However, until recently it was believed that human T cells could respond only indirectly to murine xenoantigens. We recently used the mixed leucocyte culture (MLC) to demonstrate that purified human T cells proliferate in response to direct presentation of murine xenoantigens by murine antigen-presenting cells (APC) in the presence of human cytokines. We suggested that cytokines might function poorly across species barriers. In this study, we demonstrate that although proliferation occurs in the presence of exogenously added cytokines, the precursor frequency of responding human T cells is much lower in a xenogeneic than in an allogeneic MLC. We demonstrate that human T cells also proliferate in response to murine APC in the presence of murine cytokines, and murine cytokines augment the proliferative response seen in a human anti-human MLC, ruling out the possibility that an absolute cytokine incompatibility exists between these species. We show that exogenously added human IL-1 causes maximal proliferation of human T cells in response to murine xenoantigens only when added early in the culture. We further demonstrate that murine APC preincubated in human rIL-1, and washed extensively, prior to use as stimulating cells, cause human T-cell proliferation without the need for exogenously added cytokines. Finally, we noted that during interactions of human T cells and murine APC, little to no IL-1 is produced, whereas after the addition of exogenous IL-1, a marked increase in the production of IL-1 is seen. These data suggest that during interactions between human T cells and murine APC, the murine cells do not receive adequate stimulation to produce sufficient costimulatory signals to allow proliferation of the human T cells.

Cellular basis of the proliferative response of human T cells to mouse xenoantigens.

Purified human T cells respond proliferatively to allogenic peripheral blood mononuclear (PBMC) stimulating cells but show no response to murine splenic stimulating cells. Two possible explanations for the lack of xenogeneic response are that human T cells, educated in a human thymus, cannot directly recognize a molecule as disparate as mouse antigen encoded by H-2 and/or that a cytokine(s) produced by the APCs is needed to allow a proliferative response and that the cytokine(s) produced by murine APC do not provide an adequate stimulus to the human T cells under these conditions. We show here that highly purified human T cells can respond directly in an antigen-specific manner to murine stimulating cells if human rIL-1 or rIL-2 or a T cell growth factor (TCGF) preparation are present in the culture. These findings demonstrate that human T cells can recognize murine antigens and that a highly significant response can be obtained if a human cytokine is present to permit that response.

Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, beta-IL 1, interferon-gamma and -beta.

Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.

Long-term culture of LAK cells: expansion and activation signals.

Long-term culture of cells in rIL2-containing medium increases LAK activity on a per cell basis and produces an average 30-100-fold expansion over 14-21 days. The stimulation of PBL with anti-CD3 results in a 300-1000-fold increase in cell number while maintaining LAK activity. Cells stimulated with anti-CD3 and cultured in rIL2 for 12 days) can be further stimulated with beta IL1, or beta IFN, producing a further increase in LAK activity. These findings have allowed us to begin understanding the role of different signals in the activation of cells with LAK activity and together with biotechnological advances will allow for the culture of large numbers of cells for experimental and therapeutic purposes.

The stepwise activation of cytotoxic T lymphocytes.

The recognition of antigen is the first stage of T-lymphocyte maturation in vivo. Monoclonal antibodies directed against the T-cell receptor/CD3 complex and against other surface antigens can also provide this stimulus. Gianni Gromo and his colleagues are attempting to characterize the steps leading from this initial activation to full functional maturation. They suggest that while numerous pathways of activation exist, the particular pathway followed and to what extent activation occurs depend on both the stimulating ligand and its cell surface receptor.

Sequence polymorphism of HLA DR beta 1 alleles relating to T-cell-recognized determinants.

HLA class II molecules are a highly polymorphic family of dimeric cell-surface proteins primarily involved in regulating T-cell responses to extrinsic antigens. To define regions of class II molecules involved in T-cell recognition, we have now compared sequences of three HLA DR beta cDNA clones obtained from cells that all express the same serologically defined determinants but differ in terms of T-cell-recognized specificities. The comparisons indicate that very few (one to four) nucleotides differ between what are almost certainly alleles of the DR beta 1 locus. All differences were in the first domain of the molecule and all localized to a region from amino acids 71-86. Because all differences were found only in this region of the molecule, and because DR alpha-chains seem to be relatively non-polymorphic, these positions in the DR beta-chain must have a major role in influencing T-cell recognition of the DR molecule.

Histocompatibility and isoenzyme differences in commercially supplied "BALB/c" mice.

BALB/c mice obtained commercially were found to differ significantly from the standard phenotype of BALB/c strain mice. Isoenzyme tests and H-2 haplotype analyses indicated that the majority of mice from two of the three sources tested appeared mixed, frequently heterozygous, and did not consistently express either the expected H-2 or glucose phosphate isomerase type.

Early detection and specificity analysis of human cytolytic T lymphocyte (CTL) colonies generated in soft agarose culture: a potential assay for definition of CTL defined (CD) determinants.

In this communication we describe and early, large-scale screening assay for the detection of colonies with varied cytolytic specificity. The colonies are generated in soft agarose culture from day 3 mixed lymphocyte culture (MLC) alloactivated cells. A cell mediated lympholysis (CML) assay utilizing as few as 500 target cells has made it possible to prescreen for cytolytic T lymphocyte (CTL) colonies and to test for antigen specificities as early as 11 and 14 days, respectively, after MLC priming. Large numbers of colonies, from 80 to over 150, have been prescreened against a specific sensitizing target cell, and as many as 30 CTL colonies have been simultaneously tested against a panel of multiple targets carrying defined HLA-A, -B, -C, -DR antigens to evaluate antigen specificity. All CTL colonies are lytic against the specific sensitizing target cell and do not lyse the target autologous to the responder. Some are found to be operationally specific in that they lyse only those target cells which share HLA serologically defined (SD) antigens with the sensitizing cells, and other show cytolytic patterns which are not correlated with known HLA-SD antigens. These observations support, at a much finer level of analysis, the possible distinction between SD and CTL defined (CD) determinants.

Allo-responsive T lymphocytes and their differentiation markers.

Functionally disparate subpopulations of T lymphocytes that respond to alloantigens, primarily as studied in vitro in the mixed leukocyte culture and cell mediated lympholysis assays, have been separated most usefully by cell surface markers that can be identified with antiserums. Based on the data available to date there are at least two, and perhaps three, T lymphocyte subpopulations responsive to alloantigens in vitro, which include helper, cytotoxic, and perhaps suppressor T lymphocytes. Reviewed in this paper are data pertaining to the question of whether two types of cytotoxic T lymphocytes exist and whether true memory cytotoxic T lymphocytes are generated following in vitro or in vivo priming. Further, two systems are discussed that served as differentiation markers on lymphocytes: the Ly antigens and a family of large cell surface membrane proteins (LMPs).

Cloned primed-lymphocyte-test reagents in the dissection of HLA-D.

Human T lymphocytes obtained as blasts on day 4 from a primary mixed leukocyte culture (MLC) were cloned in the presence of T cell growth factor (TCGF) and feeder cells. Parameters important in producing higher-specific-activity TCGF were evaluated; irradiation of the responding cells as well as removal of adherent cells or inclusion of indomethacin in the culture was important. In addition, the presence of an irradiated lymphoblastoid cell line (LCL) cell in the TCGF-producing system enhanced activity in the supernate. The long-term maintenance of progeny from clones was achieved by utilizing the LCL autologous with either the responding or sensitizing cells from the initial MLC as feeder cells. Under those conditions, clones could be expanded for 7 or more wk with the maintenance of PLT reactivity. Had all the cells in each clone been maintained for the full 7 wk, more than 1 X 10(10) cells could have been developed in each clone. The cloned reagents provide a higher degree of antigen-specific reactivity than do normal PLT cells. It is to be anticipated that as the requirements for cloning are made more stringent, including the recloning of the cells, these reagents will aid greatly in the dissection of the complexity attendant to HLA-D.

Long-term maintenance of "cloned" human PLT cells in TCGF with LCL cells as a feeder layer.

The long-term maintenance of T cells "cloned" by limiting dilution in TCGF was enhanced by the use of irradiated autologous lymphoblastoid cell line (LCL) cells as well as irradiated LCL cells of the individual to which the T cells were originally primed. It was possible to obtain more than 1 X 10(12) cells from a "clone" seeded at one cell per well. Some of the clones tested express primed LD-typing activity.