Angus W. Thomson

FK-506 — How much potential?

FK-506 is a recently discovered immunosuppressive agent that, although structurally quite distinct from cyclosporin A (CsA), shares many of its properties. These include interference with production of interleukin 2 (IL-2) and other lymphokines, lack of myelotoxicity and the induction of transplantation tolerance following administration of short courses to allografted rats. Compared with CsA, however, FK-506 is much more potent and the two agents can act synergistically both in vivo and in vitro. However, severe side effects of FK-506 have been reported in dogs and baboons, although not in rats. On the basis of recently published findings. Angus Thomson reviews the immunological properties of FK-506 and assesses its potential in the light of the impact already made by CsA in both the laboratory and clinic.

Serum cyclosporin levels, hepatic drug metabolism and renal tubulotoxicity

The present study was designed to examine inter-relationships between serum cyclosporin (CsA) levels, hepatic drug metabolising enzyme activity and CsA induced nephrotoxicity. CsA (25 mg/kg p.o.) was administered daily to male Sprague-Dawley rats: groups of animals were killed on days 0, 4, 7, 10 and 14 and thereafter at weekly intervals over the 7-week course of the experiment. Nephrotoxicity was evaluated by measuring tubular enzymuria and by light microscopy and serum CsA levels (parent drug plus certain metabolites) were determined by radioimmunoassay. The hepatic microsomal mono-oxygenase enzyme system was monitored by measurement of cytochrome P-450, aminopyrine N-demethylase and NADPH-cytochrome c reductase. Nephrotoxicity appeared within 4 days of starting treatment and continued for 4 weeks. Between weeks 4 and 6 there was a period of complete remission followed by the return of renal damage. Aminopyrine N-demethylase activity fell during the first 4 weeks. During the period of remission, however, N-demethylase activity rose to a point significantly higher than pretreatment values and serum CsA levels fell to their lowest concentration. With relapse, hepatic N-demethylase activity again fell below normal and serum drug levels rose to their pre-remission values. From the third week onward, changes in NADPH-cytochrome c reductase activity paralleled those in N-demethylase activity. The hepatic microsomal concentration of cytochrome P-450 did not, however, change significantly during the 7-week period of CsA treatment. Our results suggest that the spontaneous remission of CsA-induced nephrotoxicity is due to a reduction in circulating drug levels caused by increased hepatic CsA metabolism.

Rosette inhibition levels during early human gestation

The inhibitory effect of antilymphocyte globulin on spontaneous rosette formation by peripheral blood T cells between 10 and 21 weeks gestation was examined. No evidence was obtained from either a cross-sectional or a longitudinal study that increased rosette inhibition levels occur during early human pregnancy. Comparable values were found in oestrogen-treated women. The results are consistent with the view that the intrinsic reactivity of human T lymphocytes is unimpaired during gestation and oestrogen treatment.

The influence of enalapril or spironolactone on experimental cyclosporin nephrotoxicity

Adult Sprague-Dawley rats treated daily for 14 days with 50 mg/kg cyclosporin A (CsA) exhibited nephrotoxicity, characterized by reduced glomerular filtration rate, decreased urinary sodium and potassium flow, tubular enzymuria and proximal tubular structural damage. Elevations in plasma renin activity (PRA) were observed on day 4, but returned to normal within 7 days. Co-treatment of animals for the 14 day period with enalapril (8 mg/kg/day), a potent inhibitor of angiotensin converting enzyme (ACE), or spironolactone (25 mg/kg/day), the distal tubular antagonist of aldosterone, reduced the nephrotoxicity, although PRA remained elevated. Neither enalapril nor spironolactone affected circulating CsA levels. These data suggest that the action of aldosterone on the distal tubule may be important in the pathogenesis of CsA nephrotoxicity.

Differential effects of cyclosporine A on Langerhans cells and regulatory T-cell populations in severe psoriasis: An immunohistochemical and flow cytometric analysis

Systemic administration of cyclosporine A (Cy-A; initial dose 5 or 2.5 mg/kg/day) to patients with severe chronic plaque psoriasis produced marked reductions in psoriasis area and severity index within 4 weeks. The clinical response was accompanied, within 1 week, by progressive reductions in T-cell subpopulations (CD3+ and CD4+) and in numbers of interleukin-2 receptor (IL-2-R)-positive (CD25+) cells within lesional skin. Over the first 4 weeks of treatment, these changes were accompanied by reductions in DR+ cells within the epidermis (minor) and dermis (substantial). In contrast, numbers of epidermal CD1+ cells increased substantially during resolution of the skin lesions. Unlike lesional skin, however, no significant changes in absolute numbers of circulating immunoregulatory T-cell populations, including helper/inducer (CD45R) and suppressor/inducer (CD29W) subsets, quantified by dual immunofluorescence labelling, were detected. Moreover, numbers of blood-borne HLA-DR, IL-2-R and transferrin receptor (CD71) positive lymphocytes were unaffected by Cy-A therapy, nor were any differences detected between psoriatic patients and normal controls using these cell markers. Our data suggest that the immunoregulatory effects of Cy-A in psoriasis are mediated via lesional T lymphocytes and that epidermal CD1+ DR- dendritic cells may play an influential role in the regulation of T-cell function and keratinocyte growth during resolution of the skin lesions.

The influence of cyclosporin A on T cell activation, cytokine gene expression and cell-mediated immunity

The potential of cyclosporin A (CsA) for the immunotherapy of allograft rejection and certain autoimmune disorders was evident in the first account by Borel et al. (1976) of the immunosuppressive properties of the drug. It was clear from such early publications that CsA exerted a powerful and selective inhibitory action against T lymphocytes. Whilst the capacity of CsA to abrogate the activation of CD4+ (T helper/inducer) lymphocytes and the secretion of interleukin-2 (IL-2) and other cytokines in vitro is now well recognized, the molecular basis of this remarkably sophisticated selectivity remains to be elucidated. Although the generation of specific cytotoxic T cells is impaired by CsA, the activity of natural cytotoxic cells (NK cells) appears unaffected.

Flow cytometric analysis of lymphocyte populations in guinea pig blood and spleen: Ia antigen expression and the effects of immunosuppressive agents

A panel of novel mouse monoclonal antibodies was used, in an indirect immunofluorescence procedure, to identify subsets of guinea pig lymphoid cells which were quantified by flow cytometric analysis. The staining characteristics of the antibodies were also examined by alkaline phosphatase-anti-alkaline phosphatase staining of cryostat sections of spleen and cytocentrifuge preparations of blood and spleen mononuclear cells. The values obtained for T and B cell populations were similar to those previously described using rosetting procedures, and the study also confirmed the high incidence of constitutive Ia antigen expression on guinea pig lymphocytes. Compared with animals receiving immunization (ovalbumin) alone, guinea pigs treated with ciclosporin A showed only minor changes in lymphocyte subsets, whereas those pretreated with cyclophosphamide revealed striking reductions in circulating T and B lymphocytes and in splenic B cells. Both ciclosporin A and cyclophosphamide markedly reduced Ia antigen expression on lymphocytes in blood, whilst cyclophosphamide also inhibited Ia antigen expression in the spleen.

The potential of cyclosporin a as an anti-tumour agent

Cyclosporin A (CsA) has become established as the agent of choice for the prevention of organ allograft rejection and has shown considerable promise in the clinical management of certain autoimmune disorders. The impact of CsA as an immunotherapeutic agent of major importance is attributable to its powerful, selective inhibitory action on T-lymphocyte activation and proliferation. Moreover, CsA lacks the myelotoxic and other major side effects associated with cytotoxic immunosuppressive agents, such as cyclophosphamide or azathioprine. It is now clear that CsA has a potential therapeutic role in the treatment of malignancies, especially T-cell cancers. Recent studies suggest that there may be several areas of application for CsA, either as a direct antiproliferative agent or in combination with other drugs, including inhibitors of polyamine biosynthesis or cytotoxic anti-tumour agents, including vincristine and adriamycin. In addition, CsA and non-immunosuppressive analogues have been shown to restore multi-drug sensitivity in cancer cells with acquired drug resistance. A further application of CsA may be to prevent the induction of human immune responses to therapeutic mouse monoclonal antibodies directed against tumour antigens, thereby enhancing the efficiency and safety of this form of cancer immunotherapy. Due to our incomplete understanding of the antiproliferative properties of CsA, further exploration of its potential as an anti-tumour agent must be accompanied by detailed studies aimed at elucidating its action on subcellular molecular events in both normal and malignant cells.

Generation of procoagulant activity by mononuclear phagocytes: Optimisation of an in vitro assay

Leucocyte procoagulant activity (PCA) has previously been advocated as a useful measurement of cell-mediated immune reactivity. The assay is, however, susceptible to inter- and intra-experimental variation. This investigation identifies several factors which influence the stability and reproducibility of the test. Major factors which affect the recalcification time of plasma, include plasma lability, medium/buffer pH and both the nature and concentration of the indicator cells used in the assay. C. parvum-induced mouse peritoneal exudate cells have been used as a novel source of mononuclear phagocytes in the generation of PCA. Their sensitivity as indicator cells has been demonstrated by their responsiveness to stimulation by phytomitogen, endotoxin, and lymphokine (macrophage procoagulant-inducing factor). A simple test based on antigen-induced PCA of these cells, has provided an in vitro index of in vivo sensitisation to sheep red blood cells. Optimisation and standardisation of conditions detailed in this report for estimating PCA, renders the assay of value in monitoring lymphocyte and macrophage activation.

Pregnancy-associated alpha-2-glycoprotein in recurrent oral ulceration and Behçet's syndrome.

Estimation of the serum concentration of pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) in male patients with recurrent oral ulcers (ROU) and Behçets syndrome (BS) revealed significant increases in both groups of patients. The concentrations of alpha 2-PAG in 24 control subjects were less than 10 micrograms/ml, whereas 11/26 (42%; p less than 0.001) of patients with ROU and 20/33 (61%; p less than 0.0005) with BS had concentrations greater than 10 micrograms/ml. Indirect immunofluorescence staining for alpha 2-PAG on the surface membrane of blood mononuclear cells suggests that there is an increase in the number of alpha 2-PAG-positive cells in BS. It is not clear at present whether the increased concentrations of alpha 2-PAG might exert an inhibitory effect on the immune response. No correlation was observed between the levels of alpha 2-PAG in ROU and BS and concentrations of alpha 1-acid glycoprotein, C9, factor B or lysozyme. It is therefore unlikely that the increase in alpha 2-PAG is a manifestation of an increase in acute phase protein.