Summary α1,3‐Galactosyltransferase gene‐knockout pigs transgenic for porcine cytotoxic T‐lymphocyte antigen 4 immunoglobulin (pCTLA4‐Ig) have been produced to reduce T‐cell‐mediated rejection following xenotransplantation. The level of soluble pCTLA4‐Ig in their blood was greatly in excess of the therapeutic level in patients, rendering the pigs immune‐incompetent. Soluble pCTLA4‐Ig produced by these transgenic pigs was evaluated for binding to porcine and human (h) B7 molecules, and for its inhibitory effect on allogeneic and xenogeneic human T‐cell responses. Porcine CTLA4‐Ig‐expressing peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (AECs) were evaluated for their direct inhibitory effect on hCD4+ T‐cell responses. Soluble pCTLA4‐Ig and purified hCTLA4‐Ig showed similar binding to pB7 molecules, but pCTLA4‐Ig showed significantly less binding to hB7 molecules. The pCTLA4‐Ig and hCTLA4‐Ig inhibited the response of hCD4+ T cells to pAECs equally, but pCTLA4‐Ig was less successful in inhibiting the human allogeneic response. The hCD4+ T‐cell response to PBMCs from pCTLA4‐Ig pigs was significantly lower than that of non‐pCTLA4‐Ig pigs. Although pCTLA4‐Ig was detected in the cytoplasm of pCTLA4‐Ig‐expressing pAECs, only a minimal level of soluble pCTLA4‐Ig was detected in the supernatant during culture, and pCTLA4‐Ig‐expressing pAECs did not inhibit the xenogeneic direct human T‐cell response. High‐level tissue‐specific production of pCTLA4‐Ig may be required for sufficient immunosuppression for organ or cell (e.g. islets) transplantation.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P1–S1P5). S1P1 is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P1. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P1 and S1P5. ASP4058 preferentially activates S1P1 and S1P5 compared with S1P2, 3, 4 in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.

The novel effects of FK506 on shock induced by lipopolysaccharide and phorbol myristate acetate (LPS/PMA) were studied using beagles. Five groups were studied: endotoxin shock control group (both 0.5 mg/kg of LPS and 30 microg/kg of PMA, n = 6); methylprednisolone-treated endotoxin shock group (n = 5); FK506-treated endotoxin shock groups in which intravenous infusions of FK506 at 2.5 microg/kg/h (low dose, n = 5), 8 microg/kg/h (medium dose, n = 5), and 25 microg/kg/h (high dose, n = 5) were administered. In the control group, the survival rate was 33%. Also, arterial hypoxemia, systemic hypotension, and marked increases in pulmonary vascular resistance (PVR) and wet-to-dry weight ratio (W/D) were observed. FK506 treatment at both medium and high doses significantly attenuated these LPS/PMA-induced physiological changes, and the survival rates were 80 and 100%, respectively. On the other hand, in the methylprednisolone group, no obvious effects were observed. The present study suggests that FK506 could have prophylactic potential against acute lung injury in endotoxin shock.

Abstract.The aim of the current study was to characterize comparatively the binding of muscarinic receptor in the lung of rats intratracheally administered anticholinergic agents (tiotropium, ipratropium, glycopyrrolate) used clinically to treat chronic obstructive pulmonary disease (COPD) and asthma. Binding parameters of [N-methyl-3 H]scopolamine methyl chloride ([ 3 H]NMS) were determined in tissues (lung, bladder, submaxillary gland) of rats intratracheally administered tiotropium, ipratropium, and glycopyrrolate. The in vitro binding affinity of tiotropium for the receptors was 10 -11-fold higher than those of ipratropium and glycopyrrolate. Intratracheal administration of tiotropium (0.6 -6.4 nmol/kg) caused sustained (lasting at least 24 h) increase in the apparent dissociation constant (K d 3 H]NMS binding. The effect by ipratropium was observed at 2 h but not 12 h, and that by glycopyrrolate lasted for 24 h. Both agents had little influence on the muscarinic receptors in the bladder and submaxillary gland. The present study provides the first evidence that tiotropium, ipratropium, and glycopyrrolate administered intratracheally in rats selectively bound muscarinic receptors of the lung, and tiotropium and glycopyrrolate had a much longer-lasting effect than ipratropium.

No abstract