Kaori Hanaoka

Molecular identification and pharmacological characterization of adenosine receptors in the guinea‐pig colon

The aim of this study is to elucidate the role of adenosine in the motor function of the guinea‐pig distal colon. To determine whether adenosine A1 receptors and A2B receptors are expressed in the guinea‐pig colon, we employed the reverse transcription‐polymerase chain reaction (RT–PCR). The gene expression of A1 receptor and A2B receptor was found for the first time in the guinea‐pig proximal and distal colon. Adenosine A1 agonist N6‐cyclopentyladenosine (CPA), and A1/A2 agonist 5′‐N‐ethylcarboxamidoadenosine (NECA) concentration‐dependently inhibited neurogenic responses to electrical field stimulation (EC50=1.07×10−8 and 2.12×10−8 M) in the longitudinal muscle, but A2A agonist 2‐p‐(2‐carboxyethyl)phenylethylamino‐5′‐N‐ethycarboxamido‐adenosine (CGS21680) had only a slight inhibitory effect (25.9%, 1 μM). A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 10 nM: A1 selective concentration) antagonized responses to CPA and NECA. Furthermore, the affinity order of antagonists at inhibiting the effect NECA was: DPCPX>8‐phenyltheophylline (8‐PT: A1/A2 antagonist). In the presence of tetrodotoxin (TTX, 0.3 μM), CPA and NECA relaxed myogenic precontraction induced by KCl (50 mM) (EC50=1.26×10−5 and 1.04×10−5 M, respectively), but CGS21680 (1 μM) did not cause any relaxation. DPCPX did not affect responses to CPA and NECA at a concentration of 10 nM, but a higher concentration (1 μM) of DPCPX and 10 μM of 8‐PT antagonized those responses. These data lead us to the hypothesis that adenosine may mediate relaxation through two different inhibitory receptor subtypes; A1 receptors on the enteric neuron and A2B receptor on the smooth muscle in the guinea‐pig distal colon.

FR255734, a Humanized, Fc-Silent, Anti-CD28 Antibody, Improves Psoriasis in the SCID Mouse-Psoriasis Xenograft Model

In psoriasis, CD28/B7 costimulatory molecules are well characterized. Here, using the severe combined immunodeficient (SCID) mouse-psoriasis xenograft model, we report therapeutic efficacy of a humanized anti-CD28 monoclonal antibody (FR255734; Astellas Pharmaceuticals Inc., Tokyo, Japan). Transplanted psoriasis plaques on the SCID mouse were treated weekly for 4 weeks with intraperitoneal injections of FR255734 at 10, 3, and 1-mg kg(-1) doses. Groups treated with doses of 10 and 3 mg kg(-1) had significant thinning of the epidermis and reduced HLA-DR-positive lymphocytic infiltrates. The length of the rete pegs changed from 415.2+/-59.6 to 231.4+/-40.4 microm (P<0.005) in the 10-mg kg(-1) group, and from 323.4+/-69.6 to 237.5+/-73.6 microm in the 3-mg kg(-1) group (P=0.002). Positive controls treated with CTLA4-Ig and cyclosporine had significant histological improvement, whereas plaques treated with saline and isotype controls (human and mouse IgG2) remained unchanged. In vitro studies have shown that FR255734 effectively blocked T-cell proliferation and proinflammatory cytokine production. These observations warrant studies to evaluate the efficacy of FR255734 in human autoimmune diseases.

Adenosine A1 receptor blockade reverses experimental postoperative ileus in rat colon

Inhibition of colonic propulsive motility is the main contributor to postoperative ileus in humans. Experimental models for investigating colonic propulsion in surgically induced postoperative ileus have not previously been available. This study was designed to assess whether adenosine A(1) receptor antagonists (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) reverse the colonic motility disorder in newly developed rat experimental ileus models. When rats underwent anesthesia (pentobarbital) or surgical traumas (partial gastrectomy, cecectomy or gentle touching of the colon with fingers), colonic propulsive motility was evaluated by migration of intracolonically injected dye in awake unrestrained rats. Propulsive motility resulted in significant decrease after the treatment of the anesthesia or partial gastrectomy. Intravenous administration of either adenosine A(1) receptor antagonist reversed the slowed colonic propulsion in these experimental ileus models. The present study suggests that the blockade of adenosine A(1) receptors has therapeutic potential for postoperative ileus.

Adenosine A1 receptor blockade reverses dysmotility induced by ischemia–reperfusion in rat colon

This study was designed to assess whether adenosine A1 receptor antagonists [(R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)] reverse dysmotility induced by ischemia-reperfusion in the rat colon. The gene of adenosine A1 receptor was expressed in the colon. Clamping (30 min) of the colonic marginal vessels was followed by reperfusion, and the propulsive colonic motility was evaluated. Propulsion was significantly slowed by ischemia-reperfusion, while FK352 and DPCPX abolished this delay. In contrast, the non-selective adenosine receptor antagonist, 8-phenyltheophylline, failed to affect the dysmotility. Thus, adenosine A1 receptor antagonists have potent therapeutic potential against ischemia-reperfusion-induced dysmotility in the colon.

Novel CD3‐specific antibody induces immunosuppression via impaired phosphorylation of LAT and PLCγ1 following T‐cell stimulation

The activation of T cells is known to be accompanied by the temporary downmodulation of the TCR/CD3 complex on the cell surface. Here, we established a novel monoclonal antibody, Dow2, that temporarily induces downmodulation of the TCR/CD3 complex in mouse CD4+ T cells without activating T cells. Dow2 recognized the determinant on CD3ε; however, differences were observed in the binding mode between Dow2 and the agonistic anti‐CD3ε Ab, 145–2C11. An injection of Dow2 in vivo resulted in T‐cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC‐γ1 and LAT in Dow2‐induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLCγ1 in Dow2‐induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG‐132. These results suggest that proteasome‐mediated degradation is involved in hypophosphorylated LAT and PLCγ1 in Dow2‐induced anergic T cells. The novel CD3‐specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression.

ASP0028 in combination with suboptimal-dose of tacrolimus in Cynomolgus monkey renal transplantation model

FTY720, a S1P-receptor modulator, has shown to be effective in several transplant and autoimmune disease models, via modulating lymphocyte homing into secondary lymphoid organs (SLOs), and thereby reducing these cells in peripheral blood. ASP0028, a newly developed S1P1/S1P5-selective agonist, presented comparable efficacy to FTY720 and wider safety margins than FTY720. In this study, we assessed the efficacy and safety of ASP0028 co-administered with suboptimal-dose of tacrolimus in the Cynomolgus monkey renal transplantation model. Seven animals in group-1 or group-2 received mono-tacrolimus 1.0mg/kg once a day (QD), or ASP0028 0.6mg/kg plus tacrolimus 1.0mg/kg QD, respectively. Eight animals in group-3 received ASP0028 1.2mg/kg plus tacrolimus 1.0mg/kg QD. The allograft median survival time (MST) in group-2 and group-3 were significantly extended to 41 and 61.5days, versus that of 28days in group-1 (p=0.036 and 0.001, respectively). ASP0028 administration remarkably reduced absolute numbers of peripheral lymphocytes, particularly subsets of CD4+/ or CD8+/naive and central memory cells, CD4+/Treg cells, and to a lesser extent on B cells, but not CD4+/ or CD8+/effector memory cells and NK cells. These data show ASP0028 combined with suboptimal-dose of tacrolimus effectively prolongs renal allograft survival in nonhuman primates (NHPs) with well tolerated safety, supporting its further investigation to optimize CNI-sparing regimens.

Prevention of chronic renal allograft rejection by AS2553627, a novel JAK inhibitor, in a rat transplantation model

BACKGROUND Janus kinase (JAK) inhibitors are thought to be promising candidates to aid renal transplantation. However, the effectiveness of JAK inhibitors against features of chronic rejection, including interstitial fibrosis/tubular atrophy (IF/TA) and glomerulosclerosis, has not been elucidated. Here, we investigated the effect of AS2553627, a novel JAK inhibitor, on the development of chronic rejection in rat renal transplantation. METHODS Lewis (LEW) to Brown Norway (BN) rat renal transplantation was performed. Tacrolimus (TAC) at 0.1mg/kg was administered intramuscularly once a day for 10 consecutive days starting on the day of transplantation (days 0 to 9) to prevent initial acute rejection. After discontinuation of TAC treatment from days 10 to 28, AS2553627 (1 and 10mg/kg) was orally administered with TAC. At 13weeks after renal transplantation, grafts were harvested for histopathological and mRNA analysis. Creatinine and donor-specific antibodies were measured from plasma samples. Urinary protein and kidney injury markers were also evaluated. RESULTS AS2553627 in combination with TAC exhibited low plasma creatinine and a marked decrease in urinary protein and kidney injury markers, such as tissue inhibitor of metalloproteinase-1 and kidney injury molecule-1. At 13weeks, histopathological analysis revealed that AS2553627 treatment inhibited glomerulosclerosis and IF/TA. In addition, upregulation of cell surface markers, fibrosis/epithelial-mesenchymal transition and inflammation-related genes were reduced by the combination of AS2553672 and TAC, particularly CD8 and IL-6 mRNAs, indicating that AS2553627 prevented cell infiltration and inflammation in renal allografts. CONCLUSIONS These results indicate the therapeutic potential of JAK inhibitors in chronic rejection progression, and suggest that AS2553627 is a promising agent to improve long-term graft survival after renal transplantation.

A chronic renal rejection model with a fully MHC-mismatched rat strain combination under immunosuppressive therapy☆

BACKGROUND The Fischer-to-Lewis (LEW) rat model of kidney transplantation is a widely accepted and well-characterized model of chronic rejection. In contrast to transplantation in a clinical setting, however, the absence of treatment with immunosuppressants and only minor mismatch of major histocompatibility complexes (MHCs) are critical discrepancies. Here, we established a rat model of chronic rejection using fully MHC-mismatched strains in which kidney disease progresses even under immunosuppressive therapy. METHODS LEW (RT1(l)) rats were used as donors and Brown Norway (BN, RT1(n)) rats as recipients. Intramuscular administration of 0.1mg/kg of tacrolimus was initiated on the day of transplantation. Post-transplantation, this dose was maintained until Day 9, suspended until Day 28 and then resumed from Day 29. Renal function, histopathology, and levels of donor-specific antibody (DSA) and several biomarkers of renal injury were assessed. RESULTS On Day 91 post-transplantation, recipients received tacrolimus treatment with short-term suspension exhibited reduced renal function and changes in histology. Those were characteristics of chronic rejection including glomerulosclerosis, interstitial fibrosis, and tubular atrophy in human transplantation recipients. Urinary protein excretion increased in a linear fashion, and elevated levels of several biomarkers of renal injury and DSA were observed even under administration of an immunosuppressant. CONCLUSIONS We established an allograft rejection model with impaired renal function and typical histopathological changes of chronic rejection in fully MHC-mismatched rats by controlling administration of an immunosuppressant. These findings suggest that this model more accurately reflects transplantation in a clinical setting than existing models and enables the evaluation of therapeutic agents.

Integrin, alpha9 subunit blockade suppresses collagen-induced arthritis with minimal systemic immunomodulation

Abstract Integrin, alpha9 subunit (hereinafter, alpha9) has been identified as a novel putative therapeutic target for rheumatoid arthritis (RA). Support for this target comes from the observations that alpha9 is overexpressed both in the joints of RA patients and in animal models of arthritis. In the experimental models, the increase in alpha9 expression precedes the onset of arthritic symptoms. The current study presents data on the pharmacological profile of an anti‐alpha9 antibody in a collagen‐induced arthritis (CIA) mouse model. Administration of an alpha9‐blocking antibody in CIA mice suppressed the development of arthritis and significantly decreased plasma level of activated fibroblast‐like synoviocyte (FLS)‐derived biomarkers without reducing the formation of anti‐type II collagen antibodies. While anti‐alpha9 antibody administration significantly suppress the accumulation of immune cells in arthritic joints it had no effect on immune cell number in the spleen. Furthermore, in non‐arthritic mice, alpha9 had no inhibitory effect in either a mixed lymphocyte reaction (MLR) or in a delayed type hypersensitivity (DTH) reaction. These results suggest that blocking alpha9 exerts its anti‐arthritic effect through suppression of FLS‐activation via a non‐immune mediated mechanism. Finally, therapeutic administration of anti‐alpha9 antibody alleviated established arthritis in CIA mice. Our data provide evidence that alpha9 blockade is a promising therapy for joint inflammation with minimal systemic immunomodulation.

Effective suppression of donor specific antibody production by Cathepsin S inhibitors in a mouse transplantation model

Abstract Donor‐specific antibodies (DSA) are a major risk factor for antibody‐mediated rejection (ABMR) in solid organ transplantation, and ABMR remains a medical challenge. Therefore, effective anti‐ABMR therapies are needed to improve overall graft survival. Cathepsin S (Cat S) is an essential protease for antigen peptide loading onto lysosomal/endosomal major histocompatibility complex (MHC) class II molecules to promote antigen presentation. Cat S deficiency produces immuno‐deficient phenotypes including a suppressed humoral immune response, and Cat S inhibition reportedly prevents autoimmunity. However, little is known about the effects of Cat S inhibitors on organ transplantation, especially ABMR. Here, we report the pharmacological profile of novel Cat S inhibitors, AS2761325 and AS2863995, and explore their preventive potential on DSA production and acute rejection in a mouse cardiac transplantation model. Cat S inhibitors potently inhibited upregulation of antigen peptide loading MHC class II expression on the surface of splenic B cells and suppressed ovalbumin‐induced T cell‐dependent antibody production in mice. In a mouse cardiac transplantation model, oral administration of AS2761325 monotherapy inhibited DSA production without affecting graft survival. When combined with a suboptimal dose of tacrolimus, AS2761325 significantly prolonged graft survival. The more potent Cat S inhibitor AS2863995 also prolonged graft survival and almost completely suppressed DSA production. These results suggest that Cat S inhibitors may be promising ABMR prophylaxis drug candidates. Combination therapy comprising a Cat S inhibitor and calcineurin inhibitors may be a more effective immunosuppressive maintenance therapy for controlling both cell‐mediated and antibody‐mediated rejection.