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Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine kinase, has been identified as a potential therapeutic target in human bladder cancer. In the present study, we investigated the antitumor effect of a small molecule GSK-3β inhibitor, 9-ING-41, currently in clinical studies in patients with advanced cancer, in bladder cancer cell lines. We found that treatment with 9-ING-41 leads to cell cycle arrest, autophagy and apoptosis in bladder cancer cells. The autophagy inhibitor chloroquine potentiated the antitumor effects of 9-ING-41 when tested in combination studies. Our findings also demonstrate that 9-ING-41 enhanced the growth inhibitory effects of gemcitabine or cisplatin when used in combination in bladder cancer cells. Finally, we found that 9-ING-41 sensitized bladder cancer cells to the cytotoxic effects of human immune effector cells. Our results provide a rationale for the inclusion of patients with advanced bladder cancer in clinical studies of 9-ING-41.

Background Our initial studies utilizing a galactosyl-α1-3-galactosyltransferase gene knockout (GalTKO) pig-to-baboon renal transplant model demonstrated that the early development of nephrotic syndrome has been a significant obstacle to the long-term survival of baboon recipients. We have recently documented that sphingomyelin phosphodiesterase-3 (SMPDL3b) and CD80 expressed on podocytes in porcine kidney grafts contribute to this complication. We have hypothesized that one regulator of immune function is CD47 and that incompatibilities in CD47 between pig and baboon could potentially affect macrophage function, increasing the susceptibility of the kidney grafts to immunologically induced injury. Methods In order to address this hypothesis in vitro, we isolated and cultured porcine podocytes and ECs from GalTKO alone, human CD47 (hCD47)/hCD55 expressing transgenic (Tg) GalTKO swine, and GalTKO hCD46/hCD55 Tg swine along with baboon or human macrophages. Results We found that baboon macrophages phagocytosed porcine ECs in a similar manner to human macrophages, and this response was significantly reduced when porcine ECs and podocytes expressed hCD47/hCD55 but not hCD46/hCD55 without hCD47. Furthermore, masking hCD47 by anti-hCD47 antibody on hCD47/hCD55Tg ECs restored phagocytosis. These results are consistent with the hypothesis that CD47 incompatibility plays an important role in promoting macrophage phagocytosis of endogenous cells from the transplanted kidney. Conclusions The similar levels of phagocytosis of porcine cells by baboon and human macrophages suggest that the expression of hCD47Tg on glomerular cells in donor porcine kidneys may prove to be a key strategy for preventing proteinuria following kidney xenotransplantation in a pig-to-human as well as a pig-to-baboon model.

Purpose: The purposes of the present study were to evaluate growth rate of nonfunctioning adrenal incidentalomas (AIs) and their development to hormonal hypersecretion on follow-up. Materials and methods: A retrospective study was conducted from the electronic medical records. A total of 314 patients were diagnosed with adrenal tumors between 2000 and 2016. After excluding patients who had overt adrenal endocrine disorders or whose adrenal tumors were clinically diagnosed as metastatic malignancies, we investigated 108 patients with nonfunctioning AIs including characteristics, the treatment, the way of follow-up and pathology. Results: Fifteen patients received immediate adrenalectomy because of the initial tumor size or patient's preference. Pathological examination revealed malignancy in 2 patients. In the remaining 93 patients, radiological examinations were performed periodically. Tumor enlargement of ≥ 1.0cm was observed in 8.6% of the patients who were followed up as nonfunctioning AIs with a median follow-up period of 61.5 months (range: 4-192). Eleven patients underwent adrenalectomy. On the pathological examinations, all of the tumors, which showed a size increase, were diagnosed as benign tumors. Regarding the followed up patients without adrenalectomy, only 2.4% of the patients had tumor enlargement during the prolonged follow-up. Furthermore, none of the patients developed hormonal hypersecretion or clinical signs such as obesity, glucose intolerance or poorly controlled hypertension. Conclusions: Tumor enlargement of AIs did not correlate with malignancy. The value of repeat radiological and hormonal examinations may be limited in the long-term follow-up of patients whose AIs are not enlarged.

We have previously reported that co-transplantation of the kidney with vascularized donor thymus from α-1,3-galactosyltransferase gene knockout pigs with an anti-CD154 with rituximab-based regimen led to improved xenograft survival in baboons with donor-specific unresponsiveness. However, nephrotic syndrome emerged as a complication in which the glomeruli showed mild mesangial expansion with similarities to minimal change disease (MCD) in humans. Since MCD is associated with CD80 expression in glomeruli and elevated urinary excretion, we evaluated a potential role for CD80 in xenograft nephropathy. Study 1 confirmed high urinary CD80 excretion in nephrotic animals with renal xenografts showing CD80 expression in glomeruli. In Study 2, baboons receiving xenografts received CTLA4-Ig once a week from the second postoperative week or no CTLA4-Ig. The non-CTLA4-Ig group developed severe proteinuria with modest mesangial expansion with high urinary excretion of CD80 and documented CD80 expression in glomerular podocytes. All of the recipients in non-CTLA4-Ig groups had to be euthanized before POD 60. In contrast, CTLA4-Ig group showed a marked reduction in proteinuria and survived significantly longer, up to 193 days. These results demonstrate that anti-CD80 targeted therapy represents a promising strategy for reduction of proteinuria following renal xeno-transplantation with improved survival.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naive donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naive kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naive donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

Background We have previously demonstrated that the juvenile thymus plays an essential role in tolerance induced by both renal transplantation and a short course of calcineurin inhibitors. Aged thymi have a decreased ability to induce tolerance. Luteinizing hormone-releasing hormone (LHRH) is known to pharmacologically rejuvenate the thymus in rodents. In order to develop a clinically applicable regimen of transplantation tolerance in adults, we sought to determine if thymic rejuvenation would occur with LHRH agonism in non-human primates. Methods and results Thymic rejuvenation was evaluated by magnetic resonance imaging (MRI), histology, as well as in-vitro cellular and molecular tests. Four aged male hamadryas baboons underwent subcutaneous injection of a 3-month depot of Lupron (11.25 mg; LI) and were followed for 3 months. Thymi increased volumetrically by MRI. After LI, thymic cellularity markedly increased within the cortical and medullary thymus. Additionally, a significant increase in the CD4+/CD45RAhi + population in the peripheral blood occurred for 50 days after LI, and flow cytometry of thymic tissue revealed a large increase in the percentage of CD4+/CD8+ cells. TREC assay corroborated enhancement in thymic function. Conclusion These data indicate that LI is associated with thymic rejuvenation in baboons, and further confirm that extrinsic factors play an important role in thymic rejuvenation in a non-human primate model.

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12-day course of high-dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC-mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC-mismatched recipients treated with high-dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor-specific responsiveness in cell-mediated lympholysis and mixed-lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third-party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor-specific unresponsiveness in vitro. While kidney-induced cardiac allograft tolerance (KI-CAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance-resistant organs.

Diphtheria toxin (DT)-based anti-CD3 immunotoxins have clinical relevance in numerous applications including autoimmune disease therapies and organ transplantation tolerance protocols. Pre-existing anti-DT antibodies acquired either by vaccination against diphtheria toxin or infections with C. diphtheriae may interfere or inhibit the function of these anti-CD3 immunotoxins. Previously, a full-length anti-rhesus monkey CD3 immunotoxin, FN18-CRM9, was shown to be less effective at depleting circulating T cells in animals with pre-existing anti-DT antibody titers than in animals without antibodies, and subsequent doses were ineffective. In this study, the T cell depletion function of a truncated DT based recombinant anti-monkey CD3 immunotoxin, A-dmDT390-scfbDb (C207), as part of a reduced intensity conditioning regimen prior to hematopoietic cell transplantation, was compared between two groups of monkeys: those with and without pre-existing anti-diphtheria titers. T cell depletion was comparable in both groups of monkeys, and therefore appeared to be unaffected by the presence of moderate levels of pre-existing anti-diphtheria antibodies.

Basiliximab is a recently developed immunosuppressive agent for the prevention of acute allograft rejection in renal transplant recipients. The combination use of basiliximab and a calcineurin inhibitor was suggested to be more effective in comparison to immunosuppressive therapy using calcineurin inhibitor without basiliximab. Cyclosporine has been generally administered with basiliximab for renal transplant recipients. However, in cases of tacrolimus-based immunosuppressive regimen, the clinical efficacy and safety of combined use of tacrolimus and basiliximab remains to be elucidated. This study evaluated the tacrolimus pharmacological efficacy using a lymphocyte immunosuppressant sensitivity test (LIST) with MTT assay procedures in 16 cases of renal transplant recipients treated by tacrolimus without basiliximab and in 13 cases treated by tacrolimus in combination with basiliximab. The rate of acute rejection episodes in the recipients treated with tacrolimus plus basiliximab was 1/13 (7.7%), whereas the rate in the recipients treated with tacrolimus without basiliximab was 6/16 (37.5%). The recipients were divided into two groups according to their peripheral blood mononuclear cell (PBMC) sensitivity to tacrolimus [i.e., including a tacrolimus high sensitivity group (IC50 50 >1.0 ng/ml). In the recipients treated with tacrolimus without basiliximab, the rate of acute rejection episodes in the tacrolimus high sensitivity group was 1/10 (10.0%), which was significantly lower than the rate in the low sensitivity group of 5/6 (83.3%; p = 0.008). The incidence of cytomegalovirus infection was not significantly different between the tacrolimus high and the low sensitivity groups of the recipients treated with tacrolimus with and without basiliximab. Therefore, in the case of selected tacrolimus-based immunosuppressive therapy for renal transplant recipients, the tacrolimus pharmacological efficacy should be evaluated using LIST at a time just before the transplant procedure in order to accurately predict allograft rejection. The data also suggested that low tacrolimus sensitivity recipients should be treated with tacrolimus-based immunosuppressive therapy in combination with basiliximab.

Background Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Methods Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography–tandem mass spectrometry. Enrichment analysis was then performed. Results The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. Conclusions The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney. Electronic supplementary material The online version of this article (doi:10.1007/s10157-012-0708-1) contains supplementary material, which is available to authorized users.

Background Recruitment of cofactors in the interaction of the androgen receptor (AR) and AR ligands plays a critical role in determining androgenic/antiandrogenic effects of the AR ligand on signaling, but the functions of key cofactors, including nuclear receptor coactivator (NCOA), remain poorly understood in prostate cancer cells treated with AR ligands. Methods We examined prostate cancer cell lines LNCaP and VCaP expressing mutated and wild-type ARs, respectively, to clarify the significance of NCOAs in the effect of antiandrogens. Hydroxyflutamide showed antagonistic activity against VCaP and an agonistic effect on LNCaP. Bicalutamide served as an antagonist for both. We analyzed mRNA transcription and protein expression of NCOAs in these cells pretreated with dihydrotestosterone and thereafter treated with the mentioned antiandrogens. Transcriptional silencing of candidate NCOAs and AR was performed using small interfering RNA (siRNA). Cell proliferation was evaluated with MTT assay. Results LNCaP treated with bicalutamide showed an about four-fold increase in the expression of NCOA2 mRNA compared to those pretreated with dihydrotestosterone alone (P