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Background: Urinary tract infections (UTIs) are the most common infectious complication in kidney transplant recipients (KTRs). The aim of this study to investigate the risk factors for and causative organisms of UTI as well as to evaluate the impact these diseases on allograft function in KTRs., Methods: We analyzed patients who underwent kidney transplantation (KT) between January 2000 and December 2010. Among a total of 344 KTRs, 50 (14.5%) patients experienced 106 UTI episodes during a mean follow-up of 35.9 ± 26.0 months. Twenty three patients experiencing recurrent UTI were compared with 27 nonrecurrent UTI patients and with 50 non-UTI patients matched for age, gender, and transplantation date., Results: The number of patients with renal calculi, diabetes, or prior dialysis was significantly greater among the UTI group compared with control subjects. In addition, the number of patients with renal calculi was significantly higher among the recurrent compared with the nonrecurrent cohort (43.5 vs 7.4%; P = .003). The most common causative organism was Escherichia coli (64.1%), followed by Enterococcus species (20.5%). Higher rates of antibiotic resistance, especially Extended Spectrum Beta-Lactamasc (ESBL) production, were observed among the recurrent compared with the nonrecurrent group (53.1 vs 0%; P = .013). The rate of decline of estimated glomerular filtration rate was significantly faster in the UTI than the non-UTI group, whereas it did not differ between the recurrent and nonrecurrent group., Conclusions: Adequate treatment of an initial UTI to prevent as recurrent infection and prolong graft longevity is especially reasonable for KTRs with renal calculi or in cases of antibiotic-resistant microorganisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mycophenolic acid (MPA) is an immunosuppressive agent that is widely used in clinical therapy, including pancreas and islet transplantation. Previously, we showed that MPA induces significant apoptosis of insulin-secreting cells by downregulating RhoGDI-α and increasing JNK expression. In this study, we investigated Rac1 directly associated with RhoGDI-α during MPA-induced apoptosis in INS-1E cells (an insulin-secreting cell line). Cells were treated with MPA for 24 and 36 hours. Immunoprecipitation was used to examine physical interactions between RhoGDI-α and Rac1. Activation and immunoprecipitation assays showed expressions of Rac1 and RhoGDI-α to be directly correlated. Rac1 binding to RhoGDI-α decreased after MPA treatment, and Rac1 was induced and subsequently activated by MPA. We concluded that this novel RhoGDI-α/Rac1/JNK pathway induced apoptosis of transplanted islet cells after MPA treatment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In islet transplantation, encapsulation of immunoisolated islets may provide a way to protect the graft from immune attacks with no immunosuppression. To develop an immunodelusive immunoisolated bioartificial pancreas (BAP), chondrocyte sheets were prepared by cell sheet engineering. We made an immunoisolated BAP encapsulated with rodent-derived chondrocyte sheets and then evaluated its function. Sprague-Dawley rats were used as the source of auricular cartilage and chondrocytes were maintained and expanded by passages. Lewis rats were prepared for islet isolation. A 3-dimensional chondrocyte sheeting immunodelusive immunoisolated BAP (CSI-BAP) was created by multi-layering and unifying the chondrocyte sheets. Subsequently, islets were embedded between each multi-layer sheet. To evaluate the function of the CSI-BAP, a glucose challenge test was performed and secretion of insulin in the culture medium was measured by an enzyme-linked immunosorbent assay. When observed by phase-contrast microscopy, the CSI-BAP maintained close connections between chondrocyte sheets. Islets in the CSI-BAP maintained viability at day 10 and showed good insulin secretion, as revealed by a prompt reaction to increased concentrations of glucose at days 5 and 10. In long-term culture, the CSI-BAP maintained its ability to secrete insulin for 8 weeks. This BAP technology could be an important tool for successful islet transplantation without immunosuppressive drugs., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Porcine-specific obstacles in islet isolation frequently result from the low purity or contamination with exocrine tissues. We implemented a new technique involving as capsulation of islets with excess exocrine tissue as a beneficial material to address those difficulties. Pig islets were hand-picked as high purity (HI) or low purity (LO) islets containing significant amounts of exocrine tissue. We performed static (ST) or shaking (SK) cultures of HI and LO islets. Islet function was examined after 24 hours by a glucose challenge test. Insulin secretion into the culture media was continuously measured using ELISA during a 6-day culture period. Islet function after 24 hours exhibited better maintenance under SK than ST culture as assessed by a stimulation index. The ideal islet morphology was seen in LO islets at 3 days of SK culture with typical islet shapes of a smooth surface and a spherical configuration. In contrast, typical islet morphology was not observed in HI islets under SK culture; maintenance of typical spherical appearances was difficult. Insulin secretion from LO islets under SK culture was higher than under other conditions during the 6-day period. Under SK culture conditions, exocrine-encapsulated LO islets showed enhanced islet function by condensing loose islet aggregates into firm spheroids with native exocrine tissues as a natural scaffold.

Optimal immunosuppression after pancreas islet transplantation has not yet been established to achieve long-term graft survival. Mycophenolic acid (MPA) is widely used as an immunosuppressive drug after transplantation including among recipients of pancreas islet cells. Previously, we reported MPA-induced islet apoptosis in the HIT-T15 cell line. In this study, we confirmed the effects of MPA on cell death and its potential implications on the mitogen-activated protein kinase (MAPK) family expression levels in primary isolated rat islets. Lewis islets isolated by collagenase digestion were purified by the density gradient method. Cell death was analyzed by methylthiazoletetrazolium assay. Activation of MAPK kinase 4 (MKK4), c-jun N-terminal protein kinase (JNK), p38 MAPK, and caspase-3 cleavage was examined by Western blot analyses. MPA treatments (> 25 micromol/L) increased cell death significantly at 24 hours and in a dose-dependent manner activated MKK4, JNK, and p38 MAPK at 20 hours. Caspase-3 cleavage was also increased by MPA treatment. These results suggested that MPA induced significant cell death among primary isolated rat islets by activation of MKK4, JNK, and p38 MAPK, as well as caspase-3 cleavage.

Interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) assay is a powerful tool for measuring the frequency of alloantigen-specific T cells, reflecting cellular immunity. We correlated the pretransplant frequencies of donor-specific and third-party-specific IFN-gamma ELISPOT tests, with the posttransplant outcomes of 45 recipients of living donor renal transplantations. The mean frequency of pretransplant donor-specific ELISPOT was significantly greater among patients with acute rejection episodes (ARE) than those without ARE (18.0 [12 to 50] versus 8.8 [5 to 30.4]) spots per 200,000 peripheral blood lymphocytes (PBLs; P=.024). A cutoff level of 12 spots per 200,000 PBLs on the donor-specific ELISPOT identified an ARE-positive patient with a sensitivity of 81.8% and a specificity of 64.7%. The recipients with pretransplant donor-specific ELISPOT+showed higher serum creatinine levels and lower glomerular filtration rate (GFR) at 6 posttransplant months (P<.05). Although the pretransplant third-party-specific ELISPOT results correlated with the donor-specific ELISPOT results (r=.783; P<.001), there was no significant difference in the third-party ELISPOT results between the ARE-positive and ARE-negative recipients. In conclusion, an analysis of pretransplant donor-specific IFN-gamma ELISPOT may identify the posttransplant risk of developing ARE and displaying decreased GFR at 6 months.

The antibody monitoring system (AMS, GTI Inc) is a solid enzyme-linked immunosorbent assay (ELISA) crossmatch test for the detection of immunoglobulin G (IgG) antibody to donor-specific solubilized HLA class I and class II antigens. The objective of this study was to compare the results of the AMS assay with donor-specific anti-HLA IgG antibodies (DS-HLA Abs), as determined by ELISA panel reactive antibody (PRA) and the flow cytometric crossmatch test (FCXM). A total of 107 sera were screened for the presence of HLA Abs by ELISA PRA (LAT-M, One-Lambda Inc), the DS-HLA Abs were determined in 34 serum samples (31.8%) by an ELISA panel (LAT class I and class II, One-Lambda Inc) and FCXM. The FCXM and AMS assays were performed with matched lymphocytes from 56 donors. There was a significant degree of concordance (89.7%) between the two tests (P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of AMS assay to detect DS-HLA Abs was 88.2%, 94.5%, 88.2%, and 94.5%, respectively. The AMS is a simple, objective test, which has several advantages over the cell-based crossmatch test, such as elimination of non-HLA antibody reactivity, elimination of non-donor-specific antibody reactivity, no need for viable cells, and preparation of the donor's HLA antigens in advance. In summary, this study suggested that AMS may be useful as a supportive crossmatch test or as a monitoring test after transplantation to detect class I or class II DS-HLA Abs.

The aim of the present study was to identify whether the serum interferon-gamma (IFNgamma), a Th1 cytokine, or soluble CD30 (sCD30), a marker for activation of Th2 cytokine-producing T cells, predict acute cellular rejection episodes among liver graft patients. Pretransplant and posttransplant sera from 32 living donor liver transplant recipients obtained on days 1, 3, and 7 after surgery were tested for serum IFNgamma and sCD30 concentrations using commercial enzyme-linked immunosorbent assay kits. Recipients with an acute rejection episode (ARE) (n=14) displayed significantly higher IFNgamma concentrations pretransplant than did the patients with no ARE (n=18) (P<.05). The pretransplant serum levels of sCD30 were not different between the non-ARE and ARE groups. However, in comparison with the non-ARE group, who showed steadily decreasing serum sCD30 levels after transplantation, 12 among the 14 patients in the ARE group showed increasing sCD30 levels from day 1 to day 3 after transplantation (P<.05). These results suggest that the sCD30 increment during the early period after liver transplantation affects the immune response of rejection. This observation emphasizes the clinical relevance of serum sCD30, in addition to serum IFNgamma, as predictive markers for acute liver graft rejection.

Introduction: We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine whether the Fas/FasL pathway participates in apoptosis within grafts during rejection., Materials and Methods: Orthotopic small bowel transplantation with portocaval drainage was performed from Brown Norway to Lewis (LEW) rats. Isografted (LEW --> LEW) and nontransplanted animals served as the controls. Animals were sacrificed on days 3, 5, on 7 after SBT (each n = 5). An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas or Fas-L., Results: The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7. Similarly, in the PPs of the allografts, the number of ISEL-positive mononuclear cells increased significantly on days 3, 5, and 7. On day 7 the number of Fas- and FasL-positive enterocytes were increased significantly in the allografts compared with the nontransplanted controls. Similarly, in the PPs, Fas- and FasL-positive mononuclear cells also increased significantly on day 7 in the allograft., Conclusion: Although an increase, number of apoptotic enterocytes and lymphocytes were observed in the early phase, activation of Fas/FasL system occurred during the late phase of small bowel graft rejection. These findings suggest that both rejection-associated and sepsis-induced forms of apoptosis may be associated with small bowel graft rejection.

Aim: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated lymphoid tissues (GALT). We performed a semiquantative analysis of MAdCAM-1 expression during small bowel graft rejection., Methods: Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Animals were sacrificed on days 3, 4, 5, 6, and 7 after SBT. Cryostat sections were prepared from grafts, including Peyer's patches (PPs). Indirect immunoperoxidase staining was performed using mAbs against MAdCAM-1. The degree of vascular endothelial staining on high endothelial venules (HEV) in the PPs was graded from 1 (low levels) to 5 (high levels), and in the vessels of the lamina propria from 1 (faint), 2 (low at the base of villi), 3 (low to the middle of villi), 4 (high to the middle of villi), to 5 (high to villus tip)., Results: MAdCAM-1 expression on HEVs in PPs was down-regulated during rejection. In contrast its expression on endothelial cells of vessels in the lamina propria was up-regulated during rejection., Conclusion: Alteration in MAdCAM-1 expression may be associated with the development of SB graft rejection. The vessels at the base of villi, which are associated with lymphocyte recruitment, may become sites of intense immune reactivity during the early phase of small bowel allograft rejection.