RNA expression profiling of nonhuman primate renal allograft rejection identifies tolerance.

Tolerance induction to prevent allograft rejection is a long-standing clinical goal. However, convincing and dependable tolerance identification remains elusive. Hypothesizing that intragraft RNA expression is informative in both rejection and tolerance, we profile intrarenal allograft RNA expression in a mixed chimerism renal allograft model of cynomolgus monkeys and identify biologically significant tolerance. Analysis of 67 genes identified 3 dominant factors, each with a different pattern of gene expressions, relating to T cell-mediated rejection (TCMR), chronic antibody-mediated rejection (CAMR), or Tolerance. Clustering these 3 factors created 9 groups. One of the 9 clustered groups, the Tolerance cluster, showed the lowest probability of terminal rejection, the longest duration of allograft survival, and the lowest relative risk of terminal rejection. The Tolerance factor consists of a novel set of gene expressions including cytokine and immunoregulatory genes adding mechanistic insights into tolerance. The Tolerance factor could not be identified within current pathologic diagnostic categories. The TCMR and CAMR factors are dominant to the Tolerance factor, causing rejection even if the Tolerance factor is present. These 3 factors determine the probability of terminal rejection or tolerance. This novel a posteriori approach permits identification of pathways of rejection, including tolerance.

RNA expression profiling of renal allografts in a nonhuman primate identifies variation in NK and endothelial gene expression.

RNA transcript expression estimates are a promising method to study the mechanisms and classification of renal allograft rejections. Here we use the Nanostring platform to profile RNA expression in renal allografts in a nonhuman primate (NHP), the Cynomolgus monkey. We analyzed protocol and indication 278 archival renal allograft samples, both protocol and indication from 76 animals with diagnoses of chronic antibody-mediated rejection (CAMR), acute cellular rejection (TCMR), and MIXED (both CAMR and TCMR), plus normals and samples with no pathological rejection using a Cynomolgus-specific probe set of 67 genes. Analysis identified RNA expression heterogeneity of endothelial and NK genes within CAMR and TCMR, including the stages of CAMR. Three factors were partitioned into additional groups. One group with the longest allograft survival time is pure CAMR without NK or CD3. Three mixed groups show variation in NK and CD3. TCMR was split into 2 groups with variation in NK genes. Additional validation of the complete gene-set correlated many of the genes with diagnoses of CAMR, MIXED, and TCMR rejections and with Banff histologic criteria defined in human subjects. These NHP data demonstrate the utility of RNA expression profiling to identify additional heterogeneity of endothelial and NK RNA gene expressions.

Chimerism-based tolerance in organ transplantation: preclinical and clinical studies.

Induction of allograft tolerance has been considered the ultimate goal in organ transplantation. Although numerous protocols to induce allograft tolerance have been reported in mice, a chimerism-based approach through donor haematopoietic stem cell transplantation has been the only approach to date that induced allograft tolerance reproducibly following kidney transplantation in man. Renal allograft tolerance has been achieved by induction of either transient mixed chimerism or persistent full donor chimerism. Although the risk of rejection may be low in tolerance achieved via durable full donor chimerism, the development of graft-versus-host disease (GVHD) has limited the wider clinical application of this approach. In contrast, tolerance induced by transient mixed chimerism has not been associated with GVHD, but the risk of allograft rejection is more difficult to predict after the disappearance of haematopoietic chimerism. Current efforts are directed towards the development of more clinically feasible and reliable approaches to induce more durable mixed chimerism in order to widen the clinical applicability of these treatment regimens.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes.

Molecular testing represents a promising adjunct for the diagnosis of antibody-mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin-fixed paraffin-embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10-fold cross-validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non-AMR samples (area under the curve = 0.92). This three-gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39-0.63, p < 0.001). Principal component analysis confirmed the association between three-gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell-mediated rejection (TCMR). Elevated three-gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet-Kidney Transplantation in Nonhuman Primates.

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

Publisher's Note: Search for Majorana Neutrinos Near the Inverted Mass Hierarchy Region with KamLAND-Zen [Phys. Rev. Lett. 117, 082503 (2016)].

This corrects the article DOI: 10.1103/PhysRevLett.117.082503.

Search for Majorana Neutrinos Near the Inverted Mass Hierarchy Region with KamLAND-Zen.

We present an improved search for neutrinoless double-beta (0νßß) decay of ^{136}Xe in the KamLAND-Zen experiment. Owing to purification of the xenon-loaded liquid scintillator, we achieved a significant reduction of the ^{110m}Ag contaminant identified in previous searches. Combining the results from the first and second phase, we obtain a lower limit for the 0νßß decay half-life of T_{1/2}^{0ν}>1.07×10^{26} yr at 90% C.L., an almost sixfold improvement over previous limits. Using commonly adopted nuclear matrix element calculations, the corresponding upper limits on the effective Majorana neutrino mass are in the range 61-165 meV. For the most optimistic nuclear matrix elements, this limit reaches the bottom of the quasidegenerate neutrino mass region.

Efficacy and safety of once-weekly dulaglutide in combination with sulphonylurea and/or biguanide compared with once-daily insulin glargine in Japanese patients with type 2 diabetes: a randomized, open-label, phase III, non-inferiority study.

AIMS: To evaluate 0.75 mg of dulaglutide, a once-weekly glucagon-like peptide-1 receptor agonist, compared with once-daily insulin glargine for glycaemic control in Japanese patients with type 2 diabetes (T2D). METHODS: In this phase III, randomized, open-label, parallel-group, 26-week study, 361 patients with inadequately controlled T2D receiving sulphonylureas and/or biguanides, aged ≥20 years, with glycated haemoglobin (HbA1c) levels 7.0-10.0% (53-86 mmol/mol), inclusive, were randomized (1 : 1) to receive dulaglutide or glargine. Participants and investigators were not masked to treatment allocation. The primary measure was change from baseline in HbA1c at 26 weeks, analysed using a mixed-effects model for repeated measures, with a predefined non-inferiority margin of 0.4%. RESULTS: At week 26, least-squares (LS) mean (standard error) reductions in HbA1c were -1.44 (0.05)% [-15.74 (0.55) mmol/mol] in the dulaglutide group and -0.90 (0.05)% [-9.84 (0.55) mmol/mol] in the glargine group. The mean between-group treatment difference in HbA1c was -0.54% (95% CI -0.67, -0.41) [-5.90 mmol/mol (95% CI -7.32, -4.48)]; p < 0.001. Dulaglutide significantly reduced body weight compared with glargine at week 26 (LS mean difference -1.42 kg, 95% CI -1.89, -0.94; p < 0.001). The most frequent adverse events with dulaglutide treatment were nasopharyngitis and gastrointestinal symptoms. The incidence of hypoglycaemia was significantly lower with dulaglutide [47/181 (26%)] compared with glargine [86/180 (48%)], p < 0.001. CONCLUSION: In Japanese patients with T2D uncontrolled on sulphonylureas and/or biguanides, once-weekly dulaglutide was superior to once-daily glargine for reduction in HbA1c at 26 weeks. Although dulaglutide increased gastrointestinal symptoms, it was well tolerated, with an acceptable safety profile.

Pilot Study Evaluating Regulatory T Cell-Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model.

The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, anti-interleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor-specific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.

Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti-CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti-CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti-thymocyte globulin and a 1-month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti-CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long-term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long-term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti-CD154 mAb for promoting chimerism and renal allograft tolerance.

Long-term hepatic allograft acceptance based on CD40 blockade by ASKP1240 in nonhuman primates.

Blockade of the CD40-CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti-CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti-CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40-CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2-week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6-month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240-treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.

Identification of an ovarian clear cell carcinoma gene signature that reflects inherent disease biology and the carcinogenic processes.

Ovarian clear cell carcinoma (OCCC) shows unique clinical features including an association with endometriosis and poor prognosis. We previously reported that the contents of endometriotic cysts, especially high concentrations of free iron, are a possible cause of OCCC carcinogenesis through iron-induced persistent oxidative stress. In this study, we conducted gene expression microarray analysis using 38 ovarian cancer cell lines and identified genes commonly expressed in both OCCC cell lines and clinical samples, which comprise an OCCC gene signature. The OCCC signature reproducibly predicts OCCC specimens in other microarray data sets, suggesting that this gene profile reflects the inherent biological characteristics of OCCC. The OCCC signature contains known markers of OCCC, such as hepatocyte nuclear factor-1beta (HNF-1beta) and versican (VCAN), and other genes that reflect oxidative stress. Expression of OCCC signature genes was induced by treatment of immortalized ovarian surface epithelial cells with the contents of endometriotic cysts, indicating that the OCCC signature is largely dependent on the tumor microenvironment. Induction of OCCC signature genes is at least in part epigenetically regulated, as we found hypomethylation of HNF-1beta and VCAN in OCCC cell lines. This genome-wide study indicates that the tumor microenvironment induces specific gene expression profiles that contribute to the development of distinct cancer subtypes.

Does the permanent portacaval shunt for a small-for-size graft in a living donor liver transplantation do more harm than good?

In order to obviate a small-for-size graft syndrome (SFSGS), a portacaval (PC) shunt had been considered in a case of adult-to-adult living donor liver transplantation (AA-LDLT). In a recent AA-LDLT case, we adopted the PC shunt to resolve SFSGS; however, graft atrophy was observed in the late period of LDLT, thereby resulting in liver dysfunction. Due to the surgical closure of the PC shunt at 11 months post-LDLT, the graft regenerated gradually and resulted in the recovery of the liver function. This experience indicates that the portacaval shunt would overcome SFSGS in the early period of LDLT, while it would cause the graft atrophy and the graft dysfunction in the late period of LDLT.

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant.

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.

Quantitative Beutler test for newborn mass screening of galactosemia using a fluorometric microplate reader.

BACKGROUND: The Beutler enzyme spot test is an effective assay for newborn mass screening of galactosemia, but it is qualitative and relies on visual interpretation. We describe a quantitative, instrumental modification of the assay. METHODS: We modified the macroscopic visual Beutler enzyme spot test by adding extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microplate reader and personal computer. All handling was performed in microplates. The measurement time was 90 min. RESULTS: Fluorescence intensity (FI) of healthy controls correlated with hematocrit and galactose-1-phosphate uridyltransferase (GALT) activity. Patients with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by FI. FI decreased to 75% of the initial activity after storage at 25 degrees C for 3 days and to 40% after storage at 37 degrees C for 7 days. Screening of 46 742 newborns yielded 1 false-positive result (in a heterozygous carrier), 1 patient with glucose-6-phosphate dehydrogenase deficiency, and no apparent false negatives as judged by concurrent measurements of galactose and galactose-1-phosphate. CONCLUSIONS: The quantitative Beutler test can provide precise GALT activity in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, and anemia. This method is clinically useful, simple, automated, and highly reliable for newborn mass screening of galactosemia.

Life-long treatment for phenylketonuria.

Life-long low-phenylalanine diet is recommended as an additional determinant to achieve the maximal success in the treatment of phenylketonuria (PKU). In this context education of teenage patients with PKU is essential.

Molecular characterization of phenylketonuria in Japanese patients.

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T2781 (7.3%), E6nt-96A-->g (6.1%), Y356X (4.9%), R111X (3.7%), and 442-706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83+/-0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99+/-0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9-1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder.

Evaluation of highly sensitive thyrotropin assay for detecting thyroid diseases in neonatal screening: preliminary studies.

TSH concentrations in dried blood samples on filter paper were determined by a conventional enzyme-linked immunosorbent assay (ELISA), used for routine neonatal screening for primary hypothyroidism, and a highly sensitive bioluminescence ELISA (BL-ELISA) using firefly luciferase to examine whether central hypothyroidism and hyperthyroidism can be efficiently detected as cases of primary hypothyroidism. Samples were obtained from 3 patients with congenital central hypothyroidism, 5 patients with congenital primary hypothyroidism, 6 patients with hyperthyroidism, 31 neonatal babies with low birth weight (premature babies) and 242 newborn babies with normal birth weight from the general population (normal babies). The TSH values were low in central hypothyroidism and hyperthyroidism. Their deviations from the mean TSH value for normal babies by the BL-ELISA method (-3.12 SD and -4.79 SD in central hypothyroidism and hyperthyroidism respectively) were greater than those by the ELISA method (-2.00 SD and -2.97 SD respectively). The TSH values were high in primary hypothyroidism and normal in premature babies while deviations were the same when BL-ELISA and ELISA were used. These findings indicate that the highly sensitive TSH assay (BL-ELISA) can be used for detecting both primary and central hypothyroidism as well as hyperthyroidism in neonatal screening.

Low diastolic blood pressure, hypoalbuminemia, and risk of death in a cohort of chronic hemodialysis patients.

In a previous report, we showed that nutritional status and especially serum albumin had great predictive value for death in chronic hemodialysis patients, whereas blood pressure did not. In the present study, we analyzed the causes of death in consideration of the relationship between serum albumin and blood pressure. A total of 1,243 Okinawan patients (719 males, 524 females) undergoing hemodialysis in January 1991 were followed up through the end of 1995. Three hundred forty-two of the patients died, 45 received transplants, and 12 were transferred by the end of the follow-up period. The total duration of observation was 5,110.3 patient-years. Blood pressure as well as clinical and laboratory variables were determined immediately prior to the first dialysis session in January 1991. The crude death rate was 40.0% when the diastolic blood pressure (DBP) <70 mm Hg, 35.0% at 70 to 79 mm Hg, 25.0% at 80 to 89 mm Hg, 25.0% at 90 to 99 mm Hg, and 13.0% at >100 mm Hg. The death rate showed an inverse correlation with DBP. DBP showed a significant positive correlation with serum albumin (r = 0.137, P < 0.001) and age (r = -0.325, P < 0.0001). The adjusted odds ratio (95% confidence interval) of death was 0.84 (0.71 to 0.99) with 10 mm Hg increments in DBP when the reference DBP was less than 69 mm Hg. Low DBP may be a manifestation of malnutrition and/or cardiovascular disease in chronic hemodialysis patients. Target DBP levels may be higher levels in chronic hemodialysis patients than the general population.