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Jasn2011060543 1.10Upregulation of miR-142-3p in Peripheral BloodMononuclear Cells of Operationally Tolerant Patientswith a Renal Transplant Richard Danger,*† Annaïck Pallier,* Magali Giral,*†‡ Marc Martínez-Llordella,§Juan José Lozano, Nicolas Degauque,* Alberto Sanchez-Fueyo,§ Jean-Paul Soulillou,*†‡and Sophie Brouard*‡ *Institut National de la Santé Et de la Recherche Médicale UMR643 and Institut de Transplantation Urologie,Néphrologie, Nantes, France; †Université de Nantes, Nantes, France; ‡Centre Hospitalier Universitaire Hôtel-Dieu,Nantes, France; §Liver Unit, Hospital Clinic Barcelona, CIBEREHD, Barcelona, Spain; and Bioinformatics Platform,CIBEREHD, Barcelona, Spain ABSTRACTAchieving drug-free tolerance or successfully using only small doses of immunosuppression is a major goalin organ transplantation. To investigate the potential mechanisms by which some kidney transplantrecipients can achieve operational tolerance, we compared the expression proﬁles of microRNA inperipheral blood mononuclear cells of operationally tolerant patients with those of stable patients treatedwith conventional immunosuppression. B cells from operationally tolerant patients overexpressed miR-142-3p. The expression of miR-142-3p was stable over time and was not modulated by immunosuppres-sion. In Raji B cells, overexpression of miR-142-3p modulated nearly 1000 genes related to the immuneresponse of B cells, including potential miR-142-3p targets and molecules previously identiﬁed in theblood of operationally tolerant patients. Furthermore, our results suggested that a negative feedbackloop involving TGF-b signaling and miR-142-3p expression in B cells may contribute to the maintenance oftolerance. In summary, miR-142-3p expression in peripheral blood mononuclear cells correlates with op-erational tolerance. Whether upregulation of miR-142-3p modulates inﬂammatory responses to promotetolerance or is a result of this tolerance state requires further study.
J Am Soc Nephrol 23: ccc–ccc, 2012. doi: 10.1681/ASN.2011060543 The use of minimal doses of immunosuppression or performing biopsies in such patients can be even achievement of drug-free tolerance is a major challenging in terms of ethical considerations and goal in organ transplantation.1,2 Although the kid- patient adherence. In these different studies, several ney is less susceptible to successful immunosup- key pathways were highlighted, such as a pathway pressive drug withdrawal than the liver, where implicating the TGF-b gene;10 in addition, several around 20% of transplant patients can be success- other genes have been highlighted as "key leader fully weaned off immunonsuppression,3,4 an in- genes," such as BANK-1 (B-cell scaffold protein creasing number of kidney transplant recipientswho continue to display good graft function in Received June 7, 2011. Accepted November 18, 2011.
the absence of immunosuppressive drugs havebeen described in the literature.5–8 We and others J.-P.S. and S.B. contributed equally to this work.
have looked at the gene expression proﬁle in Published online ahead of print. Publication date available at PBMCs of such "operationally tolerant" kidney transplant recipients.7–12 The blood is a popular Correspondence: Dr. Sophie Brouard, INSERM UMR 643- 30 Bd choice for analysis because it provides a noninva- Jean Monnet, 44093 Nantes Cedex 93, France. Email: sive means for potential biomarker discovery, which is important in the case of tolerance; Copyright 2012 by the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 ISSN : 1046-6673/2304-ccc with ankyrin repeats 1),11 a modulator of B-cell hyperactiva- in PBMCs from healthy volunteers before and 24 hours after tion through AKT upon CD40 activation.13 The implication of phytohemagglutinin A (PHA) and IL-2 stimulation B-cell–related genes correlated with a high number of B cells14 Fifty-two miRNAs displayed a fold change and to a gene signature enriched in B-cell–related genes in the (FC) superior to 2 (i.e., log2FC.1) and 59 others, an FC in- blood of operationally tolerant kidney recipients, which was sub- ferior to 2 (i.e., log2FC.-1), indicating a strong modulation of sequently conﬁrmed in three different studies.7,8,12 The role of B expression after PHA/IL-2 stimulation cells has also been reported in experimental rodent models, in and Among the eight miRNAs dif- which transfer of B cells from tolerant rats prolonged graft ferentially expressed between operationally tolerant and STA survival when administered to untreated recipients.15 recipients, miR-450b-5p, miR-142-3p, and miR-324-5p were The mechanisms involved in the maintenance of this downregulated (FC=0.21, 0.29, and 0.42, respectively; Figure phenomenon remain elusive, and the identiﬁcation of related 1B) and miR-876-3p was upregulated (FC=3.4; Figure 1B) biomarkers remains instrumental to achieving safe drug after PHA/IL-2 stimulation. We conﬁrmed the downregula- minimization or complete weaning in clinical practice.
tion of miR-142-3p as early as 2 hours and at 24 hours after miRNA are small, endogenous, noncoding RNAs that can PHA/IL-2 stimulation, using individual Taqman microRNA regulate the expression of a variety of genes by directly destabi- assays (Figure 2A).
lizing their target mRNA.16,17 Furthermore, the incomplete Because miR-142-3p (1) appeared as the highest differen- pattern of target recognition allows a single miRNA to target tially expressed miRNA between operationally tolerant and hundreds of mRNAs, and conversely, a single mRNA to be STA recipients, (2) was underexpressed after PHA/IL-2 stim- targeted by multiple miRNAs, thus affecting a broad range of ulation (Figure 2A), (3) is speciﬁc to the hematopoietic line- gene networks.18 Numerous studies have reported on the dif- age,24–26 and (4) plays a role in lymphocyte functions,27 we ferential expression of miRNA in physiologic disorders or dis- focused on the potential role of this miRNA in our study. This eases,19,20 and miRNA has been found to be modulated in biopsy miRNA is overexpressed in operationally tolerant compared specimens from kidney transplant recipients.21–23 In this with STA patients according to TLDA assays (Figure 1C).
study, we investigated whether miRNA is modulated in theblood of patients with operational tolerance compared with a Validation of miR-142-3p Overexpression and cohort of patients with stable graft function under classic Stability over Time in PBMCs from Operationally Tolerant RecipientsIndividual miR-142-3p Taqman quantitative PCR (qPCR)assays displays a good correlation (r=0.885; P,0.0001) (Figure 1D). Furthermore, we validated the overexpression of miR-142-3p in operationally tolerant recipients on 16 independent miRNA Proﬁling in PBMC from Kidney PBMC samples (6 operationally tolerant and 10 STA patients) Transplant Recipients (P=0.02; FC=1.58) (Figure 2B). The post-transplantation We ﬁrst searched for a global miRNA proﬁle in PBMCs from time was signiﬁcantly higher in operationally tolerant than kidney transplant recipients using miRNA Taqman low- in STA patients (P=0.02; Table 1); however, we did not found density arrays (TLDAs). The expression of 381 miRNAs was any correlation between this time and miR-142-3p expression measured in PBMCs from 9 operationally tolerant patients and value (r=0.0342; P=0.845; . No signif- 10 STA recipients. A total of 266 miRNAs were expressed with a icant difference was observed between the subgroups of pa- quantiﬁcation cycle (Cq) inferior to 35 in at least half of sam- tients for any of the clinical variables tested (age, sex, creatine ples from each group. We selected the eight top-ranked miRNAs level in blood, proteinuria, number of HLA mismatches). Fi- on the basis of Mann–Whitney tests between the two groups of nally, the expression of mir-142-3p was stable over time, as patients (Figure 1, A and B). According to the expression values tested in PBMCs collected from three operationally tolerant of these eight miRNAs, the clear separation of the two groups of recipients at two different time points (5.5, 11, and 13 months) patients by clinical status was further observed using principal (Figure 2C).
component analysis Among the eightdifferentially expressed miRNAs, four were overexpressed Overexpression of miR-142-3p in the Blood of (miR-450b-5p, miR142-3p, miR-876-3p, and miR-106b) and Operationally Tolerant Recipients Is Not Due four were underexpressed (miR508-3p, miR-148b, miR-324-5p, to the Absence of Immunosuppression and miR-98) in PBMCs from operationally tolerant compared Because operationally tolerant patients display stable graft with STA patients.
function but no longer receive immunosuppression andbecause healthy volunteers also displayed an increased expres- miRNA Proﬁling in PBMCs from Healthy Volunteers sion of miR-142-3p compared with STA patients (P=0.0038; after Polyclonal Activation FC=1.54) (data not shown), we hypothesized that immuno- To further analyze the basal expression and modulation of suppression treatment may modulate the blood expression of these eight speciﬁc miRNAs, we performed miRNA proﬁling miR-142-3p. We thus measured its expression in PBMCs from Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012
Figure 1. Differential miRNA in PBMCs from operationally tolerant compared with STA kidney transplant recipients. (A) The eight top-ranked miRNAs according to Mann–Whitney tests are represented in this heat map, in which blue bars represent STA patients (n=10)and green bars represent operationally tolerant (n=10) patients. The heat map represents normalized and color-coded relative ex-pression values (22DDCq), in which red values indicate overexpression and green values indicate underexpression. (B) The eight miRNAare classiﬁed according to their uncorrected P values, which were used only for ranking miRNA rather than for an absolute identiﬁcationof differential miRNA. FCs of miRNA after PHA/IL-2 activation of PBMCs from healthy volunteers are displayed in last column. (C)Relative expression values of miR-142-3p from TLDA assays are displayed in a scatter plot. (D) Correlation between TLDA assays andindividual Taqman qPCR assays for miR-142-3p (9 operationally tolerant and 10 STA patients).
tolerant liver transplant recipients before and after the patients compared with STA recipients (P=0.01; FC= 2.84) (Figure 3B), entered an immunosuppressive drug weaning protocol whereas no difference was observed compared with healthy (n=27), a population not available in the context of kidney volunteers (data not shown). Therefore, puriﬁed B cells from transplantation. No difference was found in the PBMC expres- healthy volunteers were cultured over 24 hours with physio- sion of miR-142-3p with or without immunosuppression regimen logic and high doses of cyclosporine A (40 and 400 ng/ml).
in these liver recipients (Figure 2D).
As shown in Figure 3D, no difference was observed for eitherdose of cyclosporine A used. These results are concordant with miR-142-3p Is Overexpressed in B Cells from the absence of modulation of miR-142-3p in blood from liver- Operationally Tolerant Patients tolerant recipients before and after withdrawal of the treatment We then analyzed the expression of miR-142-3p in puriﬁed and suggest that miR-142-3p is not affected by immunosup- blood leukocyte subpopulations (T and B lymphocytes, pressive drugs in total PBMC or puriﬁed B cells.
monocytes, and NK cells) from healthy volunteers (n=3). Pu-riﬁcation was typically greater than 95%. Figure 3A shows that miR-142-3p Transfection in the Raji B-Cell Line miR-142-3p is expressed in all blood cell subsets, conﬁrming Because miRNA regulate mRNA levels,16,17 we overexpressed previous reports.24–26 Because miR-142-3p was highly ex- mir-142-3p in the Raji B-cell line using synthetic mimics and pressed in T and B lymphocytes from healthy volunteers, we performed gene expression proﬁling using microarrays next analyzed its expression in these two populations in trans- 24 hours after transfection. A total of 22,332 spots were ﬁltered plant recipients. No difference was observed in the expression and the overexpression of miR-142-3p was found to induce of miR-142-3p in the T lymphocyte subset (Figure 3C). In the up- and downregulation of 492 and 489 transcripts, re- contrast, the expression of miR-142-3p was signiﬁcantly spectively. To provide a more comprehensive biologic inter- higher in total B cells puriﬁed from operationally tolerant pretation of our ﬁnding, GOminer software was used to J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients network related to "Inﬂammation re-sponse, antimicrobial response and cell de-velopment" with 49 of these genes wasfound (Withinthis gene network, we identiﬁed a subgroupof TGF-b–related genes in which the TGF-breceptor 1 gene (TGFBR1), a possible target ofmiR-142-3p, plays a central role (Figure 4A),together with a subgroup of IFN-g–relatedgenes (Figure 4B), and a subgroup of genesrelated to B cells (Figure 4C).
The analysis of the detailed B-cell–related gene network after miR-142-3p transfectionhighlighted the upregulation of genes previ-ously identiﬁed in PBMCs from operationallytolerant recipients, such as the key moleculesM S 4 A 1 ( C D 2 0 ) , C R 2 , C D 3 8 , 1 0 a n dBANK-1.11,12 We next validated the array expression patterns of a few select targets and miRNAexpression. The upregulation of miR-142-3p,MS4A1, and BANK1 in Raji cells transfected Figure 2. miR-142-3p expression in PBMCs. (A) miR-142-3p was downregulated in with miR-142-3p mimics was conﬁrmed PBMCs from healthy volunteers after PHA/IL-2 stimulation as early as 2 hours and using individual Taqman assays (Figure 5A).
remained low for up to 24 hours. Mean6 SEM of miR-142-3p relative expression(22DDCq relative to RNU6) in total PBMCs from three healthy volunteers stimulated Implication of miR-142-3p in TGF-b with PHA (2 mg/ml) and IL-2 (150 U/ml) are represented. (B) miR-142-3p was signiﬁ- cantly overexpressed in PBMCs from operationally tolerant (Op-Tol) kidney transplant Using individual qPCR, we also conﬁrmed the recipients compared with STA patients. qPCR measurements were performed using signiﬁcant downregulation of TGFBR1 gene individual Taqman qPCR assays with independent PBMC samples from TLDA assays transcripts subsequent to miR-142-3p trans- (10 STA and 6 operationally tolerant patients). Means 6 SEM of miR-142-3p relative fection in the Raji cell line compared with con- expression (22DDCq relative to miR-374b) are represented. (C) miR-142-3p expression did not differ in PBMCs from liver transplant recipients before (n=11) and after (n=16) P=0.03; FC=0.47; Figure 5A). We then immunosuppression weaning. Means 6 SEM of miR-142-3p relative expression noted that the addition of TGF-b to the Raji (22DDCq relative to miR-374b) are represented. (D) miR-142-3p expression was stable culture media (5 ng/ml over 24 hours) in- between the two time points (5.5, 11, and 13 months between blood collections) for duced an increase in miR-142-3p expression, three operationally tolerant patients. Means 6 SEM of miR-142-3p relative expression whereas PHA stimulation did not (Figure 5B).
(22DDCq relative to miR-374b) are represented. *P,0.05.
Similarly, puriﬁed B cells from healthy volun-teers were activated with independent and de- identify the over-represented gene ontology (GO) categories pendent B-cell receptor signaling for 24 hours (Figure 3D). As based on the differential gene lists compared with all other observed after PHA stimulation in Raji cells (Figure 5B), there is expressed genes in the microarray.28 Among the 492 over- only nonsigniﬁcant and marginal regulation of miR-142-3p. This expressed genes, the GO categories "immune response" ﬁnding reinforces the fact that miR-142-3p needs speciﬁc stimu- (GO:0006955) and "B-cell activation" (GO:0042113) were lation, such as TGF-b, for its modulation (Figure 5B).
identiﬁed with a total of 25 genes. Among the 489 underex- We then measured TGF-b1 and TGFBR1 transcript levels in pressed genes, GO categories related to cell communication puriﬁed B cells from operationally tolerant and STA patients. We (GO:0007154), "vesicle-mediated transport" (GO:0016192), found that TGF-b1 expression is increased in B cells from opera- and "small GTPase [guanosine triphosphatase] mediated sig- tionally tolerant patients (FC=1.4 compared with STA; P=0.04) nal transduction" (GO:0007264) were identiﬁed. We then (Figure 5C), whereas the level of TGFBR1 did not signiﬁcantly identiﬁed 66 potential miR-142-3p targets downregulated differ between operationally tolerant and STA patients (Figure 5D).
subsequent to the miR-142-3p overexpression among the242 genes predicted to be miR-142-3p targets (among at least4 of 11 established miRNA target prediction databases com- puted by miRecords software).29 Mixing these 66 potentialtarget genes and the 25 upregulated immune-related genes The achievement of long-term drug-free tolerance in solid and using Ingenuity pathways analysis software, a gene organ transplantation is thought to be possible on the basis of Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 Table 1. Summary of clinical data for the 35 kidney recipients used for measurement of miR-142-3p expression Proteinuria Donor/Living Immunosuppression Mismatches 121.1632.5 0.2360.17 Operationally tolerant 110.1622.1 0.2560.34 Mean, minimum (min), and maximum (max) values apply only to recipient age, recipient gender, time between graft and analysis, creatine level, and proteinuria.
observations in both liver4 and kidney3,5transplantation. We and others have reportedon an increased number of peripheral B cellsand have identiﬁed modiﬁcations in bloodgene expression in operationally tolerantkidney transplant recipients that involveTGF-b signaling and B-cell–related path-ways.7,8,10,12,14 However, a clear mechanismor biologic process of peripheral regulationhas yet to be identiﬁed in these patients.
miRNAs are small RNA molecules with im-portant roles in immune modulation, homeo-stasis, the development of immune diseasesand the regulation of physiologic pro-cesses.19 Deregulated miRNA expressionhas been shown to be involved in severalhuman immune-related diseases, such asmultiple sclerosis,30,31 cancer,32,33 and rheu-matoid arthritis,34 but their function andregulation processes are still far from beingtotally understood. In renal transplantation,two studies reported on the identiﬁcation of Figure 3. miR-142-3p expression in puriﬁed blood cell populations. (A) miR-142-3p miRNA proﬁle signatures in biopsy samples expression in isolated subpopulations from PBMCs of three healthy volunteers (except from kidney transplant patients with acute NK cells; n=2). B ly, B lymphocytes; mono, mononuclear cells; T ly, T lymphocytes. (B) rejection episodes,21,23 suggesting that miR-142-3p expression exhibited increased expression in total B lymphocytes isolated miRNA expression proﬁling may be used from kidney operationally tolerant (Op-Tol) recipients (n=5) compared with STA pa- to monitor allograft status. To our knowl- tients (n=12) (P=0.0098; FC= 2.84). (C) miR-142-3p expression was similar in total T edge, and particularly in operationally toler- lymphocytes isolated from kidney operationally tolerant recipients (n=5) compared ant kidney transplant recipients, no miRNA with STA patients (n=8). (D) miR-142-3p expression was not signiﬁcantly modulated in expression analyses have been performed in puriﬁed B lymphocytes from healthy volunteers after B-cell–independent and B-cell–dependent stimulation and after cyclosporine A incubation during 24 hours of culture (n=4). Means 6 SEM of miR-142-3p relative expression (22DDCq relative to RNU6) are In this study, we report on the modulation represented. BCR, B-cell receptor. *P,0.05.
of expression of eight miRNAs in PBMCs J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients
Figure 4. Three subgroups of genes with modulated function after miR-142-3p overexpression in Raji cells. (A–C) These three sub-groups of genes were extracted from the gene network created using IPA software . Genes in red are upregulatedand genes in green are downregulated in Raji transfected by miR-142-3p compared with control mimic. PP, protein–protein binding;PD, protein–DNA binding; MB, group/complex membership; TR, translocation.
from kidney graft recipients with drug-free operational toler- described for other miRNAs, such as miR-125b, miR-16b, ance compared with patients with stable graft function under or miR-148b.35–37 miR-142-3p expression has been reported immunosuppression. Our choice to compare operationally as playing a role in CD4+CD25+Treg function.27 Although tolerant patients with patients who have stable graft function miR-142-3p was highly expressed in T lymphocytes, we did under immunosuppression was based on the fact that the latter not observe any differential expression in T lymphocytes be- population is the most likely to beneﬁt from immunosuppres- tween operationally tolerant and STA recipients (Figure 3C).
sion minimization, whereas patients with chronic rejection or In contrast, we found a signiﬁcant overexpression of miR- healthy volunteers would not. Unsupervised hierarchical clus- 142-3p in the B-lymphocyte subset of operationally tolerant tering analysis based on the expression of these eight miRNAs compared with STA patients (P=0.0098; FC=2.84) (Figure only led to the clustering of operationally tolerant patients 3B) and also compared with patients with signs of chronic together (Figure 1A). Among these eight miRNA, miR-142-3p antibody-mediated rejection (data not shown). We also found was highly expressed in PBMCs from operationally tolerant that its expression was not modulated by immunosuppressive patients (Figure 1C and Figure 2B). This miRNA has been treatment in tolerant liver transplant recipients (Figure 2D) described as a hematopoietic-restricted lineage miRNA.24–27 or when puriﬁed B cells from human volunteers were cultured We found that miR-142-3p was decreased after PHA/IL-2 with cyclosporine A in vitro (Figure 3D), indicating that its activation, further favoring a regulatory loop, as already overexpression in operationally tolerant kidney transplant Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 between miR-142-3p expression and post-transplantation time and did ﬁnd miR-142-3p expression to be stable over time (Figure2C); these results indicate that miR-142-3p isindependent of post-transplantation time.
Recently, Guo and colleagues demon- strated that miRNA decreased protein pro-duction mostly by lowering mRNA levels.17Thus, we used gene microarrays to measurethe effect of miR-142-3p overexpression inB cells, looking at the effect of experimentalmiR-142-3p transfection in a stable RajiB-cell line. Whereas the overexpression ofthis single miRNA repressed many genes, weobserved the paradoxical biologic effect ofmiR-142-3p, which also induced gene up-regulation, as already described for othermiRNAs.39 A large number of genes relatedto B cells, IFN-g, and TGF-b signaling wereupregulated after overexpression of miR-142-3p in the Raji B-cell line. Of note, inprevious studies we had already observedsome of these genes, such as MS4A1 Figure 5. miR-142-3p expression in Raji cells. (A) Conﬁrmation of the overexpression of (CD20); this gene was part of the 49-gene miR-142-3p, BANK1, and CD20 and the underexpression of TGFBR1 in Raji cells signature that correctly classiﬁed kidney transfected with miR-142-3p mimic compared with a control mimic (n=4). (B) miR-142- operationally tolerant patients10 and was 3p expression was increased by TGF-b in Raji cells, whereas PHA had marginal effects part of the best-classiﬁer genes in the blood in Raji cells, compared with control (PBS), after 24 hours of culture. Means 6 SEM of and urine of operationally tolerant patients miR-142-3p FC (control/stimulation) are represented. (C) TGF-b expression was sig- in the study by Newell and colleagues.7 niﬁcantly increased (P=0.04) in puriﬁed B cells from operationally tolerant (Op-Tol) Similarly, miR-142-3p overexpression led (n=4) and STA (n=6) patients, whereas TGFBR1 was not. (D) Means 6 SEM of gene to the upregulation of BANK1 transcripts, relative expression (22DDCq relative to RNU6 for miR-142-3p or relative to HPRT1 for one of the key leader genes upregulated in other genes) are represented. *P,0.05.
the blood of kidney operationally tolerantrecipients,11 independent of immunosup- recipients was not just a consequence of the absence of im- pressive treatment.12 BANK1 is an inhibitory adaptor protein highly expressed in peripheral B cells and is a modulator of Interestingly, these data are in accordance with an increased hyperactive B-cell responses by inhibiting AKT activation number of peripheral B cells14 and with the speciﬁc B-cell– upon CD40 signaling.13 enriched gene proﬁle that we and others previously reported Finally, the TGF-b signaling pathway was also affected after in the blood of these patients.7,8,10,12 Although the increased miR-142-3p overexpression in the Raji cell line and in B cells expression of miR-142-3p in PBMCs from operationally tol- from operationally tolerant patients. Interestingly, miR-106b, erant kidney recipients is probably in part due to the increased one of the eight differentially overexpressed miRNA in PBMCs number of blood B cells in these patients, because miR-142-3p from operationally tolerant kidney transplant recipients, also expression in PBMCs is correlated with B cell number (data not affects downstream effector molecules of TGF-b signaling.40,41 shown), we report that puriﬁed B cells from operationally tol- In a previous report, we showed that among the speciﬁc and erant also expressed more miR-142-3p. This miRNA has already unique blood signature of 49 genes associated with tolerance, been reported to be expressed speciﬁcally in hematopoietic tis- 27% of the genes modulated in blood from operationally tol- sues and particularly in B cells, but a precise role in the B-cell erant patients could be regulated by the TGF-b even though compartment has yet to be attributed.24–27,38 TGF-b was not signiﬁcantly increased in total PBMCs from miR-142-3p expression has also been associated with tubular operationally tolerant patients (increased from 30% only).10 atrophy and interstitial ﬁbrosis of renal transplants.22 Moreover, TGF-b is involved in various animal models of tolerance,42–44 miR-142-3p was overexpressed in B lymphocytes from the blood plays a role in immune regulation and homeostasis of Treg of operationally tolerant patients, and this analysis performed in cells,45 and is known for its intrinsic suppressive properties.46 peripheral blood does not exclude a different expression proﬁle We also report here that TGF-b stimulation of Raji cells within the graft itself. In addition, we did not ﬁnd any correlation induced an increased expression of miR-142-3p and that J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients TGF-b1 expression is increased in B cells from operationally protocol (Agilent Technologies Inc., Palo Alto, CA). A total of tolerant patients, whereas TGFBR1 is not; this ﬁnding 22,332 spots were ﬁltered. GOminer software and Ingenuity Pathway suggests a negative feedback loop between this cytokine and Analysis 6.5 software (Ingenuity Systems Inc.) were used to assess miR-142-3p in B cells. Such a process of regulation, already biologic signiﬁcance of genes selected with Mann–Whitney tests. Raw described for different miRNAs,40 suggests that miRNAs act as microarray data were deposited in the Gene Expression Ominbus key gene switches and as ﬁne-tuning molecules, depending on (GEO) database (accession number GSE28456).
the compartment and the speciﬁc biologic context.20 There-fore, our data suggest that mir-142-3p is also implicated in the Statistical Analyses TGF-b pathway. However, because the modulation of one The nonparametric Mann–Whitney test, Kruskal Wallis test, or miRNA may affect multiple mRNAs that are also regulated paired Wilcoxon test was used for group comparisons using Graph by several other miRNAs, a direct link between these two mol- PadPrism software, version 4. Differences were deﬁned as statistically ecules cannot be predicted at this stage.
signiﬁcant with P,0.05 and highly signiﬁcant with P,0.01.
Our ﬁndings show that overexpression of miR-142-3p in B Additional details can be found in cells correlates to the state of operational tolerance in kidney transplant recipients. They also point toward a possiblenegative feedback loop between TGF-b and miR-142-3p inB cells. The mechanism driving and maintaining spontaneoustolerance in which TGF-b could be implicated remains un- clear. Further investigations are now needed to ﬁnd outwhether this overexpression of miR-142-3p in B cells contrib- We thank all the patients who participated in this study and the utes to controlling inﬂammatory responses and tolerance physicians who helped us recruit patients: J.F. Subra, F. Villemain, maintenance or is only a consequence of this tolerance state.
C. Legendre, E. Thervet, F.J. Bemelman, G. Roussey, G. Orlando,A. Garnier, H. Jambon, H. Le Monies De Sagazan, L. Braun, C. Noël,E. Pillebout, M.C. Moal, C. Cantarell, A. Hoitsma, M. Ranbant, A.
Testa. We thank the transcriptome core facility of Nantes for technicalassistance with gene expression microarrays. We also thank Yohann Foucher for critical review of the manuscript.
A total of 86 individuals were enrolled in this study: 15 operationally The Institut de Transplantation Urologie belongs to the Fondation tolerant patients, 34 STA patients, 10 healthy volunteers, 11liver recipients Centaure, which supports a French research network in trans- with stable graft function, and 16 drug-free liver recipients from the plantation. R.D. was supported by the Fondation Centaure and by a Nantes hospital in France and the Barcelona hospital in Spain. The two grant from the Fondation pour la Recherche Médicale.
local ethics committees approved all aspects of the study, and all patientsgave informed consent. The clinical information is described in the , clinical data are summarized in Table 1, and detailed clinical data are provided in .
miRNA ProﬁlingmiRNA proﬁling was performed using TLDA microRNACards pool Aset, version 2.0 (Applied Biosystems, Foster City, CA), in accordance with the manufacturer's recommendations. Normalization was per-formed by subtracting the mean Cq of the measured miRNA.47 After 1. Danger R, Giral M, Soulillou JP, Brouard S: Rationale and criteria of eligibility for calcineurin inhibitor interruption following kidney trans- normalization, miRNA were ranked using P values from nonparametric plantation. Curr Opin Organ Transplant 13: 609–613, 2008 Mann–Whitney tests, which do not require normal assumptions or 2. Ashton-Chess J, Giral M, Brouard S, Soulillou JP: Spontaneous opera- asymptotic conditions. These uncorrected P values for multiple testing tional tolerance after immunosuppressive drug withdrawal in clinical were used only for ranking miRNA and are not an absolute identiﬁ- renal allotransplantation. Transplantation 84: 1215–1219, 2007 cation of differential miRNA.
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Orebody 32 East AWT Vertebrate Fauna Environmental Impact Assessment BHP Billiton Iron Ore 129 Royal Street East Perth WA 6004 Phone: (08) 9421 9600 Report Reference: 2451-15-BISR-1Rev0_150505 Fax: (08) 9421 9699 Orebody 32 East AWT Vertebrate Fauna Environmental Impact Assessment Prepared for
Lecture no. 15 Esophageal Motility Disorders - 3 Gastroesophageal Reflux Disease This chapter could begin like this: the esophageal mucosa is unable to endure contents coming from the stomach. These contents may be fluids secreted by the stomach itself (the most oft-occurring possibility), or even duodenal-biliary in origin. These events lead to a anatomical-clinical condition that generally goes by the name of esophagitis.