Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also ...been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coil-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COI1 -dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular "stand-by mode" by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COI1 -dependent leaf growth inhibition.
RituxiCAN-C4 combined an open-labeled randomized controlled trial (RCT) in 7 UK centers to assess whether rituximab could stabilize kidney function in patients with chronic rejection, with an ...exploratory analysis of how B cell-depletion influenced T cell anti-donor responses relative to outcome. Between January 2007 and March 2015, 59 recruits were enrolled after screening, 23 of whom consented to the embedded RCT. Recruitment was halted when in a pre-specified per protocol interim analysis, the RCT was discovered to be significantly underpowered. This report therefore focuses on the exploratory analysis, in which we confirmed that when B cells promoted CD4+ anti-donor IFNγ production assessed by ELISPOT, this associated with inferior clinical outcome; these patterns were inhibited by optimized immunosuppression but not rituximab. B cell suppression of IFNγ production, which associated with number of transitional B cells and correlated with slower declines in kidney function was abolished by rituximab, which depleted transitional B cells for prolonged periods. We conclude that in this patient population, optimized immunosuppression but not rituximab promotes anti-donor alloresponses associated with favorable outcomes.
Registered with EudraCT (2006-002330-38) and www.ClinicalTrials.gov, identifier: NCT00476164.
Abstract
Background and Aims
Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that ...methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described.
Methods
Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels.
Key Results
Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses.
Conclusions
This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.
Kidney transplant recipients (KTRs) with “operational tolerance” (OT) maintain a functioning graft without immunosuppressive (IS) drugs, thus avoiding treatment complications. Nevertheless, IS drugs ...can influence gene-expression signatures aiming to identify OT among treated KTRs.
We compared five published signatures of OT in peripheral blood samples from 18 tolerant, 183 stable, and 34 chronic rejector KTRs, using gene-expression levels with and without adjustment for IS drugs and regularised logistic regression.
IS drugs explained up to 50% of the variability in gene-expression and 20–30% of the variability in the probability of OT predicted by signatures without drug adjustment. We present a parsimonious consensus gene-set to identify OT, derived from joint analysis of IS-drug-adjusted expression of five published signature gene-sets. This signature, including CD40, CTLA4, HSD11B1, IGKV4–1, MZB1, NR3C2, and RAB40C genes, showed an area under the curve 0⋅92 (95% confidence interval 0⋅88–0⋅94) in cross-validation and 0⋅97 (0⋅93–1⋅00) in six months follow-up samples.
We advocate including adjustment for IS drug therapy in the development stage of gene-expression signatures of OT to reduce the risk of capturing features of treatment, which could be lost following IS drug minimisation or withdrawal. Our signature, however, would require further validation in an independent dataset and a biomarker-led trial.
FP7-HEALTH-2012-INNOVATION-1 305147:BIO-DrIM (SC,IR-M,PM,DSt); MRC G0801537/ID:88245 (MPH-F); MRC MR/J006742/1 (IR-M); Guy's&StThomas’ Charity R080530&R090782; CONICYT-Bicentennial-Becas-Chile (EN-L); EU:FP7/2007–2013 HEALTH-F5–2010–260687: The ONE Study (MPH-F); Czech Ministry of Health NV19–06–00031 (OV); NIHR-BRC Guy's&StThomas' NHS Foundation Trust and KCL (SC); UK Clinical Research Networks portfolio:7521.
BACKGROUNDThe development of spontaneous kidney transplant tolerance has been associated with numerous B cell–related immune alterations. We have previously shown that tolerant recipients exhibit ...reduced B-cell receptor signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether cluster of differentiation (CD)4 T cells from tolerant recipients also display an anti-inflammatory profile that could contribute to graft maintenance.
METHODSCD4 T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic rejection or healthy volunteers. CD4 T cells from the 3 groups were compared in terms of their gene expression profile, phenotype, and functionally upon activation.
RESULTSGene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired T-cell receptor signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients.
CONCLUSIONSThese data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.
Steroid conversion (HSD11B1, HSD11B2, H6PD) and receptor genes (NR3C1, NR3C2) were examined in kidney-transplant recipients with “operational tolerance” and chronic rejection (CR), independently and ...within the context of 88 tolerance-associated genes. Associations with cellular types were explored. Peripheral whole-blood gene-expression levels (RT-qPCR-based) and cell counts were adjusted for immunosuppressant drug intake. Tolerant (n = 17), stable (n = 190) and CR patients (n = 37) were compared. Healthy controls (n = 14) were used as reference. The anti-inflammatory glucocorticoid receptor (NR3C1) and the cortisol-activating HSD11B1 and H6PD genes were up-regulated in CR and were lowest in tolerant patients. The pro-inflammatory mineralocorticoid gene (NR3C2) was downregulated in stable and CR patients. NR3C1 was associated with neutrophils and NR3C2 with T-cells. Steroid conversion and receptor genes, alone, enabled classification of tolerant patients and were major contributors to gene-expression signatures of both, tolerance and CR, alongside known tolerance-associated genes, revealing a key role of steroid regulation and response in kidney transplantation.
•Steroid conversion and receptor genes in blood are altered in kidney transplantation.•Steroid activation (HSD11B1,H6PD) and response (NR3C1) are not increased in tolerance.•Upregulated NR3C1 and HSD11B1 indicate unmet cortisol demand in chronic rejection.•Drug-adjusted NR3C1 expression is associated with neutrophils and NR3C2 with T-cells.•Drug adjustment enables evaluation of endogenous factors influencing gene expression.
Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for ...non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage.
We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first post-transplant year from rejectors, non-rejectors and stable patients.
A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve 0.84 (0.77–0.88) (median, 2.5th–97.5th centile of fifty cross-validation cycles), sensitivity 0.67 (0.59–0.74) and specificity 0.85 (0.75–0.89). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive.
Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel T-cell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results.
The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the ...functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells.
To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD.
As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response.
Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis.
NCT02749630.
BACKGROUNDAcute rejection (AR) is often indicated by an increase in creatinine levels, after a considerable graft damage has already occurred. There is, therefore, a need for early identification of ...AR. Monitoring of RNA gene signatures in peripheral blood and urine samples offers the opportunity for surveillance of the recipient immune system and earlier detection of acute rejection.
SAMPLESWe examined RT-qPCR expression of 22 literature-based candidate genes in 335 longitudinal whole-blood samples from 27 stable kidney transplant recipients (KTRs) from the KALIBRE study (Kidney Allograft Immune Biomarkers of Rejection Episodes) and 237 samples from 27 KTRs with a biopsy-proven cellular rejection during the first post-transplant year. Creatinine in stable KTRs reached no higher than 20% above the reference range and had less than 15% standard deviation.
METHODSTo account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene expression as the residuals of linear mixed-effects models with stable patients. To select a parsimonious AR signature, we used penalised logistic regression with a pre-biopsy sample in rejectors and the median gene expression for each stable patient. As a test set we used the longitudinal data. We were also able to examine longitudinal expression of genes as well as effect of different anti-rejection therapies. We subsequently tested the performance of our signature in a second cohort.
RESULTSA 7-gene signature of AR showed area under the curve (AUC) 0.95 (95% confidence interval 0.91-1.00), sensitivity 0.85, specificity 0.93, positive and negative predictive values 0.92 and 0.86 and cross-validated AUC 0.85 (0.75-0.95). The estimated probability of rejection increased as early as six weeks prior to the AR biopsy and decreased after administration of treatment. Notably, gene expression in stable KTRs, as well as in rejectors, showed a pronounced variability, with 22% AR-positives longitudinal samples.(Figure is included in full-text article.)
CONCLUSIONMolecular marker alterations in blood emerge well ahead of the time of clinically overt AR. Monitoring of a gene-expression signature of AR in peripheral blood could unravel AR-related pro-inflammatory activity and provide information on subclinical processes and further justification for a biopsy in cases of sustained kidney dysfunction with no marked change in creatinine.
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