Effect of everolimus-based drug regimens on CMV-specific T-cell functionality after renal transplantation: 12-month ATHENA subcohort-study results

Post-transplant cytomegalovirus (CMV) infections and increased viral replication are associated with CMV-specific T-cell anergy. In the ATHENA-study, de-novo everolimus (EVR) with reduced-exposure tacrolimus (TAC) or cyclosporine (CyA) showed significant benefit in preventing CMV infections in renal transplant recipients as compared to standard TAC + mycophenolic acid (MPA). However, immunomodulatory mechanisms for this effect remain largely unknown. Ninety patients from the ATHENA-study completing the 12-month visit on-treatment (EVR + TAC n = 28; EVR + CyA n = 19; MPA + TAC n = 43) were included in a posthoc analysis. Total lymphocyte subpopulations were quantified. CMV-specific CD4 T cells were determined after stimulation with CMV-antigen, and cytokine-profiles and various T-cell anergy markers were analyzed using flow cytometry. While 25.6% of MPA + TAC-treated patients had CMV-infections, no such events were reported in EVR-treated patients. Absolute numbers of lymphocyte subpopulations were comparable between arms, whereas the percentage of regulatory T cells was significantly higher with EVR + CyA versus MPA + TAC (p = 0.019). Despite similar percentages of CMV-specific T cells, their median expression of CTLA-4 and PD-1 was lower with EVR + TAC (p < 0.05 for both) or EVR + CyA (p = 0.045 for CTLA-4) compared with MPA + TAC. Moreover, mean percentages of multifunctional CMV-specific T cells were higher with EVR + TAC (27.2%) and EVR + CyA (29.4%) than with MPA + TAC (19.0%). In conclusion, EVR-treated patients retained CMV-specific T-cell functionality, which may contribute to enhanced protection against CMV infections

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons

Onset and progression of diabetes in kidney transplant patients receiving everolimus or cyclosporine therapy: an analysis of two randomized, multicenter trials

Background: Conversion from calcineurin inhibitor (CNI) therapy to a mammalian target of rapamycin (mTOR) inhibitor following kidney transplantation may help to preserve graft function. Data are sparse, however, concerning the impact of conversion on posttransplant diabetes mellitus (PTDM) or the progression of pre-existing diabetes. Methods: PTDM and other diabetes-related parameters were assessed post hoc in two large open-label multicenter trials. Kidney transplant recipients were randomized (i) at month 4.5 to switch to everolimus or remain on a standard cyclosporine (CsA)-based regimen (ZEUS, n = 300), or (ii) at month 3 to switch to everolimus, remain on standard CNI therapy or convert to everolimus with reduced-exposure CsA (HERAKLES, n = 497). Results: There were no significant differences in the incidence of PTDM between treatment groups (log rank p = 0.97 [ZEUS], p = 0.90 [HERAKLES]). The mean change in random blood glucose from randomization to month 12 was also similar between treatment groups in both trials for patients with or without PTDM, and with or without pre-existing diabetes. The change in eGFR from randomization to month 12 showed a benefit for everolimus versus comparator groups in all subpopulations, but only reached significance in larger subgroups (no PTDM or no pre-existing diabetes). Conclusions: Within the restrictions of this post hoc analysis, including non-standardized diagnostic criteria and limited glycemia laboratory parameters, these data do not indicate any difference in the incidence or severity of PTDM with early conversion from a CsA-based regimen to everolimus, or in the progression of pre-existing diabetes. Trial registration: clinicaltrials.gov , NCT00154310 (registered September 2005) and NCT00514514 (registered August 2007); EudraCT ( 2006-007021-32 and 2004-004346-40 )

A role for microRNAs during plasma cell differentiation and in multiple myeloma

B-Zellen differenzieren nach Aktivierung entweder direkt zu IgM-sezernierenden Plasmazellen oder vollziehen im Keimzentrum durch somatische Hypermutation der Immunglobulin (Ig)-Gene die Affinitätsreifung ihres B-Zellrezeptors und durch Klassenwechsel den Sprung zu sekundären Ig-Isotypen. Die beiden Transkriptionsfaktoren IRF-4 und Blimp-1 sind essentiell für die Initiation, Durchführung und Termination der Plasmazelldifferenzierung. In Plasmazellen sorgen sie für die Expression von stadienspezifischen Genen und reprimieren die Expression von B-Zell-Identitätsgenen wie Pax5 und Bcl6. Für die Affinitätsreifung und den Ig-Klassenwechsel sind Bcl6, Bach2 und Mitf notwendig. Sie inhibieren die Transkriptionsfaktoren Blimp-1 und IRF-4 während der Keimzentrumsreaktionen, um eine vorzeitige Plasmazelldifferenzierung zu verhindern. Wie es zur Aufhebung der Bach2-vermittelten Repression von Blimp-1 und der Mitf-vermittelten Repression von IRF-4 kommt, ist noch ungeklärt. Das Multiple Myelom ist ein maligner Plasmazelltumor, der durch sekretorisch hyperaktive Plasmazellen gekennzeichnet ist, die das Knochenmark infiltrieren. Die Pathogenese-Mechanismen sind vielfältig. MicroRNAs (miRNAs) sind nicht-Protein-kodierende, kleine RNA-Moleküle, die auf posttranskriptioneller Ebene nach Bindung an ihre spezifische Zielsequenz in der 3'UTR einer mRNA die Genexpression negativ regulieren. Ob miRNAs an der terminalen Differenzierung der B-Zellen zu Plasmazellen beteiligt sind, ist bisher unbekannt. Ebenso ist die Rolle der miRNAs im Multiplen Myelom nur wenig erforscht. Welche Rolle miRNAs bei der Plasmazelldifferenzierung und im Multiplen Myelom spielen, war Gegenstand der Untersuchungen dieser Arbeit. Durch deep sequencing Analysen humaner naiver B-Zellen, Plasmablasten und Knochenmarks-Plasmazellen sowie primärer Myelome und verschiedener muriner Plsmazellpopulationen konnten sowohl einige während der Plasmazelldifferenzierung als auch im Myelom differentiell exprimierte miRNAs identifiziert werden. In silico und in vitro Zielgen-Analysen dieser miRNAs zeigten, dass sie eine potentielle regulatorische Rolle in Plasmazellen und während der Plasmazelldifferenzierung spielen könnten und in die onkogene Transformation von Myelomzellen involviert sein könnten.After antigen encounter B cells differentiate either directly into IgM-secreting plasma cells or enter germinal center reactions during which somatic hypermutation of the immunoglobulin-genes and class switch recombination lead to affinity maturation and generation of highly specific antibodies of isotypes different from IgM. The differentiation of mature B cells into plasma cells is tightly controlled by a regulatory network of transcription factors. IRF-4 and Blimp-1 have been shown to be essential for initiation and termination of plasma cell differentiation as well as for maintenance of plasma cell phenotype as they provide expression of critical plasma cell genes and as they negatively regulate B cell identity genes like Pax5 and Bcl6. During germinal centre reaction Bcl6, Bach2 and Mitf are crucial for affinity maturation and class switch recombination as they repress expression of IRF-4 and Blimp-1 and thereby inhibit initiation of plasma cell differentiation. How Bach2- and Mitf- mediated repression of Blimp-1 and IRF-4 is alleviated to allow onset of plasma cell differentiation is thus far not known. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. Whether miRNAs are crucial during plasma cell differentiation is barely known. Dysregulation of miRNAs contributes to the pathogenesis of various hematopoetic malignancies. Multiple Myeloma is a plasma cell malignancy characterized by hypersecretory plasma cells that infiltrate the bone marrow. Currently only little is known about the role of miRNAs in Multiple Myeloma. The specific aim of this work was to investigate the role of miRNAs during plasma cell differentiation and their involvement in pathogenesis of Multiple Myeloma. By deep sequencing of cloned miRNAs we established the miRNA expression profile of human naîve B cells, plasmablasts and normal as well as malignant bone marrow-derived plasma cells and of murine plasma cell subsets. This analysis identified numerous differentially expressed miRNAs during plasma cell differentiation as well as in malignant plasma cells from Myeloma patients. Target analysis revealed potential functions of these miRNAs during normal plasma cell differentiation and malignant transformation

Everolimus in de novo kidney transplant recipients participating in the Eurotransplant senior program: Results of a prospective randomized multicenter study (SENATOR).

Early conversion to everolimus was assessed in kidney transplant recipients participating in the Eurotransplant Senior Program (ESP), a population in whom data are lacking. The SENATOR multicenter study enrolled 207 kidney transplant recipients undergoing steroid withdrawal at week 2 post-transplant (ClinicalTrials.gov [NCT00956293]). At week 7, patients were randomized (1:2 ratio) to continue the previous calcineurin inhibitor (CNI)-based regimen with mycophenolic acid (MPA) and cyclosporine or switch to a CNI-free regimen with MPA, everolimus (5-10 ng/mL) and basiliximab at weeks 7 and 12, then followed for 18 weeks to month 6 post-transplant. The primary endpoint was estimated GFR (eGFR). At week 7, 77/207 (37.2%) patients were randomized (53 everolimus, 24 control). At month 6, eGFR was comparable: 36.5±10.8ml/min with everolimus versus 42.0±13.0ml/min in the control group (p = 0.784). Discontinuation due to adverse events occurred in 27.8% of everolimus-treated patients and 0.0% of control patients (p = 0005). Efficacy profiles showed no difference. In conclusion, eGFR, safety and efficacy outcomes at month 6 post-transplant showed no difference between groups. The everolimus group experienced a higher rate of discontinuation due to adverse events. However, the high rate of non-randomization is highly relevant, indicating this to be a somewhat unstable patient population regardless of treatment

Characterization of the domain swapping mechanism of the forkhead domain of human FoxP1 at a single-molecule level

Fondecyt 1130510 11140601 2113047

Five-year outcomes in kidney transplant patients randomized to everolimus with cyclosporine withdrawal or low-exposure cyclosporine versus standard therapy.

HERAKLES was a 1-year randomized, multicenter trial. Patients were randomized at 3 months after kidney transplantation to remain on cyclosporine-based therapy, switch to everolimus without a calcineurin inhibitor (CNI), or switch to everolimus with low-exposure cyclosporine. Overall, 417 of 497 (83.9%) patients from the core study entered a 4-year extension study. The randomized regimen was continued to year 5 in 75.9%, 41.9% and 24.6% of patients in the standard-CNI, CNI-free and low-CNI groups, respectively. Adjusted estimated GFR at year 5 was significantly higher in the CNI-free group versus standard CNI (difference 7.2 mL/min/1.73 m , P < .001) or low CNI (difference 7.6 mL/min/1.73 m , P < .001). For patients who continued randomized therapy for 5 years, differences were 14.4 mL/min/1.73 m  and 10.1 mL/min/1.73 m , respectively. Biopsy-proven acute rejection occurred during the 4-year extension study in 7.6%, 8.6%, and 9.0% of patients in the standard-CNI, CNI-free and low-CNI groups, respectively (P = .927). In conclusion, conversion to a CNI-free everolimus regimen 3 months after kidney transplantation improved long-term graft function, particularly in patients who continued the CNI-free regimen. Low CNI with everolimus did not improve renal function. Efficacy was comparable between groups but frequent immunosuppression changes should be taken into account

Additional file 1: of Histological findings to five years after early conversion of kidney transplant patients from cyclosporine to everolimus: an analysis from the randomized ZEUS study

Table S1. Baseline characteristics (safety population). Table S2. Immunosuppression at 5 years post-transplant (safety population), n (%). Table S3. Pathology assessment of biopsies according to Banff criteria in patients with ≥1 biopsy not categorized as ‘protocol-specified’ or ‘investigator-initiated’. Figire S1. CsA, cyclosporine. (DOCX 129 kb