Ythdf2 regulates cardiac remodeling through its mRNA target transcripts.

m6A mRNA methylation controls cardiomyocyte function and increased overall m6A levels are a stereotyping finding in heart failure independent of the underlying etiology. However, it is largely unknown how the information is read by m6A reader proteins in heart failure. Here we show that the m6A reader protein Ythdf2 controls cardiac function and identified a novel mechanism how reader proteins control gene expression and cardiac function. Deletion of Ythdf2 in cardiomyocytes in vivo leads to mild cardiac hypertrophy, reduced heart function, and increased fibrosis during pressure overload as well as during aging. Similarly, in vitro the knockdown of Ythdf2 results in cardiomyocyte growth and remodeling. Mechanistically, we identified the eucaryotic elongation factor 2 as post-transcriptionally regulated by Ythdf2 using cell type specific Ribo-seq data. Our study expands our understanding on the regulatory functions of m6A methylation in cardiomyocytes and how cardiac function is controlled by the m6A reader protein Ythdf2.

A novel approach to genetic engineering of T-cell subsets by hematopoietic stem cell infection with a bicistronic lentivirus.

Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck-(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.

Increased incidence of acute graft rejection on calcineurin inhibitor-free immunosuppression after heart transplantation.

BACKGROUND: Calcineurin inhibitor (CNI)-free immunosuppression is used increasingly after heart transplantation to avoid CNI toxicity, but in the absence of a randomized trial, concerns remain over an increased rejection risk. METHODS: We studied the incidence of graft rejection episodes among all cardiac graft recipients, beginning with the first introduction of CNI-free protocols. We compared events during CNI-free and CNI-containing immunosuppression among 231 transplant recipients of overall mean age 55.2 ± 11.8 years, from a mean 5.2 ± 5.4 years after transplantation through a mean follow-up of 3.1 ± 1.4 years. We considered as acute rejection episodes requiring treatment those of International Society for Heart and Lung Transplantation. RESULTS: During the total follow-up of 685 patient years (CNI-containing, 563; CNI-free, 122), we performed 1,374 biopsies which diagnosed 78 rejection episodes. More biopsies were performed in CNI-free patients: biopsies/patient-month of CNI-containing, 0.13 versus CNI-free, 0.22 (P < .05). The incidence of rejection episodes per patient-month was significantly higher on CNI-free compared with CNI therapy, among patients switched both early and later after heart transplantation, namely, within 1 year, 0.119 versus 0.035 (P = .02); beyond 1 year, 0.011 versus 0.004 (P = .007); beyond 2 years, 0.007 versus 0.003 (P = .04); and beyond 5 years: 0.00578 versus 0.00173 (P = .04). CONCLUSIONS: Rejection incidence during CNI-free immunosuppression protocols after heart transplantation was significantly increased in both early and later postoperative periods. Given the potentially long delay to rejection occurrence, patients should be monitored closely for several months after a switch to CNI-free immunosuppressive protocols.

Rosuvastatin reduces neointima formation in a rat model of balloon injury.

BACKGROUND: Processes of restenosis, following arterial injury, are complex involving different cell types producing various cytokines and enzymes. Among those enzymes, smooth muscle cell-derived matrix metalloproteinases (MMPs) are thought to take part in cell migration, degrading of extracellular matrix, and neointima formation. MMP-9, also known as gelatinase B, is expressed immediately after vascular injury and its expression and activity can be inhibited by statins. Using an established in vivo model of vascular injury, we investigated the effect of the HMG-CoA reductase inhibitor rosuvastatin on MMP-9 expression and neointima formation. MATERIALS AND METHODS: 14-week old male Sprague Dawley rats underwent balloon injury of the common carotid artery. Half of the animals received rosuvastatin (20 mg/kg body weight/day) via oral gavage, beginning 3 days prior to injury. Gelatinase activity and neointima formation were analyzed 3 days and 14 days after balloon injury, respectively. 14 days after vascular injury, proliferative activity was assessed by staining for Ki67. RESULTS: After 14 days, animals in the rosuvastatin group showed a decrease in total neointima formation (0.194±0.01 mm2 versus 0.124±0.02 mm2, p<0.05) as well as a reduced intima/media ratio (1.26±0.1 versus 0.75±0.09, p<0.05). Balloon injury resulted in increased activity of MMP-9 3 days after intervention for both rosuvastatin treated animals and controls with no significant difference observed between the groups. There was a trend towards a reduction in the number of Ki67-positive cells 14 days after injury. CONCLUSIONS: Rosuvastatin attenuates neointima formation without affecting early MMP-9 activity in a rat model of vascular injury.

Increased adherence after switch from twice daily calcineurin inhibitor based treatment to once daily modified released tacrolimus in heart transplantation: a pre-experimental study.

BACKGROUND: Modified release tacrolimus (TAC) is a new, once-daily oral formulation of the established immunosuppressive agent TAC. Simplification of regimen has been associated with better adherence. This study evaluated patient adherence, as well as safety and efficacy among chronic stable heart transplantation (HT) patients switched from a conventional twice daily calcineurin inhibitor-based regimen (TAC or cyclosporine A [CsA]) to (once daily) modified release TAC. METHODS: We switched 54 chronic stable patients (41 males and 13 females) from twice daily dosing with conventional TAC or CsA to once daily dosing with modified release TAC. Self-reported adherence was assessed at baseline and at 4 months after the switch using the Basel Assessment of Adherence with Immunosuppressive Medication Scale [BAASIS]), a 4-item validated questionnaire including also a Visual Analogue Scale (VAS). Nonadherence was defined as any self-reported nonadherence on any item. RESULTS: Modified release TAC was discontinued in 4 patients because of diarrhea (n = 1) or gastrointestinal discomfort (n = 3) leaving 50 evaluable patients. Overall nonadherence at baseline for any of the 4 items was 74% versus 38% after 4 months (P = .0001). Thereafter, adherence improved in 28 patients (56.0%), was unchanged in 18 (36.0%), and decreased in 4 subjects (8.0%). The VAS score improved from 82.3% ± 2.6% to 97.5% ± 4.8% (P < .0001). No significant changes were observed after 4 months regarding hematologic, renal, or liver function parameters (all P = NS). CONCLUSIONS: Therapeutic regimens for transplant recipients are often complex, contributing to a high incidence of medication nonadherence. This study in chronic, stable, heart transplantation patients demonstrated a significant improvement in patient adherence after a switch to modified release TAC, which was generally well tolerated.

Proton pump inhibitor co-medication reduces active drug exposure in heart transplant recipients receiving mycophenolate mofetil.

BACKGROUND: Proton pump inhibitors (PPIs) are often prescribed for gastrointestinal discomfort after heart transplantation. This study investigated the impact of PPI use on mycophenolic acid (MPA) pharmacokinetics in heart transplant recipients receiving mycophenolate mofetil (MMF) in combination with a calcineurin inhibitor (tacrolimus [TAC]/cyclosporine [CsA]) or mammalian target of rapamycin inhibitor (sirolimus/everolimus). METHODS: Abbreviated MPA areas under the curve (AUCs; 0, 30, and 120 minutes after morning intake) were obtained in 19 patients on a PPI (initial examination) and 1 month after PPI discontinuation (follow-up). Mean patient age was 58.2 ± 8.8 years, and mean time after transplantation was 2.3 ± 4.0 years (range, 0.2-13.0 years). RESULTS: At initial examination mean daily MMF dose was 2.2 ± 0.8 g. MMF dose was kept unchanged for the duration of study (P = ns). Mean predose (C0) MPA serum concentrations were insignificantly lower with PPI comedication (2.5 ± 2.2 mg/L vs 2.8 ± 1.7 mg/L; P = .15). Dose-adjusted abbreviated MPA AUCs (adjusted to morning dose) were significantly lower during PPI therapy (45.2 ± 20.3 vs 65.2 ± 38.8 mg·h/L·g [MMF]; P = .02). CONCLUSIONS: Patients with PPI comedication during MMF therapy show significantly lower exposure to mycophenolic acid determined by dose-adjusted abbreviated MPA AUCs. Although the clinical relevance of this pharmacokinetic interaction was not determined in this study, MPA drug monitoring by limited sampling strategies might be helpful during changes in antacid comedication in patients on MMF.

Malignancies after heart transplantation: incidence, risk factors, and effects of calcineurin inhibitor withdrawal.

The objectives of the present study were to evaluate the incidence of malignancies and to describe the effects of immunosuppression on survival and recurrence of malignancies after heart transplantation (HTX). Data were analyzed in 211 cardiac allograft recipients, in whom HTX was performed between 1989 and 2005. All of these patients survived for more than 2 years after HTX and received induction therapy with antithymocyte globulin (RATG) guided by T-cell monitoring since 1994. An immunosuppressive regimen consisting of cyclosporine A (CsA) combined with azathioprine was followed by CsA and mycophenolate mofetil (MMF) in 2001; mammalian target of rapamycin (mTOR) inhibitors (everolimus/sirolimus) were used since 2003. Mean patient age at HTX was 51.4 ± 10.5 years; mean follow-up time after HTX 9.2 ± 4.7 years. Overall incidence of neoplasias was 30.8%. Individual risk factors associated with a higher risk of malignancy after HTX were higher age at transplantation (P = .003), male gender (P = .005) and ischemic cardiomyopathy before HTX (P = .04). Administration of azathioprine (P < .0001) or a calcineurin inhibitor (CNI) (P = .02) for more than 1 year was associated with development of malignancy, whereas significantly fewer malignancies were noticed in patients receiving an mTOR-inhibitor (P < .0001). Kaplan-Meier analysis demonstrated a strong statistical trend toward an improved survival in patients with a noncutaneous neoplasia switched to a CNI-free protocol (P = .05). This study demonstrated the impact of a variety of individual risk factors and immunosuppressive drugs on development of malignancy after HTX. Markedly fewer patients with noncutaneous malignancies died after switch to a CNI-free regimen, not quite reaching statistical significance by Kaplan-Meier analysis, however.

Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells.

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.

Fatal varicella in an immunocompromised adult associated with a European genotype E2 variant of varicella zoster virus.

Varicella zoster virus (VZV) seronegative patients under immunosuppressive therapy are at risk for severe life-threatening varicella. A 25-year-old male patient presented with rash and hepatitis. He had been known to suffer from Crohn's disease and received immunosuppressive treatment with azathioprine. The patient showed dyspnoea, and presented with a generalized rash with vesicular lesions typical for herpesvirus infections. He was started immediately on acyclovir therapy. Varicella infection was determined in this VZV seronegative patient in rash vesicles, blood and tracheal secretions and a high VZV viral load was detected in these specimens. The causative agent was genotyped by sequencing of several genes as a variant of the European genotype E2 containing several unique single nucleotide polymorphisms. Despite all measures, there was progressive septic shock, and the patient died due to multi-organ failure. Immunocompromised adults without varicella history are at high risk to develop life-threatening complications of varicella. Antiviral therapy with acyclovir can only be successful when administered as early as possible on suspicion of varicella infection in this group of patients. The most effective method to prevent severe varicella in immunocompromised patients is active immunization prior to immunosuppressive therapy.

A novel flow-cytometry-based assay for quantification of affinity and avidity changes of integrins.

Cell adhesion plays an important role in cell-cell contact formation and cell migration. Thus, the assessment of cellular adhesiveness is one important feature when studying cell-mediated immune responses. The interaction of lymphocytes with other cell types such as antigen-presenting cells or vascular-endothelial cells occurs via adhesion molecules including L-selectin, VCAM-1 or ICAM-1. There are principally two mechanisms by which cell adhesion can be enhanced: namely changes in the affinity or avidity of receptor interactions. Conventional plate-based adhesion assays detect both forms. However, they do not permit discrimination between affinity- and avidity-mediated changes in the adhesiveness. Moreover, analysis of cell subpopulations requires cell separation prior to performance of the adhesion assay. Conventional flow-cytometry-based tests make it possible to determine changes in the affinity of integrins at the single cell level. However, they fail to quantify avidity-mediated adhesiveness. Here we describe a novel flow-cytometry-based assay, which allows the detection of both integrin-mediated affinity as well as avidity changes at the single cell level. This opens up the possibility of precisely characterizing the adhesive capacity of subpopulations in heterogeneous cell populations.

The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes.

Cofilin, an actin-depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is phosphorylated and localized in the cytoplasm. Following co-stimulation through accessory receptors (e.g. CD2 or CD28) - however, not following TCR/CD3 stimulation alone - cofilin undergoes dephosphorylation. The subcellular localization as well as the actin-binding activity of cofilin are regulated by the phosphorylation state of serine-3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM-kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM-kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19-kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506-resistant co-stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co-stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).