Predictive Modeling of Tacrolimus Dose Requirement Based on High‐Throughput Genetic Screening

Any biochemical reaction underlying drug metabolism depends on individual gene–drug interactions and on groups of genes interacting together. Based on a high‐throughput genetic approach, we sought to identify a set of covariant single‐nucleotide polymorphisms predictive of interindividual tacrolimus (Tac) dose requirement variability. Tac blood concentrations (Tac C₀) of 229 kidney transplant recipients were repeatedly monitored after transplantation over 3 mo. Given the high dimension of the genomic data in comparison to the low number of observations and the high multicolinearity among the variables (gene variants), we developed an original predictive approach that integrates an ensemble variable‐selection strategy to reinforce the stability of the variable‐selection process and multivariate modeling. Our predictive models explained up to 70% of total variability in Tac C₀ per dose with a maximum of 44 gene variants (p‐value <0.001 with a permutation test). These models included molecular networks of drug metabolism with oxidoreductase activities and the multidrug‐resistant ABCC8 transporter, which was found in the most stringent model. Finally, we identified an intronic variant of the gene encoding SLC28A3, a drug transporter, as a key gene involved in Tac metabolism, and we confirmed it in an independent validation cohort.     

MicroRNA‐494 Promotes Cyclosporine‐Induced Nephrotoxicity and Epithelial to Mesenchymal Transition by Inhibiting PTEN

A major complication associated with cyclosporine (CsA) treatment is nephrotoxicity. In this study, we examined whether microRNAs play a role in cyclosporine‐induced nephrotoxicity. Treatment of mice with CsA resulted in nephrotoxicity that was associated with an early increase in expression of microRNA mmu‐miR‐494 (miR‐494). Similarly, tubular epithelial cell epithelial‐mesenchymal transition (EMT) induced by CsA toxicity resulted in the upregulation of microRNA‐494 and a decrease in PTEN levels in vitro. miR‐494 directly targeted Pten and negatively regulated its expression. Preventing Pten targeting by miR‐494 was sufficient to prevent CsA induced EMT. Knockdown of miR‐494 prevented the downregulation of PTEN in tubular epithelial cells following CsA treatment and also prevented CsA induced EMT. Thus, miR‐494 plays a major role in promoting CsA induced nephrotoxicity through its ability to target Pten thereby contributing to EMT. We suggest that manipulating miR‐494 expression may represent a novel approach to preventing EMT associated with CsA induced nephrotoxicity.     

A Novel Rodent Orthotopic Forelimb Transplantation Model That Allows for Reliable Assessment of Functional Recovery Resulting From Nerve Regeneration

Improved nerve regeneration and functional outcomes would greatly enhance the utility of vascularized composite allotransplantation (VCA) such as hand and upper extremity transplantation. However, research aimed at achieving this goal has been limited by the lack of a functional VCA animal model. We have developed a novel rat midhumeral forelimb transplant model that allows for the characterization of upper extremity functional recovery following transplantation. At the final end point of 12 weeks, we found that animals with forelimb transplantation including median, ulnar and radial nerve coaptation demonstrated significantly improved grip strength and forelimb function as compared to forelimb transplantation without nerve approximation (grip strength: 1.71N ± 0.57 vs. no appreciable recovery; IBB scale: 2.6 ± 0.7? vs. 0.8 ± 0.40; p = 0.0005), and similar recovery to nerve transection‐and‐repair only (grip strength: 1.71N ± 0.57 vs. 2.03 ± 0.42.6; IBB scale: 2.6 ± 0.7 vs. 2.8 ± 0.8; p = ns). Moreover, all forelimb transplant animals with nerve coaptation displayed robust axonal regeneration with myelination and reduced flexor muscle atrophy when compared to forelimb transplant animals without nerve coaptation. In conclusion, this is the first VCA small‐animal model that allows for reliable and reproducible measurement of behavioral functional recovery in addition to histologic evaluation of nerve regeneration and graft reinnervation.     

Circulating delta‐like Notch ligand 1 is correlated with cardiac allograft vasculopathy and suppressed in heart transplant recipients on everolimus‐based immunosuppression

Cardiac allograft vasculopathy (CAV) causes heart failure after heart transplantation (HTx), but its pathogenesis is incompletely understood. Notch signaling, possibly modulated by everolimus (EVR), is essential for processes involved in CAV. We hypothesized that circulating Notch ligands would be dysregulated after HTx. We studied circulating delta‐like Notch ligand 1 (DLL1) and periostin (POSTN) and CAV in de novo HTx recipients (n = 70) randomized to standard or EVR‐based, calcineurin inhibitor‐free immunosuppression and in maintenance HTx recipients (n = 41). Compared to healthy controls, plasma DLL1 and POSTN were elevated in de novo (P < .01; P < .001) and maintenance HTx recipients (P < .001; P < .01). Use of EVR was associated with a treatment effect for DLL1. For de novo HTx recipients, a change in DLL1 correlated with a change in CAV at 1 (P = .021) and 3 years (P = .005). In vitro, activation of T cells increased DLL1 secretion, attenuated by EVR. In vitro data suggest that also endothelial cells and vascular smooth muscle cells (VSMCs) could contribute to circulating DLL1. Immunostaining of myocardial specimens showed colocalization of DLL1 with T cells, endothelial cells, and VSMCs. Our findings suggest a role of DLL1 in CAV progression, and that the beneficial effect of EVR on CAV could reflect a suppressive effect on DLL1. Trial registration numbers— SCHEDULE trial: ClinicalTrials.gov NCT01266148; NOCTET trial: ClinicalTrials.gov NCT00377962.     

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse‐1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2‐photon microscopy, we analyzed the effects of DNAse‐1 on NETs in mouse orthotopic lung allografts damaged by ischemia‐reperfusion injury. Although DNAse‐1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4⁺ T cells and donor‐derived antigen presenting cells. DNAse‐1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen‐specific CD4⁺ T cell proliferation through activating toll‐like receptor (TLR) — Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil‐specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse‐1 administration to wild‐type recipients promotes the recognition of allo‐ and self‐antigens and prevents immunosuppression‐mediated lung allograft acceptance through a MyD88‐dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.     

Serological proteome analysis reveals new specific biases in the IgM and IgG autoantibody repertoires in autoimmune polyendocrine syndrome type 1

Abstract

Objective: Autoimmune polyendocrine syndrome type 1 (APS 1) is caused by mutations in the AIRE gene that induce intrathymic T-cell tolerance breakdown, which results in tissue-specific autoimmune diseases. Design: To evaluate the effect of a well-defined T-cell repertoire impairment on humoral self-reactive fingerprints, comparative serum self-IgG and self-IgM reactivities were analyzed using both one- and two-dimensional western blotting approaches against a broad spectrum of peripheral tissue antigens. Methods: Autoantibody patterns of APS 1 patients were compared with those of subjects affected by other autoimmune endocrinopathies (OAE) and healthy controls. Results: Using a Chi-square test, significant changes in the Ab repertoire were found when intergroup patterns were compared. A singular distortion of both serum self-IgG and self-IgM repertoires was noted in APS 1 patients. The molecular characterization of these antigenic targets was conducted using a proteomic approach. In this context, autoantibodies recognized more significantly either tissue-specific antigens, such as pancreatic amylase, pancreatic triacylglycerol lipase and pancreatic regenerating protein 1α, or widely distributed antigens, such as peroxiredoxin-2, heat shock cognate 71-kDa protein and aldose reductase. As expected, a well-defined self-reactive T-cell repertoire impairment, as described in APS 1 patients, affected the tissue-specific self-IgG repertoire. Interestingly, discriminant IgM reactivities targeting both tissue-specific and more widely expressed antigens were also specifically observed in APS 1 patients. Using recombinant targets, we observed that post translational modifications of these specific antigens impacted upon their recognition. Conclusions: The data suggest that T-cell-dependent but also T-cell-independent mechanisms are involved in the dynamic evolution of autoimmunity in APS 1.     

In vitro models of pancreatic cancer for translational oncology research

Background: Pancreatic cancer is a disease of near uniform fatality and the overwhelming majority of patients succumb to their advanced malignancy in a few months of diagnosis. Despite considerable advances in our understanding of molecular mechanisms underlying pancreatic carcinogenesis, this knowledge has not yet been fully translated into clinically available treatment strategies that yield significant improvements in disease free or overall survival. Objective: Cell line-based in vitro model systems provide powerful tools to identify potential molecular targets for therapeutic intervention as well as for initial preclinical evaluation of novel drug candidates. Here, we provide a brief overview of recent literature on cell line-based model systems of pancreatic cancer and their application in the search for novel therapeutics against this vicious disease. Conclusion: Although in vitro models of pancreatic cancer are of tremendous value for genetic studies and for initial functional screenings in drug discovery, they carry several immanent drawbacks and are often poor in predicting therapeutic response in humans. Therefore, in most instances, they are successfully exploited to generate hypothesis and identify molecular targets for novel therapeutics, which are subsequently subject to further in-depth characterization using more advanced in vivo model systems and clinical trials.     

Drug discovery paradigms: past,present, future – a centennial symposium of the American Society for Pharmacology and Experimental Therapeutics

Background: The American Society for Pharmacology and Experimental Therapeutics (ASPET) celebrated its centennial during the April 2008 Experimental Biology meeting held in San Diego, CA, USA. Objectives: This report summarizes a centennial symposium on past, present and future paradigms in drug discovery. The John Langley (1905) concept of ‘receptive substances’ initiated a cascade of cardinal discoveries for pharmacology. During the following 100 years, receptors achieved the status of well-defined multifunctional cellular proteins mediating all drug effects. The G-protein-coupled receptors (GPCRs) were discussed in relation to multiple targets they represent for clinically used or new medicines. The development of inbred and transgenic animal strains is a fundamental twentieth century achievement for biological research activities. Progress in treating CNS disorders still awaits breakthrough treatments. Drug development remains a risky and expensive venture; improvements are expected from emerging biomarker and translational medicine technologies. Future clinical research has to document the benefits that new medications provide to concerned stakeholders. Conclusions: We wish ASPET a new century of great achievements and acknowledge the dedication of several generations of colleagues, many of whom are our ‘unsung heroes’. They have left us a precious heritage to be pursued with passion in the quest for better medicines.     

Mammillothalamic tract lesions disrupt dead reckoning in the rat

Debate surrounds the role of the limbic system structures’ contribution to spatial orientation. The results from previous studies have supported a role for the mammillary bodies and their projections to the anterior thalamus in rapid encoding of relationships among environmental cues; however, this work is based on behavioral tasks in which environmental and self‐movement cues could not be dissociated. The present study examines the effects of mammillothalamic tract lesions on spatial orientation in the food hoarding paradigm and the water maze. Although the food hoarding paradigm dissociates the use of environmental and self‐movement cues, both sources of information are available to guide performance in the water maze. Mammillothalamic tract lesions selectively impaired performance on both tasks. These impairments are interpreted as providing further evidence for the role of limbic system structures in processing self‐movement cues.     

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.