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1.  CaMKII oxidative activation and the pathogenesis of cardiac disease 
Calcium and redox signaling both play important roles in the pathogenesis of cardiac disease; although how these signals are integrated in the heart remains unclear. One putative sensor for both calcium and oxidative stress in the heart is CaMKII, a calcium activated kinase that has recently been shown to also be regulated by oxidation. Oxidative activation of CaMKII occurs in several models of cardiac disease, including myocardial injury and inflammation, excessive neurohumoral activation, atrial fibrillation, and sinus node dysfunction. Additionally, oxidative activation of CaMKII is suggested in subcellular domains where calcium and ROS signaling intersect, such as mitochondria. This review describes the mechanism of activation of CAMKII by oxidation, the cardiac diseases where oxidized CaMKII has been identified, and suggests contexts where oxidized CaMKII is likely to play an important role.
doi:10.1016/j.yjmcc.2014.02.004
PMCID: PMC4048820  PMID: 24530899
Ca2+/calmodulin dependent protein kinase II; Reactive oxygen species; Heart failure; Arrhythmia; Calcium; mitochondria
2.  The mitochondrial uniporter controls fight or flight heart rate increases 
Nature communications  2015;6:6081.
Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant negative (DN) MCU. Here we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment prior to each heartbeat. Our findings show the MCU is necessary for complete physiological heart rate acceleration and suggest MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate.
doi:10.1038/ncomms7081
PMCID: PMC4398998  PMID: 25603276
3.  Embryonic Stem Cell-Derived Cardiac Myocytes Are Not Ready For Human Trials 
Circulation research  2014;115(3):335-338.
Brief summary
Recently a preclinical report assessed the efficacy, safety, and feasibility of using human embryonic stem cells to regenerate infarcted myocardium in a non-human primate model. This commentary evaluates that study by pointing out key weaknesses, offering an alternative perspective, and summarizing major unresolved issues. Our conclusion is that significant challenges remain before human embryonic stem cells are ready for use in clinical trials.
doi:10.1161/CIRCRESAHA.114.304616
PMCID: PMC4133694  PMID: 24935962
4.  Handling changes in MRI acquisition parameters in modeling whole brain lesion volume and atrophy data in multiple sclerosis subjects: Comparison of linear mixed-effect models 
NeuroImage : Clinical  2015;8:606-610.
Magnetic resonance imaging (MRI) of the brain provides important outcome measures in the longitudinal evaluation of disease activity and progression in MS subjects. Two common measures derived from brain MRI scans are the brain parenchymal fraction (BPF) and T2 hyperintense lesion volume (T2LV), and these measures are routinely assessed longitudinally in clinical trials and observational studies. When measuring each outcome longitudinally, observed changes may be potentially confounded by variability in MRI acquisition parameters between scans. In order to accurately model longitudinal change, the acquisition parameters should thus be considered in statistical models. In this paper, several models for including protocol as well as individual MRI acquisition parameters in linear mixed models were compared using a large dataset of 3453 longitudinal MRI scans from 1341 subjects enrolled in the CLIMB study, and model fit indices were compared across the models. The model that best explained the variance in BPF data was a random intercept and random slope with protocol specific residual variance along with the following fixed-effects: baseline age, baseline disease duration, protocol and study time. The model that best explained the variance in T2LV was a random intercept and random slope along with the following fixed-effects: baseline age, baseline disease duration, protocol and study time. In light of these findings, future studies pertaining to BPF and T2LV outcomes should carefully account for the protocol factors within longitudinal models to ensure that the disease trajectory of MS subjects can be assessed more accurately.
Highlights
•A linear mixed-effect model is useful in modeling longitudinal neuroimaging data.•A model including baseline age and the longitudinal change with age is preferable.•Protocol specific residual variance is important in modeling brain atrophy in MS.•The inclusion of MRI protocol in modeling brain atrophy and lesion load is warranted.
doi:10.1016/j.nicl.2015.06.009
PMCID: PMC4506959  PMID: 26199872
Multiple sclerosis; MRI; Brain atrophy; T2 lesion; Mixed-effect models
5.  Implications of bevacizumab discontinuation in adults with recurrent glioblastoma 
Neuro-Oncology  2014;16(6):823-828.
Background
Patients with recurrent glioblastoma benefiting from bevacizumab are often treated indefinitely due to concerns regarding rebound tumor recurrence upon discontinuation. However, treatment is discontinued for reasons other than disease progression in a subset of these patients, the characteristics and outcomes of which are poorly defined.
Methods
Of 342 adults with recurrent glioblastoma in our database treated with bevacizumab, 82 received treatment for ≥6 months; of these, bevacizumab was discontinued for reasons other than tumor progression in 18 patients (Bev-D) and for disease progression in the remainder (Bev-S). The impact of discontinuation on outcome was assessed with discontinuation as a time-dependent covariate in a Cox hazards model for progression-free survival.
Results
There was no difference in hazard rates for progression between Bev-D and Bev-S groups; the adjusted hazard ratio for progression using discontinuation as a time-dependent covariate was 0.91 (95% CI:0.47, 1.78). The median PFS after bevacizumab-discontinuation was 27 weeks (95% CI:15-NR). At progression, a higher proportion of Bev-D patients had local progression compared with the Bev-S patients. Salvage therapy in Bev-D patients yielded a PFS-26 weeks of 47% (95% CI:23%–94%) with a median PFS of 23 weeks (95% CI:12-NR), vs. 5% (95% CI: 1%–21%) and 9 weeks (95% CI: 6–11) in Bev-S patients (HR:0.3;CI, 0.1–0.6) (P = .0007).
Conclusions
Bevacizumab discontinuation unrelated to disease progression does not appear to cause rebound recurrence or worsen PFS in patients who benefit from bevacizumab. Additionally, Bev-D patients had an improved response to salvage therapy, findings which provide a strong basis for a prospective study.
doi:10.1093/neuonc/nou021
PMCID: PMC4022226  PMID: 24596117
bevacizumab; discontinuation; glioblastoma; outcome; recurrence
6.  A prospective study of adverse drug reactions to antiepileptic drugs in children 
BMJ Open  2015;5(6):e008298.
Objectives
To prospectively determine the nature and rate of adverse drug reactions (ADRs) in children on antiepileptic drugs (AEDs) and to prospectively evaluate the effect of AEDs on behaviour.
Setting
A single centre prospective observational study.
Participants
Children (<18 years old) receiving one or more AEDs for epilepsy, at each clinically determined follow-up visit.
Primary and secondary outcomes
Primary outcome was adverse reactions of AEDs. Behavioural and cognitive functions were secondary outcomes.
Results
180 children were recruited. Sodium valproate and carbamazepine were the most frequently used AEDs. A total of 114 ADRs were recorded in 56 of these children (31%). 135 children (75%) were on monotherapy. 27 of the 45 children (60%) on polytherapy had ADRs; while 29 (21%) of those on monotherapy had ADRs. The risk of ADRs was significantly lower in patients receiving monotherapy than polytherapy (RR: 0.61, 95% CI 0.47 to 0.79, p<0.0001). Behavioural problems and somnolence were the most common ADRs. 23 children had to discontinue their AED due to an ADR.
Conclusions
Behavioural problems and somnolence were the most common ADRs. Polytherapy significantly increases the likelihood of ADRs in children.
Trail registration number
EudraCT (2007-000565-37).
doi:10.1136/bmjopen-2015-008298
PMCID: PMC4458612  PMID: 26033949
CLINICAL PHARMACOLOGY
7.  Routine issuance of clinical magnets to patients receiving implantable defibrillators: retention of information and appropriateness of use 
Open Heart  2015;2(1):e000224.
Background
The application of a clinical magnet over an implantable cardioverter defibrillator (ICD) can be used to suspend tachycardia therapies in patients receiving recurrent or inappropriate shocks. In our institution, they have been routinely issued to patients undergoing ICD implantation during the past 5 years. The purpose of this survey was to investigate how well information concerning their use had been retained, and in what circumstances the magnets had been used.
Methods
We sent a questionnaire to 476 patients, and received a response from 343 (72%). Data was collated using ‘Microsoft Excel’, cross-referenced against our own pacing database, and analysed using basic statistical methods.
Results
256 (74.6%) patients recalled being issued with a magnet. 48% of these were still in possession of their written information leaflet at the time of survey; 62% felt that they were able to remember when and how to use the magnet—with patients who had received written instructions and verbal reinforcement demonstrating the best recall. 8% of patients had used their magnets and the most common reason for use was multiple or inappropriate shocks. In addition, almost half of the patients who had suffered inappropriate shocks had been able to successfully use their magnets. No cases of harm related to magnet use were identified.
Conclusions
The results of our survey suggest that routinely issuing clinical magnets to ICD patients is a safe and effective practice, and a small but significant number of patients were able to utilise their magnets in clinically important situations.
doi:10.1136/openhrt-2014-000224
PMCID: PMC4442150  PMID: 26019880
8.  Too much of a good thing: NLRC4 and autoinflammation 
doi:10.1084/jem.21112insight5
PMCID: PMC4235639  PMID: 25403807
10.  Panostotic Expansile Bone Disease With Massive Jaw Tumor Formation and a Novel Mutation in the Signal Peptide of RANK 
Precise regulation of bone resorption is critical for skeletal homeostasis. We report a 32-year-old man with a panostotic expansile bone disease and a massive hemorrhagic mandibular tumor. Originally from Mexico, he was deaf at birth and became bow-legged during childhood. There was no family history of skeletal disease. Puberty occurred normally, but during adolescence he experienced difficulty straightening his limbs, sustained multiple fractures, and developed a bony tumor on his chin. By age 18 years, all limbs were misshapen. The mandibular mass grew and protruded from the oral cavity, extending to the level of the lower ribs. Other bony defects included a similar maxillary mass and serpentine limbs. Upon referral at age 27 years, biochemical studies showed serum alkaline phosphatase of 1760 U/L (Nl: 29-111) and other elevated bone turnover markers. Radiography of the limbs showed medullary expansion and cortical thinning with severe bowing. Although the jaw tumors were initially deemed inoperable, mandibular mass excision and staged partial maxillectomy were eventually performed. Tumor histopathology showed curvilinear trabeculae of woven bone on a background of hypocellular fibrous tissue. Fibrous dysplasia of bone was suspected, but there was no mutation in codon 201 of GNAS in samples from blood or tumor. His clinical and radiographic findings, elevated serum markers, and disorganized bone morphology suggested amplified receptor activator of NF-κB (RANK) signaling, even though his disorder differed from conditions with known constitutive activation of RANK signaling (eg, familial expansile osteolysis). We found a unique 12-base pair duplication in the signal peptide of TNFRSF11A, the gene that encodes RANK. No exon or splice site mutations were found in the genes encoding RANK ligand or osteoprotegerin. Alendronate followed by pamidronate therapies substantially decreased his serum alkaline phosphatase activity. This unique patient expands the phenotypes and genetic basis of the mendelian disorders of RANK signaling activation.
doi:10.1002/jbmr.2094
PMCID: PMC4419358  PMID: 24014458
PANOSTOTIC EXPANSILE BONE DISEASE; OSTEOCLASTS; RANK SIGNALING; OSTEOPROTEGERIN
11.  Canonical microRNAs in thymic epithelial cells promote central tolerance 
European journal of immunology  2014;44(5):1313-1319.
Medullary thymic epithelial cells (mTECs) facilitate the deletion of developing self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens, a process which largely depends on the expression of the autoimmune regulator (Aire) gene. Mature microRNAs (miRNAs) that regulate gene expression post-transcriptionally are generated in a multistep process. The microprocessor complex, including DGCR8, cleaves canonical miRNAs, but alternative DGCR8-independent miRNA biogenesis pathways exist as well. In order to study the role of canonical miRNAs in thymic epithelial cells (TECs), we ablated Dgcr8 using a FoxN1-Cre transgene. We report that DGCR8-deficient TECs are unable to maintain proper thymic architecture and exhibit a dramatic loss of thymic cellularity. Importantly, DGCR8-deficient TECs develop a severe loss of Aire+ mTECs. Using a novel immunization approach to amplify and detect self-reactive T cells within a polyclonal TCR repertoire, we demonstrate a link between the loss of Aire expression in DGCR8-deficient TECs and the breakdown of negative selection in the thymus. Thus, DGCR8 and canonical miRNAs are important in TECs for supporting central tolerance.
doi:10.1002/eji.201344079
PMCID: PMC4141217  PMID: 24515814
Aire; Central tolerance; DGCR8; MicroRNAs; Thymic epithelial cells
12.  Microtubule-Mediated Defects in Junctophilin-2 Trafficking Contribute to Myocyte T-Tubule Remodeling and Ca2+ Handling Dysfunction in Heart Failure 
Circulation  2014;129(17):1742-1750.
Background
Cardiac dysfunction in failing hearts of human patients and animal models is associated with both microtubule densification and T-tubule remodeling. Our objective was to investigate whether microtubule densification contributes to T-tubule remodeling and excitation-contraction coupling dysfunction in heart disease.
Methods and Results
In a mouse model of pressure overload-induced cardiomyopathy by transaortic banding (TAB), colchicine, a microtubule depolymerizer, significantly ameliorated T-tubule remodeling and cardiac dysfunction. In cultured cardiomyocytes, microtubule depolymerization with nocodazole or colchicine profoundly attenuated T-tubule impairment, whereas microtubule polymerization/stabilization with taxol accelerated T-tubule remodeling. In situ immunofluorescence of heart tissue sections demonstrated significant disorganization of JP2, a protein that bridges the T-tubule and sarcoplasmic reticulum membranes, in TAB hearts as well as in human failing hearts, while colchicine injection significantly preserved the distribution of JP2 in TAB hearts. In isolated mouse cardiomyocytes, prolonged culture or treatment with taxol resulted in pronounced redistribution of JP2 from T-tubules to the peripheral plasma membrane, without changing total JP2 expression. Nocodazole treatment antagonized JP2 redistribution. Moreover, overexpression of a dominant-negative mutant of Kinesin 1, a microtubule motor protein responsible for anterograde trafficking of proteins, protected against JP2 redistribution and T-tubule remodeling in culture. Finally, nocodazole treatment improved Ca2+ handling in cultured myocytes by increasing the amplitude of Ca2+ transients and reducing the frequency of Ca2+ sparks.
Conclusions
Our data identify a mechanistic link between microtubule densification and T-tubule remodeling and reveal microtubule-mediated JP2 redistribution as a novel mechanism for T-tubule disruption, loss of E-C coupling, and heart failure.
doi:10.1161/CIRCULATIONAHA.113.008452
PMCID: PMC4006305  PMID: 24519927
Cardiomyocytes; microtubules; T-tubules; Junctophilin-2; excitation-contraction coupling
13.  Heterogeneity of reactive astrocytes 
Neuroscience letters  2013;0:23-29.
Astrocytes respond to injury and disease in the central nervous system (CNS) with a process referred to as reactive astrogliosis. Recent progress demonstrates that reactive astrogliosis is not a simple all-or-none phenomenon, but is a finely gradated continuum of changes that range from reversible alterations in gene expression and cell hypertrophy, to scar formation with permanent tissue rearrangement. There is now compelling evidence that reactive astrocytes exhibit a substantial potential for heterogeneity at multiple levels, including gene expression, cell morphology, topography (distance from lesions), CNS regions, local (among neighboring cells), cell signaling and cell function. Structural and functional changes are regulated in reactive astrocytes by many different potential signaling events that occur in a context dependent manner. It is noteworthy that different stimuli of astrocyte reactivity can lead to similar degrees of GFAP upregulation while causing substantially different changes in transcriptome profiles and cell function. Thus, it is not possible to equate simple and uniform measures such as cell hypertrophy and upregulation of GFAP expression with a single, uniform concept of astrocyte reactivity. Instead, it is necessary to recognize the considerable potential for heterogeneity and determine the functional implications of astrocyte reactivity in a context specific manner as regulated by specific signaling events.
doi:10.1016/j.neulet.2013.12.030
PMCID: PMC3984948  PMID: 24361547
reactive astrocytes; reactive astrogliosis; glial scar; trauma; inflammation; autoimmune disease; heterogeneity
14.  βIV-Spectrin regulates TREK-1 membrane targeting in the heart 
Cardiovascular Research  2014;102(1):166-175.
Aims
Cardiac function depends on the highly regulated and co-ordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K+ channels have been well characterized in the heart, much less is known about regulation and/or targeting of two-pore K+ channel (K2P) family members, despite their potential importance in modulation of heart function.
Methods and results
Here, we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K2P channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that βIV-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in the heart, and is required for TREK-1 membrane targeting. Mice expressing βIV-spectrin lacking TREK-1 binding (qv4J) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal βIV-spectrin levels in human heart failure.
Conclusions
These data provide new insight into membrane targeting of TREK-1 in the heart and establish a broader role for βIV-spectrin in organizing functional membrane domains critical for normal heart function.
doi:10.1093/cvr/cvu008
PMCID: PMC3958619  PMID: 24445605
Arrhythmia; Ankyrin; Spectrin; TREK-1; Two-pore potassium channel
15.  Clinical and prognostic features of adult patients with gangliogliomas 
Neuro-Oncology  2013;16(3):409-413.
Background
Gangliogliomas (GGs) represent <1% of primary brain tumors in adults. Little is known regarding prognostic features, clinical characteristics, or the impact of treatment on patient outcomes.
Methods
Our neuro-oncology longitudinal database was screened for patients with GG from 1992 to 2012. Sixty-seven patients (age >18 y) were identified.
Results
Sixty-two patients presented with low-grade GG and 5 with anaplastic GG. The median age at diagnosis was 29 years. With a median follow-up of 4.7 years after the initial diagnosis, 23 patients had progressive disease. Range of time to progression was 0.2–20 years. Nine patients with low-grade GG progressed to a malignant tumor. The median overall survival (OS) for all patients was not reached. The 2-, 5-, and 10-year OS for patients with low-grade GG were 100%, 88% (95% confidence interval [CI]: 73%, 95%), and 84% (95% CI: 67%, 93%), respectively.
Factors identified by univariate analysis that were significantly associated with OS were age, KPS, extent of resection (EOR), and grade. Factors on univariate analysis that were significantly associated with progression-free survival were grade and EOR. On multicovariate Cox regression, lower tumor grade and younger age were significant factors for longer OS. EOR is a significant factor for progression-free survival.
Conclusions
While GG has excellent prognosis, malignant histologic grade, older age, and diagnosis with biopsy could indicate worse prognosis. The late nature and high rate of progression emphasize the importance of long-term follow-up. The role of chemotherapy and radiation therapy for incompletely resected low-grade GG remains unclear.
doi:10.1093/neuonc/not169
PMCID: PMC3922509  PMID: 24305706
adults; ganglioglioma; malignant transformation; progression; treatment
16.  The transcriptional regulator Aire co-opts the repressive ATF7ip-MBD1 complex for induction of immune tolerance 
Nature immunology  2014;15(3):258-265.
The maintenance of immune tolerance requires the deletion of self-reactive T cells in the thymus. The expression of tissue-specific antigen genes (TSAs) by thymic epithelial cells is critical for this process and depends on the activity of the Autoimmune Regulator (Aire) protein, however, the molecular mechanism(s) Aire uses to target TSA gene loci are unknown. Here we identified two Aire-interacting proteins – activating transcription factor 7 interacting protein (ATF7ip) and methyl CpG binding protein 1 (MBD1) –that are required for Aire’s targeting of TSA geneloci. Moreover, Mbd1−/− mice developed pathological autoimmunity and had a defect in Aire-dependent thymic TSA gene expression underscoring the critical importance of Aire’s interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.
doi:10.1038/ni.2820
PMCID: PMC4172453  PMID: 24464130
17.  CaMKII Is Essential for the Proasthmatic Effects of Oxidation 
Science translational medicine  2013;5(195):195ra97.
Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca2+/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl− current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyper-reactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent pro-asthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
doi:10.1126/scitranslmed.3006135
PMCID: PMC4331168  PMID: 23884469
18.  Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides 
Cardiovascular Research  2013;101(1):165-174.
Aims
Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca2+ handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined.
Methods and results
We examined the effects of digitoxin (DGT) on Ca2+ handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca2+ waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated.
Conclusion
These results suggest that the arrhythmogenic adverse effects of CGs on Ca2+ handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
doi:10.1093/cvr/cvt233
PMCID: PMC3868350  PMID: 24104877
Calcium; Reactive oxygen species; NADPH oxidase; Mitochondria; CaMKII
19.  Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance 
Modulation of central tolerance through RANKL alters thymic output and enhances anti-tumor immunity.
Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.
doi:10.1084/jem.20131889
PMCID: PMC4010907  PMID: 24752296
20.  Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice 
Nature neuroscience  2014;17(5):694-703.
Huntington's disease (HD) is characterized by striatal medium spiny neuron (MSN) dysfunction, but the underlying mechanisms remain unclear. We explored roles for astrocytes, which display mutant huntingtin in HD patients and mouse models. We found that symptom onset in R6/2 and Q175 HD mouse models is not associated with classical astrogliosis, but is associated with decreased Kir4.1 K+ channel functional expression, leading to elevated in vivo levels of striatal extracellular K+, which increased MSN excitability in vitro. Viral delivery of Kir4.1 channels to striatal astrocytes restored Kir4.1 function, normalized extracellular K+, recovered aspects of MSN dysfunction, prolonged survival and attenuated some motor phenotypes in R6/2 mice. These findings indicate that components of altered MSN excitability in HD may be caused by heretofore unknown disturbances of astrocyte–mediated K+ homeostasis, revealing astrocytes and Kir4.1 channels as novel therapeutic targets.
doi:10.1038/nn.3691
PMCID: PMC4064471  PMID: 24686787
21.  Secretion of atypical protein subtrates by the ESAT-6 Secretion System of Staphylococcus aureus 
Molecular microbiology  2013;90(4):734-743.
SUMMARY
Staphylococcus aureus encodes the specialized ESAT-6 Secretion System (ESS). EsxA and EsxB are secreted by the ESS pathway, and share sequence features of ESAT-6 and CFP-10 of the Type VII Secretion System (T7SS) of Mycobacterium tuberculosis. Unlike ESAT-6 and CFP-10, EsxA and EsxB do not interact. Instead, EsxB associates with a novel substrate, EsxD, and EsxA dimerizes with itself or EsxC (EsaC). Unlike EsxA and EsxB, EsxC and EsxD do not share obvious sequence features of WXG100 proteins nor PE/PPE and Esp families of proteins, all of which belong to the pfam EsxAB clan of mycobacterial T7SS. EsxD carries the C terminal motif YxxxD/E that has been proposed to target T7 substrates for secretion in mycobacteria. Here, we find that deletion, but not amino acid substitutions, in this motif prevent secretion of EsxA and EsxC but not EsxB or EsxD. This is unlike the genetic inactivation of esxA, esxB, esxC or esxD that leads to loss of secretion of all four substrates. Thus, substrate secretion can be uncoupled by deleting the last six amino acids of EsxD. The physical association of EsxC and EsxD with canonical WXG100 proteins suggests that these proteins belong to the EsxAB clan.
doi:10.1111/mmi.12395
PMCID: PMC3951145  PMID: 24033479
22.  Lineage tracing and cell ablation identifies a post-Aire expressing thymic epithelial cell population 
Cell reports  2013;5(1):10.1016/j.celrep.2013.08.038.
Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and MHC II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire+ mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire− mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates towards the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.
doi:10.1016/j.celrep.2013.08.038
PMCID: PMC3820422  PMID: 24095736
23.  Oxidized CaMKII Triggers Atrial Fibrillation 
Circulation  2013;128(16):10.1161/CIRCULATIONAHA.113.003313.
Background
Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF.
Methods and Results
We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion.
Conclusions
Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.
doi:10.1161/CIRCULATIONAHA.113.003313
PMCID: PMC3876034  PMID: 24030498
atrial fibrillation; arrhythmia mechanisms; calcium/calmodulin-dependent protein kinase II; angiotensin II; reactive oxygen species
24.  Critical roles of junctophilin-2 in T-tubule and excitation–contraction coupling maturation during postnatal development 
Cardiovascular Research  2013;100(1):54-62.
Aims
Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation.
Methods and results
Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TTpower) index revealed a progressive increase in TTpower between postnatal Days 10 and 19. By postnatal Day 19, TTpower was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TTpower. We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca2+ transient amplitude and marked increase in Ca2+ release dyssynchrony, Ca2+ alternans, and spontaneous Ca2+ waves, leading to contractile failure.
Conclusion
Our data identify a critical role for JP2 in T-tubule and excitation–contraction coupling maturation during development.
doi:10.1093/cvr/cvt180
PMCID: PMC3778961  PMID: 23860812
T-tubules; Junctophilin-2; Excitation–contraction coupling; Calcium; Heart development
25.  Extrathymic Aire-Expressing Cells are a Distinct Bone Marrow-Derived Population that Induce Functional Inactivation of CD4+ T Cells 
Immunity  2013;39(3):560-572.
Summary
The autoimmune regulator (AIRE) is essential for prevention of autoimmunity; its role is best understood in the thymus where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)hi, CD80lo, CD86lo, epithelial cell adhesion molecule (EpCAM)hi, CD45lo bone marrow-derived peripheral antigen presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4+ T cells through a mechanism that does not require regulatory T cells (Treg), and is resistant to innate inflammatory stimuli. Together these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.
doi:10.1016/j.immuni.2013.08.005
PMCID: PMC3804105  PMID: 23993652

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