Proteomic investigations of adult polyglucosan body disease: insights into the pathobiology of a neurodegenerative disorder.

Inadequate glycogen branching enzyme 1 (GBE1) activity results in different forms of glycogen storage disease type IV, including adult polyglucosan body disorder (APBD). APBD is clinically characterized by adult-onset development of progressive spasticity, neuropathy, and neurogenic bladder and is histologically characterized by the accumulation of structurally abnormal glycogen (polyglucosan bodies) in multiple cell types. How insufficient GBE1 activity causes the disease phenotype of APBD is poorly understood. We hypothesized that proteomic analysis of tissue from GBE1-deficient individuals would provide insights into GBE1-mediated pathobiology. In this discovery study, we utilized label-free LC-MS/MS to quantify the proteomes of lymphoblasts from 3 persons with APBD and 15 age- and gender-matched controls, with validation of the findings by targeted MS. There were 531 differentially expressed proteins out of 3,427 detected between APBD subjects vs. controls, including pronounced deficiency of GBE1. Bioinformatic analyses indicated multiple canonical pathways and protein-protein interaction networks to be statistically markedly enriched in APBD subjects, including: RNA processing/transport/translation, cell cycle control/replication, mTOR signaling, protein ubiquitination, unfolded protein and endoplasmic reticulum stress responses, glycolysis and cell death/apoptosis. Dysregulation of these processes, therefore, are primary or secondary factors in APBD pathobiology in this model system. Our findings further suggest that proteomic analysis of GBE1 mutant lymphoblasts can be leveraged as part of the screening for pharmaceutical agents for the treatment of APBD.

Gut microbe-targeted choline trimethylamine lyase inhibition improves obesity via rewiring of host circadian rhythms.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline.

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

Therapeutic Targeting of Notch Signaling: From Cancer to Inflammatory Disorders.

Over the past two decades, the Notch signaling pathway has been investigated as a therapeutic target for the treatment of cancers, and more recently in the context of immune and inflammatory disorders. Notch is an evolutionary conserved pathway found in all metazoans that is critical for proper embryonic development and for the postnatal maintenance of selected tissues. Through cell-to-cell contacts, Notch orchestrates cell fate decisions and differentiation in non-hematopoietic and hematopoietic cell types, regulates immune cell development, and is integral to shaping the amplitude as well as the quality of different types of immune responses. Depriving some cancer types of Notch signals has been shown in preclinical studies to stunt tumor growth, consistent with an oncogenic function of Notch signaling. In addition, therapeutically antagonizing Notch signals showed preclinical potential to prevent or reverse inflammatory disorders, including autoimmune diseases, allergic inflammation and immune complications of life-saving procedures such allogeneic bone marrow and solid organ transplantation (graft-versus-host disease and graft rejection). In this review, we discuss some of these unique approaches, along with the successes and challenges encountered so far to target Notch signaling in preclinical and early clinical studies. Our goal is to emphasize lessons learned to provide guidance about emerging strategies of Notch-based therapeutics that could be deployed safely and efficiently in patients with immune and inflammatory disorders.

TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis.

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1's functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.

The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation.

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM-Qa-SNARE complex, Vps45-Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45-Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Regulatory T cells differ from conventional CD4+ T cells in their recirculatory behavior and lymph node transit times.

Regulatory T cells (Tregs) continuously suppress autoreactive immune responses within tissues to prevent autoimmunity, yet the recirculatory behavior of Tregs between and within tissues enabling the maintenance of peripheral tolerance remains incompletely defined. Here, we quantified homing efficiency to and the dwell time of Tregs within secondary lymphoid organs (SLOs) and used intravital two-photon microscopy to measure Treg surveillance behavior of dendritic cells. Tregs homed substantially less efficiently to SLOs compared with conventional CD4+ T cells (Tconvs), despite similar expression of homing receptors. Tregs remained on average 2-3 times longer within the LN than Tconvs before exiting, and retained Tregs differed from recirculating Tregs in phenotype, motility and interaction duration with dendritic cells. Taken together, these data revealed fundamental differences in Treg versus conventional T cell in vivo recirculation and migration behaviors, identified a Treg population with prolonged LN dwell time, and provided quantitative insight into their spatiotemporal behavior within LNs.

Corrigendum: Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.

This corrects the article DOI: 10.1038/nm.4475.

CCL3 augments tumor rejection and enhances CD8+ T cell infiltration through NK and CD103+ dendritic cell recruitment via IFNγ.

Inflammatory chemokines are critical contributors in attracting relevant immune cells to the tumor microenvironment and driving cellular interactions and molecular signaling cascades that dictate the ultimate outcome of host anti-tumor immune response. Therefore, rational application of chemokines in a spatial-temporal dependent manner may constitute an attractive adjuvant in immunotherapeutic approaches against cancer. Existing data suggest that the macrophage inflammatory protein (MIP)-1 family and related proteins, consisting of CCL3 (MIP-1α), CCL4 (MIP-1ß), and CCL5 (RANTES), can be major determinant of immune cellular infiltration in certain tumors through their direct recruitment of antigen presenting cells, including dendritic cells (DCs) to the tumor site. In this study, we examined how CCL3 in a murine colon tumor microenvironment, CT26, enhances antitumor immunity. We identified natural killer (NK) cells as a major lymphocyte subtype that is preferentially recruited to the CCL3-rich tumor site. NK cells contribute to the overall IFNγ content, CD103+ DC accumulation, and augment the production of chemokines CXCL9 and CXCL10 for enhanced T cell recruitment. We further demonstrate that both soluble CCL3 and CCL3-secreting irradiated tumor vaccine can effectively halt the progression of established tumors in a spatial-dependent manner. Our finding implies an important contribution of NK in the CCL3 - CD103+ DC - CXCL9/10 signaling axis in determining tumor immune landscape within the tumor microenvironment.

Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

CCL3 Enhances Antitumor Immune Priming in the Lymph Node via IFNγ with Dependency on Natural Killer Cells.

Lymph node (LN) plays a critical role in tumor cell survival outside of the primary tumor sites and dictates overall clinical response in many tumor types (1, 2). Previously, we and others have demonstrated that CCL3 plays an essential role in orchestrating T cell-antigen-presenting cell (APC) encounters in the draining LN following vaccination, and such interactions enhance the magnitude of the memory T cell pool (3-5). In the current study, we investigate the cellular responses in the tumor-draining lymph nodes (TDLNs) of a CCL3-secreting CT26 colon tumor (L3TU) as compared to wild-type tumor (WTTU) during the priming phase of an antitumor response (≤10 days). In comparison to WTTU, inoculation of L3TU resulted in suppressed tumor growth, a phenomenon that is accompanied by altered in vivo inflammatory responses on several fronts. Autologous tumor-derived CCL3 (aCCL3) secretion by L3TU bolstered the recruitment of T- and B-lymphocytes, tissue-migratory CD103+ dendritic cells (DCs), and CD49b+ natural killer (NK) cells, resulting in significant increases in the differentiation and activation of multiple Interferon-gamma (IFNγ)-producing leukocytes in the TDLN. During this early phase of immune priming, NK cells constitute the major producers of IFNγ in the TDLN. CCL3 also enhances CD8+ T cell proliferation and differentiation by augmenting DC capacity to drive T cell activation in the TDLN. Our results revealed that CCL3-dependent IFNγ production and CCL3-induced DC maturation drive the priming of effective antitumor immunity in the TDLN.

Adoptive natural killer cell therapy is effective in reducing pulmonary metastasis of Ewing sarcoma.

The survival of patients with metastatic or relapsed Ewing sarcoma (ES) remains dismal despite intensification of combination chemotherapy and radiotherapy, precipitating the need for novel alternative therapies with minimal side effects. Natural killer (NK) cells are promising additions to the field of cellular immunotherapy. Adoptive NK cell therapy has shown encouraging results in hematological malignancies. Despite these initial promising successes, however, NK cell therapy for solid tumors remains to be investigated using in vivo tumor models. The purpose of this study is to evaluate the efficacy of ex vivo expanded human NK cells in controlling primary and metastatic ES tumor growth in vitro and in vivo. Using membrane-bound IL-21 containing K562 (K562-mbIL-21) expansion platform, we were able to obtain sufficient numbers of expanded NK (eNK) cells that display favorable activation phenotypes and inflammatory cytokine secretion, along with a strong in vitro cytotoxic effect against ES. Furthermore, eNK therapy significantly decreased lung metastasis without any significant therapeutic effect in limiting primary tumor growth in an in vivo xenograft model. Our data demonstrate that eNK may be effective against pulmonary metastatic ES, but challenges remain to direct proper trafficking and augmenting the cytotoxic function of eNK to target primary tumor sites.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands.

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Unique Transcompartmental Bridge: Antigen-Presenting Cells Sampling across Endothelial and Mucosal Barriers.

Potentially harmful pathogens can gain access to tissues and organ systems through body sites that are in direct contact with the outside environment, such as the skin, the gut, and the airway mucosa. Antigen-presenting cells (APCs) represent a bridge between the innate and adaptive immunity, and their capacity for constant immune surveillance and rapid sampling of incoming pathogens and other potentially harmful antigens is central for mounting an effective and robust protective host response. The classical view is that APCs perform this task efficiently within the tissue to sense invading agents intra-compartmentally. However, recent data based on high resolution imaging support an additional transcompartmental surveillance behavior by APC by reaching across intact physical barriers. In this review, we summarize intravital microscopic evidences of APC to sample antigens transcompartmentally at the gut mucosa and other body sites.

Polyphenol administration impairs T-cell proliferation by imprinting a distinct dendritic cell maturational profile.

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs-QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs-QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs-QP-exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs-QP administration suppresses antigen-specific T-cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti-inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.

Murine leukemia virus envelope gp70 is a shared biomarker for the high-sensitivity quantification of murine tumor burden.

The preclinical development of anticancer drugs including immunotherapeutics and targeted agents relies on the ability to detect minimal residual tumor burden as a measure of therapeutic efficacy. Real-time quantitative (qPCR) represents an exquisitely sensitive method to perform such an assessment. However, qPCR-based applications are limited by the availability of a genetic defect associated with each tumor model under investigation. Here, we describe an off-the-shelf qPCR-based approach to detect a broad array of commonly used preclinical murine tumor models. In particular, we report that the mRNA coding for the envelope glycoprotein 70 (gp70) encoded by the endogenous murine leukemia virus (MuLV) is universally expressed in 22 murine cancer cell lines of disparate histological origin but is silent in 20 out of 22 normal mouse tissues. Further, we detected the presence of as few as 100 tumor cells in whole lung extracts using qPCR specific for gp70, supporting the notion that this detection approach has a higher sensitivity as compared with traditional tissue histology methods. Although gp70 is expressed in a wide variety of tumor cell lines, it was absent in inflamed tissues, non-transformed cell lines, or pre-cancerous lesions. Having a high-sensitivity biomarker for the detection of a wide range of murine tumor cells that does not require additional genetic manipulations or the knowledge of specific genetic alterations present in a given neoplasm represents a unique experimental tool for investigating metastasis, assessing antitumor therapeutic interventions, and further determining tumor recurrence or minimal residual disease.

Bilateral thoracic sympathetic block for refractory polymorphic tachycardia.

INTRODUCTION: Extensive evidence has established a link between sympathetic nervous system hyperactivity, ventricular arrhythmias, and sudden cardiac death. For this reason, cardiac sympathectomy is often beneficial in the treatment of patients at high risk for ventricular ectopy, although it involves an invasive procedure associated with potential morbidity. We report a case in which we used guided lytic thoracic sympathetic block in a patient with underlying cardiomyopathy and refractory polymorphic ventricular tachycardia. CLINICAL FEATURES: A 74-yr-old African American male with ischemic cardiomyopathy presented with refractory episodes of ventricular tachycardia despite maximal medical therapy involving antiarrhythmic drugs and previous interventions, including endovascular epicardial ablation and open cryoablation via sternotomy. During his inpatient admission, the patient developed sustained ventricular tachycardia associated with cardiac depression requiring vasopressors. An open thoracoscopic sympathectomy was considered as a possible treatment, but in our view, the patient would not tolerate this procedure. As an alternative, the pain medicine team successfully performed a lytic thoracic sympathetic block. Subsequently, the patient demonstrated a period of clinical improvement with no apparent morbidity related to the procedure. CONCLUSION: Lytic thoracic sympathetic blockade is a novel technique for the treatment of sympathetically mediated ventricular tachycardia, and it is less invasive than other types of cardiac sympathectomy. Additional studies are required to evaluate this treatment as a viable alternative in patients at high risk for ventricular ectopy. This report suggests the feasibility of this approach and the potential for minimal morbidity in cases of refractory ventricular arrhythmias.

Case report: intracranial placement of a nasotracheal tube in a patient with Goldenhar syndrome associated with cribriform plate agenesis.

We describe a case of inadvertent intracranial placement of a nasotracheal tube in a patient with an undiagnosed major congenital cranial anomaly (a variant of Goldenhar syndrome, which included absence of the cribriform plate). We believe that this is the first reported case in which this complication arose as a result of a congenital abnormality rather than traumatic or iatrogenic disruption of the skull base. We conclude that patients with known craniofacial abnormalities or associated syndromes scheduled for procedures involving planned nasotracheal intubation or nasogastric tube placement should undergo preoperative cranial imaging studies to verify an intact skull base.

Parenteral and enteral metabolism of anaplerotic triheptanoin in normal rats. II. Effects on lipolysis, glucose production, and liver acyl-CoA profile.

The anaplerotic odd-medium-chain triglyceride triheptanoin is used in clinical trials for the chronic dietary treatment of patients with long-chain fatty acid oxidation disorders. We previously showed (Kinman RP, Kasumov T, Jobbins KA, Thomas KR, Adams JE, Brunengraber LN, Kutz G, Brewer WU, Roe CR, Brunengraber H. Am J Physiol Endocrinol Metab 291: E860-E866, 2006) that the intravenous infusion of triheptanoin increases lipolysis traced by the turnover of glycerol. In this study, we tested whether lipolysis induced by triheptanoin infusion is accompanied by the potentially harmful release of long-chain fatty acids. Rats were infused with heptanoate +/- glycerol or triheptanoin. Intravenous infusion of triheptanoin at 40% of caloric requirement markedly increased glycerol endogenous R(a) but not oleate endogenous R(a). Thus, the activation of lipolysis was balanced by fatty acid reesterification in the same cells. The liver acyl-CoA profile showed the accumulation of intermediates of heptanoate beta-oxidation and C(5)-ketogenesis and a decrease in free CoA but no evidence of metabolic perturbation of liver metabolism such as propionyl overload. Our data suggest that triheptanoin, administered either intravenously or intraduodenally, could be used for intensive care and nutritional support of metabolically decompensated long-chain fatty acid oxidation disorders.

Impact of mandatory resident work hour limitations on medical students' interest in surgery.

BACKGROUND: The number of US medical students applying for general surgery residency has been declining. Recent studies have shown that the issue of "controllable lifestyle" has become a critical factor in medical students' decision-making process. We postulate that widespread implementation of resident work hour limitations would bolster medical students' interest in pursuing surgical careers. STUDY DESIGN: Students from New York University School of Medicine were surveyed about their attitudes toward work hour limitations and its effect on their interest in pursuing a surgical residency. One hundred thirty-two students participated. RESULTS: Nearly 95% of respondents believed that work hour limitations were a positive change and, if all other factors were equal, they would choose a training program that used work hour limitations over one that did not. The most common reasons cited in favor of limits were improvements in resident lifestyle (42%) and patient safety (34%). Fifty-three percent of respondents indicated that presence of work hour limitations alone would increase their interest in considering a surgical residency and only 2% of medical students indicated that it would lessen their interest in surgery. Not surprisingly, intellectual interest in a specialty was the most important factor in choosing a residency for 86% of students. Nevertheless, work hour limitations were designated a higher priority than future salary by 55% of medical students. CONCLUSIONS: The presence of work hour limitations has a positive impact on medical students' interest in surgery. Widespread implementation of work hour limitations may bolster the number of applications for surgical residency.