Concise Communication

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset degeneration of motor neurons that is commonly caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Both patients and Tg mice expressing mutant human SOD1 (hSOD1) develop aggregates of unknown importance. In Tg mice, 2 different strains of hSOD1 aggregates (denoted A and B) can arise; however, the role of these aggregates in disease pathogenesis has not been fully characterized. Here, minute amounts of strain A and B hSOD1 aggregate seeds that were prepared by centrifugation through a density cushion were inoculated into lumbar spinal cords of 100-day-old mice carrying a human SOD1 Tg. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill after approximately 100 days, which is 200 days earlier than for mice that had not been inoculated or were given a control preparation. Concomitantly, exponentially growing strain A and B hSOD1 aggregations propagated rostrally throughout the spinal cord and brainstem. The phenotypes provoked by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. Together, our data indicate that the aggregate strains are prions that transmit a templated, spreading aggregation of hSOD1, resulting in a fatal ALS-like disease.

Authors

Elaheh Ekhtiari Bidhendi, Johan Bergh, Per Zetterström, Peter M. Andersen, Stefan L. Marklund, Thomas Brännström

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Abstract

The development of pathologic mucus, which is not readily cleared from the airways, is an important contributor to the morbidity and mortality associated with asthma. It is not clear how the major airway mucins MUC5AC and MUC5B are organized within the mucus gel or how this gel contributes to airway obstruction in asthma. Here, we demonstrated that mucus plugs from individuals with fatal asthma are heterogeneous gels with distinct MUC5AC- and MUC5B-containing domains. Stimulation of cultured human bronchial epithelial cells with IL-13, a key mediator in asthma, induced the formation of heterogeneous mucus gels and dramatically impaired mucociliary transport. Impaired transport was not associated with defects in ciliary function but instead was related to tethering of MUC5AC-containing mucus gel domains to mucus-producing cells in the epithelium. Replacement of tethered mucus with untethered mucus restored mucociliary transport. Together, our results indicate that tethering of MUC5AC-containing domains to the epithelium causes mucostasis and likely represents a major cause of mucus plugging in asthma.

Authors

Luke R. Bonser, Lorna Zlock, Walter Finkbeiner, David J. Erle

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Abstract

Antibodies that target the immune checkpoint receptor programmed cell death protein 1 (PD-1) have resulted in prolonged and beneficial responses toward a variety of human cancers. However, anti–PD-1 therapy in some patients provides no benefit and/or results in adverse side effects. The factors that determine whether patients will be drug sensitive or resistant are not fully understood; therefore, genomic assessment of exceptional responders can provide important insight into patient response. Here, we identified a patient with endometrial cancer who had an exceptional response to the anti–PD-1 antibody pembrolizumab. Clinical grade targeted genomic profiling of a pretreatment tumor sample from this individual identified a mutation in DNA polymerase epsilon (POLE) that associated with an ultramutator phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that the presence of POLE mutation associates with high mutational burden and elevated expression of several immune checkpoint genes. Together, these data suggest that cancers harboring POLE mutations are good candidates for immune checkpoint inhibitor therapy.

Authors

Janice M. Mehnert, Anshuman Panda, Hua Zhong, Kim Hirshfield, Sherri Damare, Katherine Lane, Levi Sokol, Mark N. Stein, Lorna Rodriguez-Rodriquez, Howard L. Kaufman, Siraj Ali, Jeffrey S. Ross, Dean C. Pavlick, Gyan Bhanot, Eileen P. White, Robert S. DiPaola, Ann Lovell, Jonathan Cheng, Shridar Ganesan

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Abstract

BACKGROUND. Clinical laboratory tests are now being prescribed and made directly available to consumers through retail outlets in the USA. Concerns with these test have been raised regarding the uncertainty of testing methods used in these venues and a lack of open, scientific validation of the technical accuracy and clinical equivalency of results obtained through these services.

METHODS. We conducted a cohort study of 60 healthy adults to compare the uncertainty and accuracy in 22 common clinical lab tests between one company offering blood tests obtained from finger prick (Theranos) and 2 major clinical testing services that require standard venipuncture draws (Quest and LabCorp). Samples were collected in Phoenix, Arizona, at an ambulatory clinic and at retail outlets with point-of-care services.

RESULTS. Theranos flagged tests outside their normal range 1.6× more often than other testing services (P < 0.0001). Of the 22 lab measurements evaluated, 15 (68%) showed significant interservice variability (P < 0.002). We found nonequivalent lipid panel test results between Theranos and other clinical services. Variability in testing services, sample collection times, and subjects markedly influenced lab results.

CONCLUSION. While laboratory practice standards exist to control this variability, the disparities between testing services we observed could potentially alter clinical interpretation and health care utilization. Greater transparency and evaluation of testing technologies would increase their utility in personalized health management.

FUNDING. This work was supported by the Icahn Institute for Genomics and Multiscale Biology, a gift from the Harris Family Charitable Foundation (to J.T. Dudley), and grants from the NIH (R01 DK098242 and U54 CA189201, to J.T. Dudley, and R01 AG046170 and U01 AI111598, to E.E. Schadt).

Authors

Brian A. Kidd, Gabriel Hoffman, Noah Zimmerman, Li Li, Joseph W. Morgan, Patricia K. Glowe, Gregory J. Botwin, Samir Parekh, Nikolina Babic, Matthew W. Doust, Gregory B. Stock, Eric E. Schadt, Joel T. Dudley

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Abstract

Asthma is one of the most common chronic diseases globally and can be divided into presenting with or without an immune response. Current therapies have little effect on nonimmune disease, and the mechanisms that drive this type of asthma are poorly understood. Here, we have shown that loss of the transcription factors forkhead box P1 (Foxp1) and Foxp4, which are critical for lung epithelial development, in the adult airway epithelium evokes a non-Th2 asthma phenotype that is characterized by airway hyperresponsiveness (AHR) without eosinophilic inflammation. Transcriptome analysis revealed that loss of Foxp1 and Foxp4 expression induces ectopic expression of neuropeptide Y (Npy), which has been reported to be present in the airways of asthma patients, but whose importance in disease pathogenesis remains unclear. Treatment of human lung airway explants with recombinant NPY increased airway contractility. Conversely, loss of Npy in Foxp1- and Foxp4-mutant airway epithelium rescued the AHR phenotype. We determined that NPY promotes AHR through the induction of Rho kinase activity and phosphorylation of myosin light chain, which induces airway smooth muscle contraction. Together, these studies highlight the importance of paracrine signals from the airway epithelium to the underlying smooth muscle to induce AHR and suggest that therapies targeting epithelial induction of this phenotype may prove useful in treatment of noneosinophilic asthma.

Authors

Shanru Li, Cynthia Koziol-White, Joseph Jude, Meiqi Jiang, Hengjiang Zhao, Gaoyuan Cao, Edwin Yoo, William Jester, Michael P. Morley, Su Zhou, Yi Wang, Min Min Lu, Reynold A. Panettieri Jr., Edward E. Morrisey

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Abstract

It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices.

Authors

Jeannette S. Kirkegaard, Philippe Ravassard, Signe Ingvarsen, Marc Diedisheim, Emilie Bricout-Neveu, Mads Grønborg, Thomas Frogne, Raphael Scharfmann, Ole D. Madsen, Claude Rescan, Olivier Albagli

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Abstract

Selenium is a trace element that is essential for human health and is incorporated into more than 25 human selenocysteine-containing (Sec-containing) proteins via unique Sec-insertion machinery that includes a specific, nuclear genome–encoded, transfer RNA (tRNA[Ser]Sec). Here, we have identified a human tRNA[Ser]Sec mutation in a proband who presented with a variety of symptoms, including abdominal pain, fatigue, muscle weakness, and low plasma levels of selenium. This mutation resulted in a marked reduction in expression of stress-related, but not housekeeping, selenoproteins. Evaluation of primary cells from the homozygous proband and a heterozygous parent indicated that the observed deficit in stress-related selenoprotein production is likely mediated by reduced expression and diminished 2′-O-methylribosylation at uridine 34 in mutant tRNA[Ser]Sec. Moreover, this methylribosylation defect was restored by cellular complementation with normal tRNA[Ser]Sec. This study identifies a tRNA mutation that selectively impairs synthesis of stress-related selenoproteins and demonstrates the importance of tRNA modification for normal selenoprotein synthesis.

Authors

Erik Schoenmakers, Bradley Carlson, Maura Agostini, Carla Moran, Odelia Rajanayagam, Elena Bochukova, Ryuta Tobe, Rachel Peat, Evelien Gevers, Francesco Muntoni, Pascale Guicheney, Nadia Schoenmakers, Sadaf Farooqi, Greta Lyons, Dolph Hatfield, Krishna Chatterjee

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Abstract

Nonalcoholic steatohepatitis (NASH) is the most common liver disease in industrialized countries. NASH is a progressive disease that can lead to cirrhosis, cancer, and death, and there are currently no approved therapies. The development of NASH in animal models requires intact TLR9, but how the TLR9 pathway is activated in NASH is not clear. Our objectives in this study were to identify NASH-associated ligands for TLR9, establish the cellular requirement for TLR9, and evaluate the role of obesity-induced changes in TLR9 pathway activation. We demonstrated that plasma from mice and patients with NASH contains high levels of mitochondrial DNA (mtDNA) and intact mitochondria and has the ability to activate TLR9. Most of the plasma mtDNA was contained in microparticles (MPs) of hepatocyte origin, and removal of these MPs from plasma resulted in a substantial decrease in TLR9 activation capacity. In mice, NASH development in response to a high-fat diet required TLR9 on lysozyme-expressing cells, and a clinically applicable TLR9 antagonist blocked the development of NASH when given prophylactically and therapeutically. These data demonstrate that activation of the TLR9 pathway provides a link between the key metabolic and inflammatory phenotypes in NASH.

Authors

Irma Garcia-Martinez, Nicola Santoro, Yonglin Chen, Rafaz Hoque, Xinshou Ouyang, Sonia Caprio, Mark J. Shlomchik, Robert Lee Coffman, Albert Candia, Wajahat Zafar Mehal

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Abstract

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2–transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A–specific (MART-1–specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy.

Authors

Matthias Leisegang, Thomas Kammertoens, Wolfgang Uckert, Thomas Blankenstein

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Abstract

Analogous behavioral assays are needed across animal models and human patients to improve translational research. Here, we examined the extent to which performance in the Morris water maze — the most frequently used behavioral assay of spatial learning and memory in rodents — translates to humans. We designed a virtual version of the assay for human subjects that includes the visible-target training, hidden-target learning, and probe trials that are typically administered in the mouse version. We compared transgenic mice that express human amyloid precursor protein (hAPP) and patients with mild cognitive impairment due to Alzheimer’s disease (MCI-AD) to evaluate the sensitivity of performance measures in detecting deficits. Patients performed normally during visible-target training, while hAPP mice showed procedural learning deficits. In hidden-target learning and probe trials, hAPP mice and MCI-AD patients showed similar deficits in learning and remembering the target location. In addition, we have provided recommendations for selecting performance measures and sample sizes to make these assays sensitive to learning and memory deficits in humans with MCI-AD and in mouse models. Together, our results demonstrate that with careful study design and analysis, the Morris maze is a sensitive assay for detecting AD-relevant impairments across species.

Authors

Katherine L. Possin, Pascal E. Sanchez, Clifford Anderson-Bergman, Roland Fernandez, Geoffrey A. Kerchner, Erica T. Johnson, Allyson Davis, Iris Lo, Nicholas T. Bott, Thomas Kiely, Michelle C. Fenesy, Bruce L. Miller, Joel H. Kramer, Steven Finkbeiner

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