Profiling helper T cell subset gene expression in deer mice

BACKGROUND: Deer mice (Peromyscus maniculatus) are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV), the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. RESULTS: We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4(+ )helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT), Th2 cells (GATA-3, STAT6, IL-4, IL-5) and regulatory T cells (Fox-p3, IL-10, TGFβ1). These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. CONCLUSION: We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice

Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus)

BACKGROUND: Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. RESULTS: To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse (Mus musculus) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. CONCLUSIONS: The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases

Software for the frontiers of quantum chemistry:An overview of developments in the Q-Chem 5 package

This article summarizes technical advances contained in the fifth major release of the Q-Chem quantum chemistry program package, covering developments since 2015. A comprehensive library of exchange–correlation functionals, along with a suite of correlated many-body methods, continues to be a hallmark of the Q-Chem software. The many-body methods include novel variants of both coupled-cluster and configuration-interaction approaches along with methods based on the algebraic diagrammatic construction and variational reduced density-matrix methods. Methods highlighted in Q-Chem 5 include a suite of tools for modeling core-level spectroscopy, methods for describing metastable resonances, methods for computing vibronic spectra, the nuclear–electronic orbital method, and several different energy decomposition analysis techniques. High-performance capabilities including multithreaded parallelism and support for calculations on graphics processing units are described. Q-Chem boasts a community of well over 100 active academic developers, and the continuing evolution of the software is supported by an “open teamware” model and an increasingly modular design

Lung Cancer Detection in Patients With Airflow Obstruction Identified in a Primary Care Outpatient Practice*

Introduction: This prospective study describes a community-based lung cancer identification project focusing on high-risk patients who receive general care in a primary care outpatient practice. Within 1 calendar year, a simple questionnaire was completed in 1,296 patients > 50 years old to identify 430 patients at high risk of lung cancer (smoking, family history of aerodigestive tract cancer, or occupational exposures). Spirometric abnormalities were found in 126 of these patients. Methods: Chest posteroanterior radiographs, thoracic CT scans, and sputum cytology were offered to subjects with airflow obstruction (n = 126). Eighty-eight patients underwent all tests. Thirty-eight patients refused or could not consent in a timely fashion. Results: Six cancers were found in the screened group, and all were treated. Two more cancers were found in the nonscreened patients with airflow obstruction. Both were treated by surgical resection or radiation therapy. Costs per cancer found were $11,925 per patient. Conclusions: Case finding in high-risk patients in a primary care population can be accomplished at a relatively low cost

Software for the frontiers of quantum chemistry: An overview of developments in the Q-Chem 5 package

This article summarizes technical advances contained in the fifth major release of the Q-Chem quantum chemistry program package, covering developments since 2015. A comprehensive library of exchange-correlation functionals, along with a suite of correlated many-body methods, continues to be a hallmark of the Q-Chem software. The many-body methods include novel variants of both coupled-cluster and configuration-interaction approaches along with methods based on the algebraic diagrammatic construction and variational reduced density-matrix methods. Methods highlighted in Q-Chem 5 include a suite of tools for modeling core-level spectroscopy, methods for describing metastable resonances, methods for computing vibronic spectra, the nuclear-electronic orbital method, and several different energy decomposition analysis techniques. High-performance capabilities including multithreaded parallelism and support for calculations on graphics processing units are described. Q-Chem boasts a community of well over 100 active academic developers, and the continuing evolution of the software is supported by an "open teamware" model and an increasingly modular design

Genomic Classification of Cutaneous Melanoma

We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four sub-types based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multidimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-makingclose3