Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity.

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.

Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses.

The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.

PTK2-associated gene signature could predict the prognosis of IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with a poor prognosis. Current/available clinical prediction tools have limited sensitivity and accuracy when evaluating clinical outcomes of IPF. Research has shown that focal adhesion kinase (FAK), produced by the protein tyrosine kinase 2 (PTK2) gene, is crucial in IPF development. FAK activation is a characteristic of lesional fibroblasts; Thus, FAK may be a valuable therapeutic target or prognostic biomarker for IPF. This study aimed to create a gene signature based on PTK2-associated genes and microarray data from blood cells to predict disease prognosis in patients with IPF. PTK2 levels were found to be higher in lung tissues of IPF patients compared to healthy controls, and PTK2 inhibitor Defactinib was found to reduce TGFß-induced FAK activation and increase α-smooth muscle actin. Although the blood PTK2 levels were higher in IPF patients, blood PTK level alone could not predict IPF prognosis. From 196 PTK2-associated genes, 11 genes were prioritized to create a gene signature (PTK2 molecular signature) and a risk score system using univariate and multivariate Cox regression analysis. Patients were divided into high-risk and low-risk groups using PTK2 molecular signature. Patients in the high-risk group experienced decreased survival rates compared to patients in the low-risk group across all discovery and validation cohorts. Further functional enrichment and immune cell proportion analyses revealed that the PTK2 molecular signature strongly reflected the activation levels of immune pathways and immune cells. These findings suggested that PTK2 is a molecular target of IPF and the PTK2 molecular signature is an effective IPF prognostic biomarker.

Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging.

Idiopathic pulmonary fibrosis (IPF) is a fatal age-associated disease with no cure. Although IPF is widely regarded as a disease of aging, the cellular mechanisms that contribute to this age-associated predilection remain elusive. In this study, we sought to evaluate the consequences of senescence on myofibroblast cell fate and fibrotic responses to lung injury in the context of aging. We demonstrated that nonsenescent lung myofibroblasts maintained the capacity for dedifferentiation, whereas senescent/IPF myofibroblasts exhibited an impaired capacity for dedifferentiation. We previously demonstrated that the transcription factor MyoD acts as a critical switch in the differentiation and dedifferentiation of myofibroblasts. Here, we demonstrate that decreased levels of MyoD preceded myofibroblast dedifferentiation and apoptosis susceptibility in nonsenescent cells, whereas MyoD expression remained elevated in senescent/IPF myofibroblasts, which failed to undergo dedifferentiation and demonstrated resistance to apoptosis. Genetic strategies to silence MyoD restored the susceptibility of IPF myofibroblasts to undergo apoptosis and led to a partial reversal of age-associated persistent fibrosis in vivo. The capacity for myofibroblast dedifferentiation and subsequent apoptosis may be critical for normal physiologic responses to tissue injury, whereas restricted dedifferentiation and apoptosis resistance in senescent cells may underlie the progressive nature of age-associated human fibrotic disorders. These studies support the concept that senescence may promote profibrotic effects via impaired myofibroblast dedifferentiation and apoptosis resistance, which contributes to myofibroblast accumulation and ultimately persistent fibrosis in aging.

Exact variance component tests for longitudinal microbiome studies.

In metagenomic studies, testing the association between microbiome composition and clinical outcomes translates to testing the nullity of variance components. Motivated by a lung human immunodeficiency virus (HIV) microbiome project, we study longitudinal microbiome data by using variance component models with more than two variance components. Current testing strategies only apply to models with exactly two variance components and when sample sizes are large. Therefore, they are not applicable to longitudinal microbiome studies. In this paper, we propose exact tests (score test, likelihood ratio test, and restricted likelihood ratio test) to (a) test the association of the overall microbiome composition in a longitudinal design and (b) detect the association of one specific microbiome cluster while adjusting for the effects from related clusters. Our approach combines the exact tests for null hypothesis with a single variance component with a strategy of reducing multiple variance components to a single one. Simulation studies demonstrate that our method has a correct type I error rate and superior power compared to existing methods at small sample sizes and weak signals. Finally, we apply our method to a longitudinal pulmonary microbiome study of HIV-infected patients and reveal two interesting genera Prevotella and Veillonella associated with forced vital capacity. Our findings shed light on the impact of the lung microbiome on HIV complexities. The method is implemented in the open-source, high-performance computing language Julia and is freely available at https://github.com/JingZhai63/VCmicrobiome.

Peripheral blood non-canonical small non-coding RNAs as novel biomarkers in lung cancer.

One unmet challenge in lung cancer diagnosis is to accurately differentiate lung cancer from other lung diseases with similar clinical symptoms and radiological features, such as pulmonary tuberculosis (TB). To identify reliable biomarkers for lung cancer screening, we leverage the recently discovered non-canonical small non-coding RNAs (i.e., tRNA-derived small RNAs [tsRNAs], rRNA-derived small RNAs [rsRNAs], and YRNA-derived small RNAs [ysRNAs]) in human peripheral blood mononuclear cells and develop a molecular signature composed of distinct ts/rs/ysRNAs (TRY-RNA). Our TRY-RNA signature precisely discriminates between control, lung cancer, and pulmonary TB subjects in both the discovery and validation cohorts and outperforms microRNA-based biomarkers, which bears the diagnostic potential for lung cancer screening.

Rat BodyMap transcriptomes reveal unique circular RNA features across tissue types and developmental stages.

Circular RNAs (circRNAs) are a novel class of regulatory RNAs. Here, we present a comprehensive investigation of circRNA expression profiles across 11 tissues and four developmental stages in rats, along with cross-species analyses in humans and mice. Although the expression of circRNAs is positively correlated with that of cognate mRNAs, highly expressed genes tend to splice a larger fraction of circular transcripts. Moreover, circRNAs exhibit higher tissue specificity than cognate mRNAs. Intriguingly, while we observed a monotonic increase of circRNA abundance with age in the rat brain, we further discovered a dynamic, age-dependent pattern of circRNA expression in the testes that is characterized by a dramatic increase with advancing stages of sexual maturity and a decrease with aging. The age-sensitive testicular circRNAs are highly associated with spermatogenesis, independent of cognate mRNA expression. The tissue/age implications of circRNAs suggest that they present unique physiological functions rather than simply occurring as occasional by-products of gene transcription.

Differential transcriptomics in sarcoidosis lung and lymph node granulomas with comparisons to pathogen-specific granulomas.

RATIONALE: Despite the availability of multi-"omics" strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures. OBJECTIVE: To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis). METHODS: Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways. RESULTS: Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level. CONCLUSION: These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.

Comparison of three anti-coccidioides antibody enzyme immunoassay kits for the diagnosis of coccidioidomycosis.

Coccidioidomycosis is a common cause of community-acquired pneumonia in endemic areas of the southwestern United States. Clinical presentations range from self-limited disease to severe, disseminated disease. As such, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic testing has variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from patients with coccidioidomycosis and controls were tested for immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies using the MVista Coccidioides antibody detection EIA and two commonly used commercial enzyme immunoassay (EIA) kits: the IMMY Omega EIA and the Meridian Premier EIA. The sensitivity of the IgG antibody detection was 87.4% using the MVista test compared to 46.6% for IMMY and 70.9% for Meridian. The sensitivity for IgM antibody detection was 61.2% for the MVista test, 22.3% for IMMY and 29.1% for Meridian. For IgG antibody detection, specificity was 90% for the MVista EIA, 94.6% for IMMY, 96.4% for Meridian. For IgM antibody detection, specificity was 95.3% for the MVista test 98.2% for IMMY and 99.1% for Meridian. The MVista Coccidioides antibody EIA offers improved sensitivity, including among high-risk patient populations, for the detection of IgG and IgM antibodies in comparison to other currently available EIAs.

Microbiological Laboratory Testing in the Diagnosis of Fungal Infections in Pulmonary and Critical Care Practice. An Official American Thoracic Society Clinical Practice Guideline.

Background: Fungal infections are of increasing incidence and importance in immunocompromised and immunocompetent patients. Timely diagnosis relies on appropriate use of laboratory testing in susceptible patients.Methods: The relevant literature related to diagnosis of invasive pulmonary aspergillosis, invasive candidiasis, and the common endemic mycoses was systematically reviewed. Meta-analysis was performed when appropriate. Recommendations were developed using the Grading of Recommendations Assessment, Development, and Evaluation approach.Results: This guideline includes specific recommendations on the use of galactomannan testing in serum and BAL and for the diagnosis of invasive pulmonary aspergillosis, the role of PCR in the diagnosis of invasive pulmonary aspergillosis, the role of ß-d-glucan assays in the diagnosis of invasive candidiasis, and the application of serology and antigen testing in the diagnosis of the endemic mycoses.Conclusions: Rapid, accurate diagnosis of fungal infections relies on appropriate application of laboratory testing, including antigen testing, serological testing, and PCR-based assays.

Pseudomonas aeruginosa increases MUC1 expression in macrophages through the TLR4-p38 pathway.

Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.

Enhanced Antibody Detection and Diagnosis of Coccidioidomycosis with the MiraVista IgG and IgM Detection Enzyme Immunoassay.

Coccidioidomycosis is a common cause of community-acquired pneumonia in areas of the southwestern United States in which the disease is endemic. Clinical presentations range from self-limited disease to severe disseminated disease. Therefore, early and accurate diagnosis is essential to ensure appropriate treatment and monitoring. Currently available diagnostic tests have variable accuracy, particularly in certain patient populations, and new tests may offer improved accuracy for the diagnosis of coccidioidomycosis. Serum samples from 103 cases of coccidioidomycosis and 373 controls were tested for IgG and IgM antibodies using the MVista anti-Coccidioides antibody enzyme immunoassay. Serum specimens from 170 controls from areas in which the disease is endemic and 44 cases were tested by immunodiffusion at MiraVista Diagnostics. The sensitivity of the MVista antibody assay was 88.3%, and the specificity was 90%. The sensitivity was maintained in the presence of immunocompromising conditions or immunosuppressive therapies. The sensitivity of immunodiffusion was 60.2%, and the specificity was 98.8%. The sensitivity of complement fixation (62 cases) was 66.1%, but the specificity could not be determined. The MVista anti-Coccidioides antibody enzyme immunoassay offers improved sensitivity, compared with immunodiffusion and complement fixation, is not impaired in immunocompromised patients, and permits highly reproducible semiquantification.

Coccidioidomycosis among persons undergoing lung transplantation in the coccidioidal endemic region.

BACKGROUND: Coccidioidomycosis, an endemic fungal infection, is more likely to be symptomatic and severe among those receiving allogeneic transplants. While several case series have been published for various transplanted organs, none has described the incidence and outcomes in those receiving lung transplants within the coccidioidal endemic region. METHODS: Patients receiving a heart-lung, single-lung, or bilateral-lung transplantation at the University of Arizona between 1985 and 2009 were retrospectively reviewed. RESULTS: Coccidioidomycosis occurred post transplantation in 11 (5.8%) of 189 patients. All but one patient was diagnosed with pulmonary coccidioidomycosis and only one had a history of prior coccidioidomycosis. Two patients received transplants from donors found to have coccidioidomycosis at the time of transplantation and one death was directly attributed to coccidioidomycosis. The risk of developing active coccidioidomycosis was significantly higher if the patient did not receive some type of antifungal therapy post transplantation (P<.001). CONCLUSION: Within the coccidioidal endemic region, post-transplantation coccidioidomycosis was a definable risk among lung transplant recipients. Use of antifungals appeared to reduce this incidence of disease. Almost all cases resulted in pulmonary disease, suggesting that the lung is the primary site of infection.

Effect of Advanced HIV Infection on the Respiratory Microbiome.

RATIONALE: Previous work found the lung microbiome in healthy subjects infected with HIV was similar to that in uninfected subjects. We hypothesized the lung microbiome from subjects infected with HIV with more advanced disease would differ from that of an uninfected control population. OBJECTIVES: To measure the lung microbiome in an HIV-infected population with advanced disease. METHODS: 16s RNA gene sequencing was performed on acellular bronchoalveolar lavage (BAL) fluid from 30 subjects infected with HIV with advanced disease (baseline mean CD4 count, 262 cells/mm(3)) before and up to 3 years after starting highly active antiretroviral therapy (HAART) and compared with 22 uninfected control subjects. MEASUREMENTS AND MAIN RESULTS: The lung microbiome in subjects infected with HIV with advanced disease demonstrated decreased alpha diversity (richness and diversity) and greater beta diversity compared with uninfected BAL. Differences improved with HAART, but still persisted up to 3 years after starting therapy. Population dispersion in the group infected with HIV was significantly greater than in the uninfected cohort and declined after treatment. There were differences in the relative abundance of some bacteria between the two groups at baseline and after 1 year of therapy. After 1 year on HAART, HIV BAL contained an increased abundance of Prevotella and Veillonella, bacteria previously associated with lung inflammation. CONCLUSIONS: The lung microbiome in subjects infected with HIV with advanced disease is altered compared with an uninfected population both in diversity and bacterial composition. Differences remain up to 3 years after starting HAART. We speculate an altered lung microbiome in HIV infection may contribute to chronic inflammation and lung complications seen in the HAART era.

Membrane-Tethered MUC1 Mucin Counter-Regulates the Phagocytic Activity of Macrophages.

MUC1 (MUC in human; Muc in animals) is a transmembrane mucin glycoprotein expressed in mucosal epithelial cells and hematopoietic cells. MUC1 is involved in the resolution of inflammation during airway Pseudomonas aeruginosa (Pa) infection by suppressing Toll-like receptor signaling in airway epithelial cells. Although alveolar macrophages are recognized as critical mediators of cell-mediated immunity against microorganisms inhaled into the airways, the role of MUC1 in regulating their response is unknown. The aims of this study were to determine whether macrophages express MUC1, and, if so, whether MUC1 expression might be associated with macrophage M0/M1/M2 differentiation or phagocytic activity. Human and mouse MUC1/Muc1 expression was drastically up-regulated in classically activated (M1) macrophages compared with nonactivated (M0) and alternatively activated (M2) macrophages. M1 polarization and Pa stimulation each increased MUC1 ectodomain shedding from the macrophage surface in a TNF-α-converting enzyme-dependent manner. MUC1/Muc1 deficiency in M0 macrophages increased adhesion and phagocytosis of Pa and Escherichia coli compared with MUC1/Muc1-expressing cells, and attenuation of phagocytosis by MUC1 was augmented after polarization into M1 macrophages compared with M0 macrophages. Finally, MUC1/Muc1 deficiency in macrophages increased reactive oxygen species production and TNF-α release in response to Pa compared with MUC1/Muc1-sufficient cells. These results indicate that MUC1/Muc1 expression by macrophages is predominantly in the M1 subtype, and that MUC1/Muc1 expression in these cells decreases their phagocytic activity in an antiinflammatory manner.

Positive (1-3) B-d-glucan and cross reactivity of fungal assays in coccidioidomycosis.

Fungal antigen testing in immunosuppressed patients has emerged as a powerful diagnostic tool. Some assays are relatively nonspecific, and misinterpretation can have severe clinical consequences. Additionally, when new assays become commercially available it is important to evaluate the potential for cross reactivity. We recently observed several immunosuppressed patients with positive (1→3)-ß-D-glucan (BG) who were eventually diagnosed with coccidioidomycosis in the endemic area of Tucson, Arizona. Although the BG assay is known to detect glucans of many fungal pathogens, reports of cross-reactivity with Coccidioides remain sparsely reported. To test the cross-reactivity of fungal antigens in detection assays, serum samples from patients with coccidioidomycosis testing positive for Coccidioides antigen were evaluated for BG. Of 12 samples positive for Coccidioides antigen (≥0.07 ng/ml), 11 (92%) were positive by BG (>80 pg/ml), and of 11 positive for Aspergillus galactomannan, 10 (91%) were positive by BG (>80 pg/ml). We conclude that the BG assay is nonspecific, detecting glucans from many fungal pathogens, including Coccidioides. In the endemic area, a positive BG warrants further specific testing.

Positron emission tomography in the evaluation of pulmonary nodules among patients living in a coccidioidal endemic region.

BACKGROUND: Within a coccidioidal endemic region, pulmonary nodules due to coccidioidomycosis are common. Uptake of (18)fluorodeoxyglucose ((18)FDG) by positron emission tomography with computed axial tomography (PET/CT) has been used to assess whether pulmonary nodules are malignant but inflammatory lesions can be positive. The purpose of this study was to compare by PET/CT the (18)FDG uptake in pulmonary nodules likely due to coccidioidomycosis to that of nodules shown to be malignant among patients living in a coccidioidal endemic region. METHODS: We retrospectively reviewed patients who underwent a PET/CT at the Southern Arizona Veterans Affairs Health Care System between January 2008 and March 2012 who were subsequently found on biopsy to have pulmonary nodules that were coccidioidal or granulomatous or were due to malignancy. RESULTS: Among 245 diagnostic biopsies where the subject had a previous PET/CT, 15 (6.1 %) were either coccidioidal (n = 12) or granulomatous without an identified organism (n = 3). The median maximum standard unit of uptake (SUV(max)) on PET/CT of coccidioidal or granulomatous lesions was 2.0 compared to 9.8 for malignant lesions (P < 0.001). The maximum diameter of the coccidioidal or granulomatous nodules was 2.1 cm compared to 3.0 cm for the malignant lesions (P = 0.009). On multivariable analysis, an elevated SUV(max) was the only distinguishing feature between the malignant and the granulomatous lesions (OR 1.28, 95 % CI 1.05-1.55; P = 0.013). CONCLUSIONS: Coccidioidal pulmonary nodules take up significantly less (18)FDG than those due to malignancies, but there is considerable overlap between granulomatous and malignant lesions at lower SUV(max).