Clinical Research and Public Health
Abstract
BACKGROUND Transrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.Methods Using whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.Results TR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.Conclusion Our data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.Funding NIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.
Authors
Chandan Bhambhani, Qing Kang, Daniel H. Hovelson, Erin Sandford, Mary Olesnavich, Sarah M. Dermody, Jenny Wolfgang, Kirsten L. Tuck, Collin Brummel, Apurva D. Bhangale, Kuang He, Marc G. Gutierrez, Ryan H. Lindstrom, Chia-Jen Liu, Melissa Tuck, Malathi Kandarpa, Michelle Mierzwa, Keith Casper, Mark E. Prince, John C. Krauss, Moshe Talpaz, N. Lynn Henry, Maria D. Giraldez, Nithya Ramnath, Scott A. Tomlins, Paul L. Swiecicki, J. Chad Brenner, Muneesh Tewari
Abstract
Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modelling and network correlation analyses identified 118 differentially expressed proteins, significant through three complementary analytical pipelines. Candidate biomarkers were subsequently analysed in two validation cohorts of differing ethnicity using antibody-based proximity extension assays. TB-specific host biomarkers were confirmed. A six-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14 and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts. This biomarker panel exceeds the World Health Organisation Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.
Authors
Hannah F. Schiff, Naomi F. Walker, Cesar Ugarte-Gil, Marc Tebruegge, Antigoni Manousopoulou, Spiros D. Garbis, Salah Mansour, Pak Ho Wong, Gabrielle Rockett, Paolo Piazza, Mahesan Niranjan, Andres F. Vallejo, Christopher H. Woelk, Robert J. Wilkinson, Liku B. Tezera, Diana Garay-Baquero, Paul Elkington
Abstract
BACKGROUND. Survivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans. METHODS. We measured the levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from a cohort of 341 patients with respiratory failure and 13 healthy control patients, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We flow-cytometry sorted neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing. RESULTS. Microglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines. CONCLUSIONS. Prolonged lung inflammation results in sustained elevations in circulating cytokines patients with SARS-CoV-2 pneumonia compared to those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction. FUNDING. SCRIPT U19AI135964.
Authors
Rogan A. Grant, Taylor A. Poor, Lango Sichizya, Estefani Diaz, Joseph I. Bailey, Sahil Soni, Karolina J. Senkow, Xóchitl G. Pérez-Leonor, Hiam Abdala-Valencia, Ziyan Lu, Helen K. Donnelly, Lacy M. Simons, Egon A. Ozer, Robert M. Tighe, Jon W. Lomasney, Richard G. Wunderink, Benjamin D. Singer, Alexander V. Misharin, G.R. Scott Budinger
Abstract
BACKGROUND COVID-19 convalescent plasma (CCP) viral specific antibody levels that translate into recipient post-transfusion antibody levels sufficient to prevent disease progression is not defined. METHODS This secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low post-transfusion antibody levels was established by two methods: 1) analyzing virus neutralization-equivalent anti-Spike-receptor-binding-domain immunoglobulin G (anti-S-RBD IgG) responses in donors or 2) receiver operated curve (ROC) analysis. RESULTS SARS-CoV-2 anti-S-RBD IgG antibody was volume diluted 21.3 fold into post-transfusion seronegative recipients from matched donor units. Viral specific antibody delivered approximated 1.2 mg. The high antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP recipient analysis for antibody thresholds correlated to reduced hospitalizations found a statistical significant association between early transfusion and high antibodies versus all other CCP recipients (or control plasma) with antibody cutoffs established by both methods-donor-based virus neutralization cutoff in post-transfusion recipients: (0/85; 0% versus 15/276; 5.6%) p=0.03 or ROC based cutoff: (0/94; 0% versus 15/267; 5.4%) p=0.01. CONCLUSION In unvaccinated, seronegative CCP recipients, early transfusion of plasma units in the upper 30% of study donors antibody levels reduced outpatient hospitalizations. High antibody level plasma units, given early, should be reserved for therapeutic use. Trial registration: NCT04373460 FUNDING Defense Health Agency and others.
Authors
Han-Sol Park, Anna Yin, Caelan Barranta, John S. Lee, Christopher A. Caputo, Jaiprasath Sachithanandham, Maggie Li, Steve Yoon, Ioannis Sitaras, Anne Jedlicka, Yolanda Eby, Malathi Ram, Reinaldo E. Fernandez, Owen R. Baker, Aarthi G. Shenoy, Giselle S. Mosnaim, Yuriko Fukuta, Bela Patel, Sonya L. Heath, Adam C. Levine, Barry R. Meisenberg, Emily S. Spivak, Shweta Anjan, Moises A. Huaman, Janis E. Blair, Judith S. Currier, James H. Paxton, Jonathan M. Gerber, Joann R. Petrini, Patrick B. Broderick, William Rausch, Marie Elena Cordisco, Jean Hammel, Benjamin Greenblatt, Valerie C. Cluzet, Daniel Cruser, Kevin Oei, Matthew Abinante, Laura L. Hammitt, Catherine G. Sutcliffe, Donald N. Forthal, Martin S. Zand, Edward R. Cachay, Jay S. Raval, Seble G. Kassaye, Christi E. Marshall, Anusha Yarava, Karen Lane, Nichol A. McBee, Amy L. Gawad, Nicky Karlen, Atika Singh, Daniel E. Ford, Douglas A. Jabs, Lawrence J. Appel, David M. Shade, Bryan Lau, Stephan Ehrhardt, Sheriza N. Baksh, Janna R. Shapiro, Jiangda Ou, Yu Bin Na, Maria D. Knoll, Elysse Ornelas-Gatdula, Netzahualcóyotl Arroyo-Currás, Thomas J. Gniadek, Patrizio Caturegli, Jinke Wu, Nelson Ndahiro, Michael J. Betenbaugh, Alyssa Ziman, Daniel F. Hanley, Arturo Casadevall, Shmuel Shoham, Evan M. Bloch, Kelly A. Gebo, Aaron A.R. Tobian, Oliver Laeyendecker, Andrew Pekosz, Sabra L. Klein, David J. Sullivan
Abstract
BACKGROUND While the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODS Eight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTS Maximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSION These findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDING NIH R01 AR071263.
Authors
Terence E. Ryan, Maria J. Torres, Chien-Te Lin, Angela H. Clark, Patricia M. Brophy, Cheryl A. Smith, Cody D. Smith, E. Matthew Morris, John P. Thyfault, P. Darrell Neufer
Abstract
BACKGROUND Identifying factors that predict the timing of HIV rebound after treatment interruption will be crucial for designing and evaluating interventions for HIV remission.METHODS We performed a broad evaluation of viral and immune factors that predict viral rebound (AIDS Clinical Trials Group A5345). Participants initiated antiretroviral therapy (ART) during chronic (N = 33) or early (N = 12) HIV infection with ≥ 2 years of suppressive ART and restarted ART if they had 2 viral loads ≥ 1,000 copies/mL after treatment interruption.RESULTS Compared with chronic-treated participants, early-treated individuals had smaller and fewer transcriptionally active HIV reservoirs. A higher percentage of HIV Gag-specific CD8+ T cell cytotoxic response was associated with lower intact proviral DNA. Predictors of HIV rebound timing differed between early- versus chronic-treated participants, as the strongest reservoir predictor of time to HIV rebound was level of residual viremia in early-treated participants and intact DNA level in chronic-treated individuals. We also identified distinct sets of pre–treatment interruption viral, immune, and inflammatory markers that differentiated participants who had rapid versus slow rebound.CONCLUSION The results provide an in-depth overview of the complex interplay of viral, immunologic, and inflammatory predictors of viral rebound and demonstrate that the timing of ART initiation modifies the features of rapid and slow viral rebound.TRIAL REGISTRATION ClinicalTrials.gov NCT03001128FUNDING NIH National Institute of Allergy and Infectious Diseases, Merck
Authors
Jonathan Z. Li, Meghan Melberg, Autumn Kittilson, Mohamed Abdel-Mohsen, Yijia Li, Evgenia Aga, Ronald J. Bosch, Elizabeth R. Wonderlich, Jennifer Kinslow, Leila B. Giron, Clara Di Germanio, Mark Pilkinton, Lynsay MacLaren, Michael Keefer, Lawrence Fox, Liz Barr, Edward Acosta, Jintanat Ananworanich, Robert Coombs, John Mellors, Steven Deeks, Rajesh T. Gandhi, Michael Busch, Alan Landay, Bernard Macatangay, Davey M. Smith, for the AIDS Clinical Trials Group A5345 Study Team
Abstract
BACKGROUND. T cell responses are impaired in Staphylococcus aureus-infected children, highlighting a potential mechanism of immune evasion. This study tested the hypotheses that toxin-specific antibodies protect immune cells from bacterial killing and are associated with improved T cell function following infection. METHODS.S. aureus-infected and healthy children (n = 33 each) were prospectively enrolled. During acute infection and convalescence, we quantified toxin-specific IgG levels by ELISA, antibody function using a cell-killing assay, and functional T cell responses by ELISpot. RESULTS. There were no differences in toxin-specific IgG levels or ability to neutralize toxin-mediated immune cell killing between healthy and acutely-infected children, but antibody levels and function increased following infection. Similarly, T cell function, which was impaired during acute infection, improved following infection. However, the response to infection was highly variable; up to half of children did not have improved antibody or T cell function. Serum from children with higher ɑ-hemolysin (Hla)-specific IgG levels more strongly protected immune cells against toxin-mediated killing. Importantly, children whose serum more strongly protected against toxin-mediated killing also had stronger immune responses to infection, characterized by more elicited antibody and greater improvement in T cell function following infection. CONCLUSIONS. This study demonstrates that, despite T cell impairment during acute infection, S. aureus elicits toxin-neutralizing antibodies. Individual antibody responses and T cell recovery are variable. These findings also suggest that toxin-neutralizing antibodies protect antigen-presenting cells and T cells, thereby promoting immune recovery. Finally, failure to elicit toxin-neutralizing antibodies may identify children at risk for prolonged T cell suppression. FUNDING. NIAID R01AI125489 and Nationwide Children’s Hospital.
Authors
Maureen Kleinhenz, Zhaotao Li, Usha V. Chidella, Walissa Picard, Amber Wolfe, Jill Popelka, Robin Alexander, Christopher P. Montgomery
Abstract
BACKGROUND. Obesity is a multi-factorial disease with adverse health implications including insulin resistance (IR). In patients with obesity, the presence of high circulating levels of leptin, deemed hyperleptinemia, is associated with IR. Recent data in mice with diet-induced-obesity (DIO) shows a partial reduction in leptin levels improves IR. Additional animal studies demonstrate IL-4 decreases leptin levels. In rodents, resident adipose tissue (AT) eosinophils (EOS) are the main source of IL-4 and are instrumental in maintaining metabolic homeostasis. A marked reduction in AT-EOS content is observed in animal models of DIO. These observations have not been explored in humans. METHODS. We analyzed AT from individuals with obesity and age-matched lean counterparts for AT-EOS content, IL-4, circulating leptin levels and measures of IR. RESULTS. Our results showed that individuals with obesity (n=15) had a significant reduction in AT-EOS content (P<0.01), decreased AT-IL-4 gene expression (P=0.02), and decreased IL-4 plasma levels (P<0.05) in addition to expected IR (P<0.001) and hyperleptinemia (P<0.01) compared to lean subjects (n=15). AT-EOS content inversely correlated with BMI (P=0.002) and IR (P=0.005). Ex vivo AT explants and in vitro cell culture of primary, human mature adipocytes exposed to either IL-4 or EOS conditioned media produced less leptin (P<0.05). CONCLUSIONS. Our results suggested for IL-4 to act as a link between EOS, AT, and leptin production. Future studies exploring this interaction may identify a new avenue for the treatment of obesity and its complications through amelioration of hyperleptinemia. TRIAL REGISTRATION. Clinicaltrials.gov NCT02378077 & NCT04234295. FUNDING. Dr. Eleanna De Filippis received support by Arizona Department of Health Services, Arizona Biomedical Research Commission (ABRC) (ADHS14-00003606), the Katryn H. and Roger Penske Career Development Award in Endocrinology in Honor of Dr. Ian Hay, and Mayo Foundation, KL2 TR002379-02-01 CTSA UL1 TR002377 NCATS/NIH. Dr. Elizabeth A. Jacobsen received support from NIAID AI132840 and Mayo Foundation
Authors
James D. Hernandez, Ting Li, Hamza Ghannam, Cassandra M. Rau, Mia Y. Masuda, James A. Madura, Elizabeth A. Jacobsen, Eleanna De Filippis
Abstract
BACKGROUND. Information about the size, airway location, and longitudinal behavior of mucus plugs in asthma is needed to understand their role in mechanisms of airflow obstruction and to rationally design muco-active treatments. METHODS. Computed tomography (CT) lung scans from 57 asthma patients were analyzed to quantify mucus plug size and airway location, and paired CT scans obtained 3 years apart were analyzed to determine plug behavior over time. Radiologist annotations of mucus plugs were incorporated in an image-processing pipeline to generate size and location information that was related to measures of airflow. RESULTS. The length distribution of 778 annotated mucus plugs was multimodal and a 12 mm length defined short (“stubby”, ≤12 mm) and long (“stringy”, >12 mm) plug phenotypes. High mucus plug burden was disproportionately attributable to stringy mucus plugs. Mucus plugs localized predominantly to airway generations 6 to 9, and 47% of plugs in baseline scans, persisted in the same airway for three years, and fluctuated in length and volume. Mucus plugs in larger proximal generations had greater effects on spirometry measures than plugs in smaller distal generations, and a model of airflow that estimates the increased airway resistance attributable to plugs predicted higher impact for proximal and more numerous mucus plugs. CONCLUSIONS. Persistent mucus plugs in proximal airway generations occur in asthma and demonstrate a stochastic process of formation and resolution over time. Proximal airway mucus plugs are consequential for airflow and are in locations amenable to treatment by inhaled muco-active drugs or bronchoscopy. TRIAL REGISTRATION. Clinicaltrials.gov NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01759186, NCT01716494, and NCT01760915 FUNDING. NIH Grants: R01 HL080414, UG1 HL139106, P01 HL107202, U01 HL146002, U10 HL109172, U10 HL109168, U10 HL109152, U10 HL109257, U10 HL109146, U10 HL109250, U10 HL109164, U10 109086, and T32 HL007185, F32 HL162422. The following companies provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up: AstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi–Genzyme– Regeneron, and TEVA. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.
Authors
Brendan K. Huang, Brett M. Elicker, Travis S. Henry, Kimberly G. Kallianos, Lewis D. Hahn, Monica Tang, Franklin Heng, Charles E. McCulloch, Nirav R. Bhakta, Sharmila Majumdar, Jiwoong Choi, Loren C. Denlinger, Sean B. Fain, Annette T. Hastie, Eric A. Hoffman, Elliot Israel, Nizar N. Jarjour, Bruce D. Levy, David T. Mauger, Kaharu Sumino, Sally E. Wenzel, Mario Castro, Prescott G. Woodruff, John V. Fahy
Abstract
BACKGROUND. Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. METHODS. An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. RESULTS. Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. CONCLUSIONS. A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients.
Authors
Evan L. Barrios, Monty B. Mazer, Patrick W. McGonagill, Christian B. Bergmann, Michael D. Goodman, Robert W. Gould, Mahil Rao, Valerie E. Polcz, Ruth Davis, Drew Del Toro, Marvin Dirain, Alexandra Dram, Lucas Hale, Mohammad Heidarian, Caleb Y. Kim, Tamara A. Kucaba, Jennifer P. Lanz, Ashley McCray, Alexandra Meszaros, Sydney M. Miles, Candace Nelson, Ivanna Rocha, Elvia E. Silva, Ricardo Ungaro, Andrew Walton, Julie Xu, Leilani Zeumer-Spataro, Anne M. Drewry, Muxuan Liang, Letita E. Bible, Tyler J. Loftus, Isaiah R. Turnbull, Philip A. Efron, Kenneth E. Remy, Scott C. Brakenridge, Vladimir P. Badovinac, Thomas S. Griffith, Lyle L. Moldawer, Richard S. Hotchkiss, Charles C. Caldwell
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