Clinical Research and Public Health
Abstract
Background: Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent among reproductive-age females worldwide. Adverse health outcomes associated with BV include an increased risk of sexually-acquired HIV, yet the immunological mechanisms underlying this association are not well understood. Methods: To investigate BV-driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, Kinga Study participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and PBMC samples. Results: High-parameter flow cytometry revealed an increased frequency of cervical conventional CD4+ T cells (Tconv) expressing CCR5. However, we found no difference in number of CD3+CD4+CCR5+ cells in the CX or VT of BV+ versus BV- individuals, suggesting that BV-driven increased HIV susceptibility may not be solely attributed to increased CVT HIV target cell abundance. Flow cytometry also revealed that individuals with BV have an increased frequency of dysfunctional CX and VT CD39+ Tconv and CX tissue-resident CD69+CD103+ Tconv, reported to be implicated in HIV acquisition risk and replication. Many soluble immune factor differences in the CVT further support that BV elicits diverse and complex CVT immune alterations. Conclusion: Our comprehensive analysis expands on potential immunological mechanisms that may underlie the adverse health outcomes associated with BV including increased HIV susceptibility.
Authors
Finn MacLean, Adino Tesfahun Tsegaye, Jessica B. Graham, Jessica L. Swarts, Sarah C. Vick, Nicole B. Potchen, Irene Cruz Talavera, Lakshmi Warrier, Julien Dubrulle, Lena K. Schroeder, Ayumi Saito, Corinne Mar, Katherine K. Thomas, Matthias Mack, Michelle C. Sabo, Bhavna H. Chohan, Kenneth Ngure, Nelly Rwamba Mugo, Jairam R. Lingappa, Jennifer M. Lund
Abstract
BACKGROUND. Hyperinsulinemia and insulin resistance often accompany elevated serum urate levels (hyperuricemia), a highly heritable condition that triggers gout; however, the underlying mechanisms are unclear. METHODS. We evaluated the association between the index of hyperinsulinemia and the fractional excretion of urate (FEUA) in 162 outpatients. The underlying mechanisms were investigated through single-cell data analysis and kinase screening combined with cell culture experiments. In 377,358 participants of the UK Biobank (UKBB), we analyzed serum urate, hyperinsulinemia, and salt intake. We also examined gene-environment interactions using single nucleotide variants in SLC22A12, which encodes urate transporter 1 (URAT1). RESULTS. The index of hyperinsulinemia was inversely associated with FEUA independently of other covariates. Mechanistically, URAT1 cell-surface abundance and urate transport activity were regulated by URAT1-Thr408 phosphorylation, which was stimulated by hyperinsulinemia via AKT. Kinase screening and single-cell data analysis revealed that SGK1, induced by high salt, activated the same pathway, increasing URAT1. Arg405 was essential for these kinases to phosphorylate URAT1-Thr408. In UKBB participants, hyperinsulinemia and high salt intake were independently associated with increased serum urate levels. We found that SLC22A12 eQTL rs475688 synergistically enhanced the positive association between serum urate and hyperinsulinemia. CONCLUSION. URAT1 mediates the association between hyperinsulinemia and hyperuricemia. Our data provide evidence for the role of gene-environment interactions in determining serum urate levels, paving the way for personalized management of hyperuricemia. FUNDING. ACRO Research Grants of Teikyo University; JSPS; the Japanese Society of Gout and Uric & Nucleic Acids; Fuji Yakuhin; Nanken-Kyoten; Medical Research Center Initiative for High Depth Omics.
Authors
Wataru Fujii, Osamu Yamazaki, Daigoro Hirohama, Ken Kaseda, Emiko Kuribayashi-Okuma, Motonori Tsuji, Makoto Hosoyamada, Yuta Kochi, Shigeru Shibata
Abstract
BACKGROUND This study examined the underlying cellular mechanisms associated with insulin resistance (IR) and metabolic disease risk within subcutaneous adipose tissue (SAT) in youth with obesity and IR compared with those without IR.METHODS Thirteen adolescents who were insulin sensitive (IS) and 17 adolescents with IR and obesity underwent a 3-hour oral glucose tolerance test and MRI to measure abdominal fat distribution and liver fat content. Lipolysis was determined by glycerol turnover ([2H5]-glycerol infusion) and adipose triglyceride lipase (ATGL) phosphorylation (Western blot) from SAT samples biopsied prior to and 30-minutes following insulin infusion during a hyperinsulinemic-euglycemic clamp (HEC).RESULTS Glycerol turnover suppression during the HEC (first step) was lower in participants with IR compared with those with IS. Prior to insulin infusion, activated ATGL (reflected by the p-ATGL (Ser406)-to-ATGL ratio) was greater in participants with IR compared with those with IS and suppressed in response to a 30-minute insulin exposure in participants with IS, but not in those with IR. Lastly, greater ATGL inactivation is associated with greater glycerol suppression and lower liver fat.CONCLUSIONS Insulin-mediated inhibition of adipose tissue lipolysis via ATGL is dysregulated among adolescents with IR compared with those with IS, thereby serving as a vital mechanism linking glucose and insulin dysregulation and ectopic lipid storage within the liver.FUNDING This work was supported by funding from the NIH (R01-HD028016-25A1, T32- DK-007058, R01-DK124272, RO1-DK119968, R01MD015974, RO1-DK113984, P3-DK045735, RO1-DK133143, and RC2-DK120534) and the Robert E. Leet and Clara Guthrie Patterson Trust Mentored Research Award.
Authors
Aaron L. Slusher, Nicola Santoro, Alla Vash-Margita, Alfonso Galderisi, Pamela Hu, Fuyuze Tokoglu, Zhongyao Li, Elena Tarabra, Jordan Strober, Daniel F. Vatner, Gerald I. Shulman, Sonia Caprio
Abstract
BACKGROUND Mucus plugs form in acute asthma and persist in chronic disease. Although eosinophils are implicated in mechanisms of mucus pathology, many mechanistic details about mucus plug formation and persistence in asthma are unknown.METHODS Using histology and spatial, single-cell proteomics, we characterized mucus-plugged airways from nontransplantable donor lungs of 14 patients with asthma (9 with fatal asthma and 5 with nonfatal asthma) and individuals acting as controls (10 with chronic obstructive pulmonary disease and 14 free of lung disease). Additionally, we used an airway epithelial cell–eosinophil (AEC-eosinophil) coculture model to explore how AEC mucus affects eosinophil degranulation.RESULTS Asthma mucus plugs were tethered to airways showing infiltration with innate lymphoid type 2 cells and hyperplasia of smooth muscle cells and MUC5AC-expressing goblet cells. Asthma mucus plugs were infiltrated with immune cells that were mostly dual positive for eosinophil peroxidase (EPX) and neutrophil elastase, suggesting that neutrophils internalize EPX from degranulating eosinophils. Indeed, eosinophils exposed to mucus from IL-13–activated AECs underwent CD11b- and glycan-dependent cytolytic degranulation. Dual-positive granulocytes varied in frequency in mucus plugs. Whereas paucigranulocytic plugs were MUC5AC rich, granulocytic plugs had a mix of MUC5AC, MUC5B, and extracellular DNA traps. Paucigranulocytic plugs occurred more frequently in (acute) fatal asthma and granulocytic plugs predominated in (chronic) nonfatal asthma.CONCLUSION Together, our data suggest that mucin-rich mucus plugs in fatal asthma form because of acute goblet cell degranulation in remodeled airways and that granulocytic mucus plugs in chronic asthma persist because of a sustaining niche characterized by epithelial cell–mucin-granulocyte cross-talk.FUNDING NIH grants HL080414, HL107202, and AI077439.
Authors
Maude A. Liegeois, Aileen Hsieh, May Al-Fouadi, Annabelle R. Charbit, Chen Xi Yang, Tillie-Louise Hackett, John V. Fahy
Abstract
BACKGROUND Immune checkpoint blockade (ICB) is an effective treatment in a subset of patients diagnosed with head and neck squamous cell carcinoma (HNSCC); however, the majority of patients are refractory.METHODS In a nonrandomized, open-label Phase 1b clinical trial, participants with recurrent and/or metastatic (R/M) HNSCC were treated with low-dose 5-azacytidine (5-aza) daily for either 5 or 10 days in combination with durvalumab and tremelimumab after progression on ICB. The primary objective was to assess the biologically effective dose of 5-aza as determined by molecular changes in paired baseline and on-treatment tumor biopsies; the secondary objective was safety.RESULTS Thirty-eight percent (3 of 8) of participants with evaluable paired tissue samples had a greater-than 2-fold increase from baseline in IFN-γ signature and CD274 (programmed cell death protein 1 ligand, PD-L1) expression within the tumor microenvironment (TME), which was associated with increased CD8+ T cell infiltration and decreased infiltration of CD4+ T regulatory cells. The mean neutrophil-to-lymphocyte ratio (NLR) decreased by greater than 50%, from 14.2 (SD 22.6) to 6.9 (SD 5.2). Median overall survival (OS) was 16.3 months (95% CI 1.9, NA), 2-year OS rate was 24.7% (95% CI: 4.5%, 53.2%), and 58% (7 of 12) of treated participants demonstrated prolonged OS of greater than 12 months.CONCLUSION Our findings suggest that low-dose 5-aza can reprogram systemic host immune responses and the local TME to increase IFN-γ and PD-L1 expression. The increased expression of these established biomarkers correlated with prolonged OS upon ICB rechallenge.TRIAL REGISTRATION ClinicalTrials.gov NCT03019003.FUNDING NIH/NCI P01 CA240239.
Authors
Tingting Qin, Austin K. Mattox, Jean S. Campbell, Jong Chul Park, Kee-Young Shin, Shiting Li, Peter M. Sadow, William C. Faquin, Goran Micevic, Andrew J. Daniels, Robert Haddad, Christopher S. Garris, Mikael J. Pittet, Thorsten R. Mempel, Anne ONeill, Maureen A. Sartor, Sara I. Pai
Abstract
Background: Despite growing preclinical evidence that glucagon-like peptide-1 receptor agonists (GLP-1RAs) could be repurposed to treat alcohol use disorder (AUD), clinical evidence is scarce. Additionally, the potential impact of dipeptidyl peptidase-4 inhibitors (DPP-4Is) on alcohol intake is largely unknown. Methods: We conducted a large cohort study using 2008-2023 electronic health records data from the U.S. Department of Veterans Affairs. Changes in Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) scores were compared between propensity-score-matched GLP-1RA recipients, DPP-4I recipients, and unexposed comparators. We further tested the effects of two DPP-4Is, linagliptin and omarigliptin, on binge-like alcohol drinking in mice and operant oral alcohol self-administration in alcohol-dependent rats, models previously used to show a significant effect of the GLP-1RA semaglutide in reducing alcohol intake. Results: GLP-1RA recipients reported a greater reduction in AUDIT-C scores than unexposed individuals [difference-in-difference: 0.09(0.03,0.14), p=0.0025] and DPP-4I recipients [difference-in-difference: 0.11(0.05,0.17), p=0.0002]. Reductions in drinking were more pronounced among individuals with baseline AUD [GLP-1RA vs. unexposed: 0.51(0.29,0.72), p<0.0001; GLP-1RA vs. DPP-4I: 0.65(0.43,0.88), p<0.0001] and baseline hazardous drinking [GLP-1RA vs. unexposed: 1.38(1.07,1.69), p<0.0001; GLP-1RA vs. DPP-4I: 1.00(0.68,1.33), p<0.0001]. There were no differences between DPP-4I recipients and unexposed individuals. The latter results were confirmed via a reverse translational approach. Specifically, neither linagliptin nor omarigliptin reduced alcohol drinking in mice or rats. The rodent experiments also confirmed target engagement as both DPP-4Is reduced blood glucose levels. Conclusion: Convergent findings across humans, mice, and rats indicate that GLP-1RAs but not DPP-4Is reduce alcohol consumption and may be efficacious in treating AUD.
Authors
Mehdi Farokhnia, John Tazare, Claire L. Pince, Nicolaus Bruns Vi, Joshua C. Gray, Vincent Lo Re III, David A. Fiellin, Henry R. Kranzler, George F. Koob, Amy C. Justice, Leandro F. Vendruscolo, Christopher T. Rentsch, Lorenzo Leggio
Abstract
BACKGROUND. Naïve cells comprise 90% of the CD4+ T-cell population in neonates and exhibit distinct age-specific capacities for proliferation and activation. We hypothesized that HIV-infected naïve CD4+ T-cell populations in children on long-term antiretroviral therapy (ART) would thus be distinct from infected memory cells. METHODS. Peripheral blood naïve and memory CD4+ T cells from 8 children with perinatal HIV on ART initiated at age 1.7-17 months were isolated by FACS. DNA was extracted from sorted cells and HIV proviruses counted, evaluated for intactness, and subjected to integration site analysis. RESULTS. Naïve CD4+ T cells containing HIV proviruses were detected in children with 95% statistical confidence. A median of 4.7% of LTR-containing naïve CD4+ T cells also contained HIV genetic elements consistent with intactness. Full-length proviral sequencing confirmed intactness of one provirus. In the participant with the greatest level of naïve cell infection, ISA revealed infected expanded cell clones in both naïve and memory T cells with no common HIV integration sites detected between subsets. Divergent integration site profiles reflected differential gene expression patterns of naïve and memory T cells. CONCLUSIONS. These results demonstrate that HIV persists in both naïve and memory CD4+ T cells that undergo clonal expansion and harbor intact proviruses, suggesting that infected memory T-cell clones do not frequently arise from naïve cell differentiation in children with perinatal HIV on long-term ART. FUNDING. Center for Cancer Research, NCI and Office of AIDS Research funding to MFK, NCI FLEX funding to JWR. Children’s and Emory JFF pilot to MM.
Authors
Mary Grace Katusiime, Victoria Neer, Shuang Guo, Sean C. Patro, Wenjie Wang, Brian Luke, Adam A. Capoferri, Xiaolin Wu, Anna M. Horner, Jason W. Rausch, Ann Chahroudi, Maud Mavigner, Mary F. Kearney
Abstract
BACKGROUND.Pneumocystis jirovecii pneumonia (PCP) is a leading cause of fungal pneumonia, but its diagnosis primarily relies on invasive bronchoalveolar lavage (BAL) specimens that are difficult to obtain. Oropharyngeal swabs and serum could improve the PCP diagnostic workflow, and we hypothesized that CRISPR could enhance assay sensitivity to allow robust P. jirovecii diagnosis using swabs and serum. Herein we describe the development of an ultrasensitive RT-PCR-coupled CRISPR assay with high active-infection specificity in infant swabs and adult BAL and serum. METHODS. Mouse analyses employed an RT-PCR CRISPR assay to analyze P. murina transcripts in wild-type and Rag2–/– mouse lung RNA, BAL, and serum at 2-, 4-, and 6-weeks post-infection. Human studies used an optimized RT-PCR CRISPR assay to detect P. jirovecii transcripts in infant oropharyngeal swab samples, adult serum, and adult BAL specimens from P. jirovecii-infected and P. jirovecii-non-infected patients. RESULTS. The P. murina assays sensitively detected Pneumocystis RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay results identified infants infected with P. jirovecii with greater sensitivity (96.3% vs. 66.7%) and specificity (100% vs. 90.6%) than RT-qPCR compared to mtLSU standard marker, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% vs. 26.7%) in adult serum specimens. CONCLUSION. Since swabs are routinely collected in pediatric pneumonia patients and serum is easier to obtain than BAL, this assay approach could improve the accuracy and timing of pediatric and adult Pneumocystis diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.
Authors
Brady M. Youngquist, Ayanda Trevor Mnguni, Dora Pungan, Rachel P.J. Lai, Guixiang Dai, Chun Fai Ng, Amy Samson, Yasmean Abdelgaliel, Christopher J. Lyon, Bo Ning, Shahid Husain, Sean Wasserman, Jay K. Kolls, Tony Y. Hu
Abstract
BACKGROUND Rapid diagnosis to facilitate urgent intervention is critical for treatment of acute spinal cord injury (SCI). We hypothesized that a multi-analyte blood biomarker would support point-of-care SCI diagnosis, correlate with injury severity, and predict long-term neurologic outcomes.METHODS Droplet digital PCR (ddPCR) assays were designed to amplify differentially hypomethylated genomic loci in spinal cord tissue. An optimized ddPCR assay was applied to cell-free DNA (cfDNA) from plasma samples collected from prospectively enrolled acute SCI patients. Targeted proteomic profiling was also performed. Spinal cord–derived cfDNA and plasma proteins were tested for their association with SCI and ability to predict conversion in American Spinal Injury Association (ASIA) score at 6 months.RESULTS A bespoke ddPCR assay detected spinal cord–derived cfDNA in plasma of 50 patients with acute SCI (AUC: 0.89, 95% CI 0.83–0.95, P < 0.0001). Levels of cfDNA were highest in patients with the most severe injury, i.e., ASIA A, compared with those with ASIA B (P = 0.04), ASIA C (P = 0.009), and ASIA D injuries (P < 0.001). Dimensionality reduction identified 4 candidate proteins (FABP3, REST, IL-6, NF-H) that were integrated with spinal cord–derived cfDNA to derive the Spinal Cord Injury Index (SCII), which has high sensitivity and specificity for SCI diagnosis (AUC: 0.91, 95% CI 0.82–0.99, P < 0.0001), correlates with injury severity (P < 0.0001), and predicts 6-month neurologic improvement (AUC: 0.77, 95% CI 0.61–0.93, P = 0.006).CONCLUSION The detection of spinal cord–derived cfDNA and plasma protein alterations as part of a multi-analyte blood test can inform SCI diagnosis and prognosis.FUNDING North American Spine Society Young Investigator Award; Morton Cure Paralysis Fund.
Authors
Tej D. Azad, Kathleen R. Ran, Joshua D. Materi, Divyaansh Raj, Timour Al-Khindi, Sameer Gabbita, Marvin Li, Elizabeth T. Wang, A. Karim Ahmed, Megan Parker, Anita L. Kalluri, Daniel Lubelski, Christopher M. Jackson, Daniel M. Sciubba, Jon D. Weingart, Ali Bydon, Timothy F. Witham, David W. Nauen, Srinivasan Yegnasubramanian, Nicholas Theodore, Chetan Bettegowda
Abstract
BACKGROUND. Current methods for detecting esophageal cancer (EC) are generally invasive or exhibit limited sensitivity and specificity, especially for the identification of early-stage tumors. METHODS. We identified potential methylated DNA markers (MDM) from multiple genomic regions in a discovery cohort and a diagnostic model was developed and verified in a model-verification cohort of 297 participants. The accuracy of the MDM panel was validated in a multicenter, prospective cohort (n = 1429). The clinical performance of identified MDMs were compared with current tumor-associated protein markers. RESULTS. From 31 significant differentially methylated EC-associated regions identified in the marker discovery, we trained and validated a 3-MDM diagnostic model that could discriminate among EC patients and Non-EC volunteers in a multicenter clinical prospective cohort with a sensitivity of 85.5% and a specificity of 95.3%. This panel showed higher sensitivity in diagnosing early-stage tumors, with sensitivities of 56% for Stage 0 and 77% for Stage I, comparing with the performance of current biochemical markers. In population with high risk for EC, the sensitivity and specificity are 85.68% and 93.61% respectively. CONCLUSION. The assessment of tumor-associated methylation status in blood samples can facilitate non-invasive, and reliable diagnosis of early-stage EC, which warrants further development to expand screening and reduce mortality rates. TRIAL REGISTRATION NUMBER. ChiCTR2400083525.
Authors
Ruixiang Zhang, Yongzhan Nie, Xiaobing Chen, Tao Jiang, Jinhai Wang, Yuhui Peng, Guangpeng Zhou, Yong Li, Lina Zhao, Beibei Chen, Yunfeng Ni, Yan Cheng, Yiwei Xu, Zhenyu Zhu, Xianchun Gao, Zhen Wu, Tianbao Li, Jie Zhao, Cantong Liu, Gang Zhao, Jiakuan Chen, Jing Zhao, Gang Ji, Xiaoliang Han, Jie He, Yin Li
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