Background. Upper body obesity (UBO) results in insulin resistance with regards to free fatty acid (FFA) release; how this differs by fat depot and sex between UBO and lean adults is unknown. We tested the hypothesis that insulin suppression of FFA release from the splanchnic bed, leg fat and upper body non-splanchnic (UBNS) adipose tissue would be impaired in UBO. Methods. Fourteen UBO (7 men, 7 women) and 14 healthy, normal weight (7 men, 7 women) volunteers participated in studies that included femoral artery, femoral vein and hepatic vein catheterization. We then measured leg and splanchnic plasma flow as well as FFA kinetics (using isotopic tracers) under overnight fasting, low- and high-dose insulin infusion using the insulin clamp technique. Results. We found the expected insulin resistance in UBO; the most quantitatively important difference between UBO and lean adults was greater FFA release from UBNS adipose tissue when plasma insulin concentrations are in the post-prandial, physiological range. There were obesity, but not sex differences in the regulation of splanchnic FFA release and sex differences in the regulation of leg FFA release. Conclusion. Reversing the defects in insulin-regulated UBNS adipose tissue FFA release would have the greatest impact on systemic FFA abnormalities in UBO. Trial Registration: (not applicable) Funding: These studies were supported by grants DK45343 and DK40484 from the U.S. Public Health Service, and the Novo Nordic Foundation (grant numbers NNF18OC0031804 and NNF16OC0021406) and the Independent Research Fund Denmark (grant number 8020-00420B).
Søren Nielsen, Michael D. Jensen
Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent anti-inflammatory and anti-tumor activity in several pre-clinical models and has proven both safe and efficacious in clinical trials for advanced gastro-intestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared to cyclophosphamide post-aHSCT (PTCy). Strikingly, we found Minnelide improved survival, weight loss and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score and colonic length. Notably, in addition to decreased donor T cell infiltration early post-HSCT, several regulatory cell populations including Tregs, ILC2s and MDSCs in the colon were increased which together may account for Minnelide’s GVHD suppression post-HSCT. Importantly, Minnelide GVHD prevention was accompanied by preservation of graft-versus-tumor (GVT) activity. As Minnelide possesses anti-AML activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for AML patients undergoing aHSCT.
Sabrina N. Copsel, Vanessa T. Garrido, Henry Barreras, Cameron S. Bader, Brent Pfeiffer, Beatriz Mateo-Victoriano, Dietlinde Wolf, Miguel Gallardo, Sophie Paczesny, Krishna V. Komanduri, Cara L. Benjamin, Alejandro Villarino, Ashok K. Saluja, Robert B. Levy
Background. Disease of the aorta varies from atherosclerosis to aneurysms with complications including rupture, dissection, and poorly characterized limited tears. We studied limited tears without any mural hematoma, termed intimomedial tears to gain insight into aortic vulnerability to excessive wall stresses. Our premise is that minimal injuries in aortas with sufficient medial resilience to prevent tear progression correspond to initial mechanisms leading to complete structural failure in aortas with significantly compromised medial resilience. Methods. Intimomedial tears were macroscopically identified in 9 of 108 ascending aortas after surgery and analyzed by histology and immunofluorescence confocal microscopy. Results. Non-hemorrhagic, non atheromatous tears correlated with advanced aneurysmal disease and most lacked distinctive symptoms or radiological signs. Tears traversed the intima and part of the subjacent media, while the resultant defects were partially or completely filled with neointima characterized by differentiated smooth muscle cells, scattered leukocytes, dense fibrosis, and absent elastic laminae despite tropoelastin synthesis. Healed lesions contained organized fibrin at tear edges without evidence of plasma and erythrocyte extravasation or lipid accumulation. Conclusion. These findings suggest a multiphasic model of aortic wall failure in which primary lesions of intimomedial tears either heal if the media is sufficiently resilient or progress as dissection or rupture by medial delamination and tear completion, respectively. Moreover, mural incorporation of thrombus and cellular responses to injury, two historically important concepts in atheroma pathogenesis, contribute to vessel wall repair with adequate conduit function but even together are not sufficient to induce atherosclerosis. Funding. R01-HL146723, R01-HL168473, and Yale Department of Surgery.
Abdulrahman H.M. Hassab, David J. Hur, Prashanth Vallabhajosyula, George Tellides, Roland Assi
Recent studies have uncovered that non-coding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese ARS family and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between PITX2 and FAM241A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-seq and RT-qPCR revealed a significant reduction in Pitx2 gene expression in LOH-1–/– mice, while Foxc1 expression remained unchanged. ChIP-seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a significant downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) which is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence which is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent. 2
Yizheng Jiang, Yu Peng, Qi Tian, Zhe Cheng, Bei Feng, Junping Hu, Lu Xia, Hui Guo, Kun Xia, Liang Zhou, Zhengmao Hu
The viral kinetics of documented SARS-CoV-2 infections exhibit a high degree of inter-individual variability. We identified six distinct viral shedding patterns, which differed according to peak viral load, duration, expansion rate and clearance rate, by clustering data from 768 infections in the National Basketball Association cohort. Omicron variant infections in previously vaccinated individuals generally led to lower cumulative shedding levels of SARS-CoV-2 than other scenarios. We then developed a mechanistic mathematical model that recapitulated 1510 observed viral trajectories, including viral rebound and cases of reinfection. Lower peak viral loads were explained by a more rapid and sustained transition of susceptible cells to a refractory state during infection, as well as an earlier and more potent late, cytolytic immune response. Our results suggest that viral elimination occurs more rapidly during omicron infection, following vaccination, and following re-infection due to enhanced innate and acquired immune responses. Because viral load has been linked with COVID-19 severity and transmission risk, our model provides a framework for understanding the wide range of observed SARS-CoV-2 infection outcomes.
Katherine Owens, Shadisadat Esmaeili, Joshua Schiffer
Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), thus creating an unmet need to identify and develop non-invasive biomarkers for AH. In murine models of ethanol-induced liver injury, complement activation contributes to hepatic inflammation and injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from the human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC), alcohol use disorder (AUD) and healthy controls (HCs). Notably, serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance amongst patients with sAH, AC, AUD, and HCs. Furthermore, complement component receptor 1-like protein (CR1L) was negatively associated with pro-inflammatory cytokines. Additionally, lower serum mannose-binding lectin associated serine protease 1 and coagulation factor II were associated with and independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.
Moyinoluwa T. Taiwo, Emily Huang, Vai Pathak, Annette Bellar, Nicole Welch, Jaividhya Dasarathy, David Streem, Craig J. McClain, Mack C. Mitchell, Bruce A. Barton, Gyongyi Szabo, Srinivasan Dasarathy, Esperance A. Schaefer, Jay Luther, Le Z. Day, Xinshou Ouyang, Suyavaran Arumugam, Wajahat Z. Mehal, Jon M. Jacobs, Russell P. Goodman, Daniel M. Rotroff, Laura E. Nagy
Loss-of-Function (LoF) variants in the filaggrin (FLG) gene are the strongest known genetic risk factor for atopic dermatitis (AD), but the impact of these variants on AD outcomes is poorly understood. We comprehensively identified genetic variants through targeted region sequencing of FLG in children (n = 438) participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children (MPAACH) cohort. Twenty FLG LoF variants were identified, including one novel variant and nine variants not previously associated with AD. FLG LoF variants were found in 13.6% of the cohort. Among these children, the presence of one or more FLG LoF variants was associated with moderate/severe AD (odds ratio (OR) = 2.00 (95% CI, 1.23–3.68) compared to those with mild AD. Children with FLG LoF variants had a higher SCORAD (SCORing for Atopic Dermatitis (SCORAD); P = 0.012) and higher likelihood of food allergy within the first 2.5 years of life (OR = 2.81, 1.50–5.26). LoF variants were associated with higher transepidermal Water Loss (TEWL) in both lesional (P = 0.018) and non-lesional skin (P = 0.015). Collectively, our study identifies established and novel AD-associated FLG LoF variants and associates FLG LoF with higher TEWL in lesional and non-lesional skin.
Samuel J. Virolainen, Latha Satish, Jocelyn M. Biagini, Hassan Chaib, Wan Chi Chang, Phillip J. Dexheimer, Michael R. Dixon, Katelyn A. Dunn, David Fletcher, Carmy Forney, Marissa Granitto, Matthew S. Hestand, Makenna Hurd, Kenneth Kaufman, Lucinda P. Lawson, Lisa J. Martin, Loren D.M. Peña, Kieran J. Phelan, Molly S. Shook, Matthew T. Weirauch, Gurjit K. Khurana Hershey, Leah C. Kottyan
Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation ihas been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired re-epithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, whilst its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif knockout mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.
Ritu Bansal, Monica Torres, Matthew Hunt, Nuoqi Wang, Margarita Chatzopoulou, Mansi Manchanda, Evan P. Taddeo, Cynthia Shu, Orian S. Shirihai, Etty Bachar-Wikstrom, Jakob D. Wikstrom
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease associated with cardiomyopathy. DMD-cardiomyopathy is characterized by abnormal intracellular Ca2+ homeostasis and mitochondrial dysfunction. We used dystrophin and utrophin null (mdx:utrn–/–) mice in sarcolipin (SLN) heterozygous knockout (sln+/–) background to examine the effect of SLN reduction on mitochondrial function in the dystrophic myocardium. Germline reduction of SLN expression in mdx:utrn–/– mice improved cardiac sarco/endoplasmic reticulum (SR) Ca2+ cycling, reduced cardiac fibrosis, and improved cardiac function. At the cellular level, reducing SLN expression prevented mitochondrial Ca2+ overload, reduced mitochondrial membrane potential loss, and improved mitochondrial function. Transmission electron microscopy of myocardial tissues and proteomic analysis of mitochondria-associated membranes show that reducing SLN expression improved mitochondrial structure and SR-mitochondria interactions in dystrophic cardiac myocytes. These findings indicate that SLN upregulation plays a significant role in the pathogenesis of cardiomyopathy and that reducing SLN expression has clinical implications in the treatment of DMD-cardiomyopathy.
Satvik Mareedu, Nadezhda Fefelova, Cristi L. Galindo, Goutham Prakash, Risa Mukai, Junichi Sadoshima, Lai-Hua Xie, Gopal J. Babu
People with HIV (PWH) have a higher age-adjusted mortality due to chronic immune activation and age-related comorbidities. PWH also have higher rates of clonal hematopoiesis (CH) than age-matched non-HIV cohorts, however, risk factors influencing the development and expansion of CH in PWH remain incompletely explored. We investigated the relationship between CH, immune biomarkers, and HIV-associated risk factors (CD4, CD8 T-cells, nadir CD4 count, opportunistic infections [OIs], and immune reconstitution inflammatory syndrome [IRIS]) in a diverse cohort of 197-PWH with median age of 42-years, using a 56-gene panel. Seventy-nine percent had a CD4 nadir < 200, 58.9% had prior OIs, and 34.5% had a history of IRIS. The prevalence of CH was high (27.4%), even in younger individuals, and CD8 T-cells and nadir CD4 counts strongly associated with CH after controlling for age. A history of IRIS was associated with CH in a subgroup analysis of ≥ 35-years-old patients. Inflammatory biomarkers were higher in CH carriers compared to non-carriers supporting a dysregulated immune state. These findings suggest PWH with low nadir CD4 and/or inflammatory complications may be at high risk of CH regardless of age and represent a high-risk group that could benefit from risk reduction and potentially targeted immunomodulation.
Joseph M. Rocco, Yifan Zhou, Nicholas S. Liu, Elizabeth Laidlaw, Frances Galindo, Megan V. Anderson, Adam Rupert, Silvia Lucena Lage, Ana M. Ortega-Villa, Shiqin Yu, Andrea Lisco, Maura Manion, George S. Vassiliou, Cynthia E. Dunbar, Irini Sereti
Fibroblast Growth Factor 23 (FGF23) production has recently been shown to increase downstream of G⍺q/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding G⍺11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the impact of G⍺11 deficiency on FGF23 production in mice with heterozygous (Gna11+/–) or homozygous (Gna11–/–) ablation of Gna11. Both Gna11+/– and Gna11–/– mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11–/– mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and evidence of systemic inflammation with elevated serum IL1β levels. Furin gene expression was significantly increased in the Gna11–/– liver, suggesting enhanced FGF23 cleavage despite the observed rise in intact FGF23 levels. Gna11–/– mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, G⍺11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that FHH patients, at least those with GNA11 mutations, may be at risk for these complications.
Birol Ay, Sajin Marcus Cyr, Kaitlin Klovdahl, Wen Zhou, Christina M. Tognoni, Yorihiro Iwasaki, Eugene P. Rhee, Alpaslan Dedeoglu, Petra Simic, Murat Bastepe
Skeletal muscle wasting results from numerous pathological conditions impacting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss of function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 results in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fibre integrity. We further implicate a disruption in atrogin-1 mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We reveal that BiP is not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorates pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicates atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy, and highlights atrogin-1’s essential role in maintaining muscle homeostasis.
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
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.
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
Spine metastases can result in severe neurologic compromise and decreased overall survival. Despite treatment advances, local disease progression is frequent, highlighting the need for novel therapies. Tumor treating fields (TTFields) impair tumor cell replication and are influenced by properties of surrounding tissue. We hypothesize bone’s dielectric properties will enhance TTFields mediated suppression of tumor growth in spine metastasis models. Computational modeling of TTFields intensity was performed following surgical resection of a spinal metastasis and demonstrated enhanced TTFields intensity within the resected vertebral body. Additionally, luciferase-tagged human KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized bone grafts and exposed to TTFields. Following TTFields exposure, BLI signal decreased 10-80% of baseline while control cultures displayed 4.48-9.36 fold increase in signal. Lastly, TTFields were applied in an orthotopic murine model of spinal metastasis. After 21 days of treatment, control mice demonstrated a 5-fold increase in BLI signal compared to TTFields treated mice. TTFields similarly prevented tumor invasion into the spinal canal and development of neurologic symptoms. Our data suggest that TTFields can be leveraged as a local therapy within minimally-conductive bone of spine metastases. This provides the groundwork for future studies investigating TTFields for patients with treatment-refractory spine metastases.
Daniel Ledbetter, Romulo de Almeida, Xizi Wu, Ariel Naveh, Chirag B. Patel, Queena Gonzalez, Thomas H. Beckham, Robert North, Laurence Rhines, Jing Li, Amol Ghia, David Aten, Claudio Tatsui, Christopher Alvarez-Breckenridge
HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-β/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that Caspase 6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) leads to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment can restrain p65 NF-κB transcriptional activity by cytoplasmic sequestration NF-κB signaling component, p65 NF-κB, and attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidney of mice with unilateral ureteral obstruction and lipopolysaccharide-induced acute kidney injury. Collectively, our findings indicate that the C-terminal region of HIPK2 is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential anti-inflammatory agents to treat kidney disease.
Ye Feng, Zhengzhe Li, Heather Wang, Bi-Cheng Liu, Kyung Lee, John Cijiang He
Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, that showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks-of-age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks-of-age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.
Lisa Duvick, W. Michael Southern, Kellie A. Benzow, Zoe N. Burch, Hillary P. Handler, Jason S. Mitchell, Hannah Kuivinen, Udaya Gadiparthi, Praseuth Yang, Alyssa Soles, Carrie A. Sheeler, Orion Rainwater, Shannah Serres, Erin B. Lind, Tessa Nichols-Meade, Brennon O'Callaghan, Huda Y. Zoghbi, Marija Cvetanovic, Vanessa C. Wheeler, James M. Ervasti, Michael D. Koob, Harry T. Orr
Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement of cholesterol for activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and non-responsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells detects T cell activation and has potential to assess the success of ICI therapy.
Nicholas G. Ciavattone, Jenny Nan Guan, Alex Farfel, Jenelle Stauff, Timothy J. Desmond, Benjamin L. Viglianti, Peter J.H. Scott, Allen F. Brooks, Gary D. Luker
Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), were dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression in a destabilization of the medial meniscus (DMM) mouse model, while genetic deficiency of plasmin activator inhibitor (PAI-1), or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans, induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, PDK1, AKT, and ERK signaling cascades, and activates matrix metalloproteinases (pro-MMPs) to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.
Qian Wang, Guoqiang Shao, Xiaoyi Zhao, Heidi H. Wong, Kate Chin, Mackenzie Zhao, Audrey Bai, Michelle S. Bloom, Zelda Z. Love, Constance R. Chu, Zhen Cheng, William H. Robinson
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.
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
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.
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