Vogt-Koyanagi-Harada (VKH) disease is an important refractory uveitis mediated by pathological T cells (TCs). Tofacitinib (TOFA) is a Janus kinases (JAKs) targeted therapy for several autoimmune diseases. However, the specific pathogenesis and targeted therapeutics for VKH remain largely unknown. Based on single-cell RNA sequencing and mass cytometry, we present the first multimodal high-dimensional analysis to determine a comprehensive human immune atlas of VKH patients undergoing TOFA therapy in the context of subset composition, gene signatures, enriched pathways, and intercellular interactions. VKH patients are characterized by TCs polarization from naive to effector and memory subsets, altogether with accrued monocytes, upregulated cytokines and JAK-STAT signaling pathways. In vitro, TOFA reversed Th17/ regulatory T-cell (Treg) imbalance and inhibited IL-2-induced STAT1/3 phosphorylation. TOFA alleviated VKH symptoms by restoring pathological TCs polarization and functional marker expression and downregulating cytokine signaling and lymphocyte function. Remarkably, inflammation-related responses and intercellular interactions decreased after TOFA treatment, particularly in monocytes. Notably, we identified two inflammation- and JAK-associated monocyte subpopulations that were strongly implicated in VKH pathogenesis and mechanisms involved in TOFA treatment. Here, we provide a novel JAK-targeted therapy for VKH and elaborate on the possible therapeutic mechanisms of TOFA, expanding our knowledge of VKH pathological patterns.
Xiuxing Liu, Qi Jiang, Jianjie Lv, Shizhao Yang, Zhaohao Huang, Runping Duan, Tianyu Tao, Zhaohuai Li, Rong Ju, Yingfeng Zheng, Wenru Su
The muscular dystrophies (MDs) are genetic muscle diseases that result in progressive muscle degeneration followed by the fibrotic replacement of affected muscles as regenerative processes fail. Therapeutics that specifically address the fibrosis and failed regeneration associated with MDs represent a major unmet clinical need for MD patients, particularly those with advanced-stage disease progression. The current study investigated targeting NAD(P)H oxidase 4 (NOX4) as a potential strategy to reduce fibrosis and promote regeneration in disease-burdened muscle that models Duchenne muscular dystrophy (DMD). NOX4 was elevated in the muscles of dystrophic mice and DMD patients, localizing primarily to interstitial cells located between muscle fibers. Genetic and pharmacological targeting of NOX4 significantly reduced fibrosis in dystrophic respiratory and limb muscles. Mechanistically, NOX4 targeting decreased the number of fibrosis-depositing cells (myofibroblasts) and restored the number of muscle-specific stem cells (satellite cells) localized to their physiological niche, thereby rejuvenating muscle regeneration. Furthermore, acute inhibition of NOX4 was sufficient to induce apoptotic clearing of myofibroblasts within dystrophic muscle. These data indicate that targeting NOX4 is an effective strategy to promote the beneficial remodeling of disease-burdened muscle representative of DMD and, potentially, other MDs and muscle pathologies.
David W. Hammers
Recessive PJVK mutations that cause a deficiency of pejvakin, a protein expressed in both sensory hair cells and first-order neurons of the inner ear, are an important cause of hereditary hearing impairment. Patients with PJVK mutations garner limited benefits from cochlear implantation; thus, alternative biological therapies may be required to address this clinical difficulty. The synthetic adeno-associated viral vector Anc80L65, with its wide tropism and high transduction efficiency in various inner ear cells, may provide a solution. We delivered the PJVK transgene to the inner ear of Pjvk mutant mice using the synthetic Anc80L65 vector. We observed robust exogenous pejvakin expression in the hair cells and neurons of the cochlea and vestibular organs. Subsequent morphologic and audiologic studies demonstrated significant restoration of spiral ganglion neuron density and hair cells in the cochlea, along with partial recovery of sensorineural hearing impairment. In addition, we observed a recovery of vestibular ganglion neurons and balance function to WT levels. Our study demonstrates the utility of Anc80L65-mediated gene delivery in Pjvk mutant mice and provides insights into the potential of gene therapy for PJVK-related inner ear deficits.
Ying-Chang Lu, Yi-Hsiu Tsai, Yen-Huei Chan, Chin-Ju Hu, Chun-Ying Huang, Ru Xiao, Chuan-Jen Hsu, Luk H. Vandenberghe, Chen-Chi Wu, Yen-Fu Cheng
Deficiency of glycogen debranching enzyme in glycogen storage disease type III (GSD III) results in excessive glycogen accumulation in multiple tissues, primarily the liver, heart, and skeletal muscle. We recently reported that an adeno-associated virus (AAV) vector expressing a bacterial debranching enzyme (Pullulanase) driven by the ubiquitous CMV enhancer/chicken β-actin (CB) promoter cleared glycogen in major affected tissues of infant GSD IIIa mice. In this study, we developed a novel dual promoter consisting of a liver-specific promoter (LSP) and the CB promoter for gene therapy in adult GSD IIIa mice. Ten-week treatment with an AAV vector containing the LSP-CB dual promoter in adult GSD IIIa mice significantly increased Pullulanase expression and reduced glycogen contents in the liver (-60%), heart (-76%), and skeletal muscle (-63%), accompanied by the reversal of liver fibrosis, improved muscle function, and significant decrease in plasma biomarkers alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Compared to the CB promoter, the dual promoter effectively decreased Pullulanase-induced cytotoxic T lymphocyte responses and enabled persistent therapeutic gene expression in adult GSD IIIa mice. Future studies are needed to determine the long-term durability of the dual promoter mediated expression of Pullulanase in adult GSD IIIa mice and in large animal models.
Jeong-A Lim, Priya S. Kishnani, Baodong Sun
BACKGROUND. A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS. We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS. We reported an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed significant difference in response to standard of care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDOs cultures. CONCLUSION. While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION. Not applicable. FUNDING. This work was supported by the Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, CA265050, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, a University of Minnesota-Mayo Clinic Partnership grant, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).
Tara L. Hogenson, Hao Xie, William J. Phillips, Merih D. Toruner, Jenny J. Li, Isaac P. Horn, Devin J. Kennedy, Luciana L. Almada, David L. Marks, Ryan M. Carr, Murat Toruner, Ashley N. Sigafoos, Amanda N. Koenig-Kappes, Rachel L.O. Olson, Ezequiel J. Tolosa, Cheng Zhang, Hu Li, Jason D. Doles, Jonathan Bleeker, Michael T. Barrett, James H. Boyum, Benjamin R. Kipp, Amit Mahipal, Joleen M. Hubbard, Temperance J. Scheffler Hanson, Gloria M. Petersen, Surendra Dasari, Ann L. Oberg, Mark J. Truty, Rondell P. Graham, Michael J. Levy, Mojun Zhu, Daniel D. Billadeau, Alex A. Adjei, Nelson Dusetti, Juan L. Iovanna, Tanios S. Bekaii-Saab, Wen Wee Ma, Martin E. Fernandez-Zapico
No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials due to the unmapped interpatient heterogeneity and disease complexity. We have utilized a single-cell cytometry-by-time-of-flight (cyTOF) based platform to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with two drugs, an NF-κB pathway inhibitor, BMS-345541, and a chondroinductive small molecule, Kartogenin, that showed preclinical success in animal models for OA. cyTOF conducted with 30 metal isotope-labeled antibodies parsed the effects of the drugs on inflammatory, senescent, and chondroprogenitor populations. The NF-κB pathway inhibition decreased the expression of NF-κB, HIF2A and iNOS in multiple chondrocyte clusters and significantly depleted four p16ink4a expressing senescent populations including NOTCH1+STRO1+ chondroprogenitors. While Kartogenin also affected select p16ink4a expressing senescent clusters, there was a less discernible effect on chondroprogenitor populations. Overall, BMS-345541 elicited a uniform drug response in all patients while only a few responded to Kartogenin. These studies demonstrate that a single-cell cyTOF-based drug screening platform can provide insights into patient response assessment and their stratification.
Neety Sahu, Fiorella C. Grandi, Nidhi Bhutani
Intravenous administration of a high affinity carbon monoxide (CO)-binding molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to understand how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating three readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb), N-ethylmaleimide modified hemoglobin (NEMHb), and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls, and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values and scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.
Qinzi Xu, Jason J. Rose, Xiukai Chen, Ling Wang, Anthony W. DeMartino, Matthew R. Dent, Sagarika Tiwari, Kaitlin Bocian, Xueyin N. Huang, Qin Tong, Charles F. McTiernan, Lanping Guo, Elmira Alipour, Trevor C. Jones, Kamil Burak Ucer, Daniel B. Kim-Shapiro, Jesus Tejero, Mark T. Gladwin
Therapy with radiation plus cisplatin kills HPV+ oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why these standard treatments fail for some patients, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs affects mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlations. Ectopically expressing fl-E6 in models with low baseline levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the peroxisome proliferator–activated receptor gamma co-activator 1α/estrogen-related receptor α (PGC-1α/ERRα) pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1α transcription. Concordant observations were made in 3 clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, the highest p53 target gene expression, and an activated PGC-1α/ERRα pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively affecting patient survival. E6’s interaction with the PGC-1α/ERRα axis has implications for predicting and targeting treatment resistance in OPSCC.
Malay K. Sannigrahi, Pavithra Rajagopalan, Ling Lai, Xinyi Liu, Varun Sahu, Hiroshi Nakagawa, Jalal B. Jalaly, Robert M. Brody, Iain M. Morgan, Bradford E. Windle, Xiaowei Wang, Phyllis A. Gimotty, Daniel P. Kelly, Elizabeth A. White, Devraj Basu
Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations confirmed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is likely related to the more severe pathology and insufficient drug concentrations in the pancreas.
Zsófia G. Pesei, Zsanett Jancsó, Alexandra Demcsák, Balázs Csaba Németh, Sandor Vajda, Miklós Sahin-Tóth
Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes — the “basal-like” (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible “classical” (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.
Niklas Krebs, Lukas Klein, Florian Wegwitz, Elisa Espinet, Hans Carlo Maurer, Mengyu Tu, Frederike Penz, Stefan Küffer, Xingbo Xu, Hanibal Bohnenberger, Silke Cameron, Marius Brunner, Albrecht Neesse, Uday Kishore, Elisabeth Hessmann, Andreas Trumpp, Philipp Ströbel, Rolf A. Brekken, Volker Ellenrieder, Shiv K. Singh
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