We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population before initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single-genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) control (nonfailure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine/tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/Optimal Combined Therapy After Nevirapine Exposure (OCTANE) Trial 1 among women who had received prior single-dose nevirapine. Ultrasensitive single-genome sequencing revealed a significant association between pre-ART HIV variants with DRMs to 2 drug classes linked on the same genome (dual class) and failure of combination ART with 3 drugs to suppress viremia. In contrast, linked, single-class DRMs were not associated with ART failure. We conclude that linked dual-class DRMs present before the initiation of ART are associated with ART failure, whereas linked single-class DRMs are not.
Valerie F. Boltz, Wei Shao, Michael J. Bale, Elias K. Halvas, Brian Luke, James A. McIntyre, Robert T. Schooley, Shahin Lockman, Judith S. Currier, Fred Sawe, Evelyn Hogg, Michael D. Hughes, Mary F. Kearney, John M. Coffin, John W. Mellors
Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII–deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI’s protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.
Tine Wyseure, Tingyi Yang, Jenny Y. Zhou, Esther J. Cooke, Bettina Wanko, Merissa Olmer, Ruchi Agashe, Yosuke Morodomi, Niels Behrendt, Martin Lotz, John Morser, Annette von Drygalski, Laurent O. Mosnier
Aberrant activation of the NF-κB transcription factors underlies chemoresistance in various cancer types, including colorectal cancer (CRC). Targeting the activating mechanisms, particularly with inhibitors to the upstream IκB kinase (IKK) complex, is a promising strategy to augment the effect of chemotherapy. However, clinical success has been limited, largely because of low specificity and toxicities of tested compounds. In solid cancers, the IKKs are driven predominantly by the Toll-like receptor (TLR)/IL-1 receptor family members, which signal through the IL-1 receptor–associated kinases (IRAKs), with isoform 4 (IRAK4) being the most critical. The pathogenic role and therapeutic value of IRAK4 in CRC have not been investigated. We found that IRAK4 inhibition significantly abrogates colitis-induced neoplasm in APCMin/+ mice, and bone marrow transplant experiments showed an essential role of IRAK4 in immune cells during neoplastic progression. Chemotherapy significantly enhances IRAK4 and NF-κB activity in CRC cells through upregulating TLR9 expression, which can in turn be suppressed by IRAK4 and IKK inhibitors, suggesting a feed-forward pathway that protects CRC cells from chemotherapy. Lastly, increased tumor phospho-IRAK4 staining or IRAK4 mRNA expression is associated with significantly worse survival in CRC patients. Our results support targeting IRAK4 to improve the effects of chemotherapy and outcomes in CRC.
Qiong Li, Yali Chen, Daoxiang Zhang, Julie Grossman, Lin Li, Namrata Khurana, Hongmei Jiang, Patrick M. Grierson, John Herndon, David G. DeNardo, Grant A. Challen, Jingxia Liu, Marianna B. Ruzinova, Ryan C. Fields, Kian-Huat Lim
Nitric oxide regulates BP by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMCs). We previously showed that sGC heme iron reduction (Fe3+ → Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell–specific Cyb5r3 KO mice (SMC CYB5R3–KO) to test if SMC CYB5R3 loss affects systemic BP in normotension and hypertension via regulation of the sGC redox state. SMC CYB5R3–KO mice exhibited a 5.84-mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared with controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin II. We found that SMC CYB5R3–KO mice exhibited a 14.75-mmHg BP increase, and mesenteric arteries had diminished nitric oxide–dependent vasodilation but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin II–treated SMC CYB5R3–KO mice showed greater BP reduction compared with controls. Together, these data provide the first in vivo evidence to our knowledge that SMC CYB5R3 is an sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.
Brittany G. Durgin, Scott A. Hahn, Heidi M. Schmidt, Megan P. Miller, Neha Hafeez, Ilka Mathar, Daniel Freitag, Peter Sandner, Adam C. Straub
The cardiac hormone atrial natriuretic peptide (ANP) is a central regulator of blood volume and a therapeutic target in hypertension and heart failure. Enhanced ANP activity in such conditions through inhibition of the degradative enzyme neprilysin has shown clinical efficacy but is complicated by consequences of simultaneous accumulation of a heterogeneous array of other hormones. Targets for specific ANP enhancement have not been available. Here, we describe a cis-acting antisense transcript (NPPA-AS1), which negatively regulates ANP expression in human cardiomyocytes. We show that NPPA-AS1 regulates ANP expression via facilitating NPPA repressor RE1-silencing transcription factor (REST) binding to its promoter, rather than forming an RNA duplex with ANP mRNA. Expression of ANP mRNA and NPPA-AS1 was increased and correlated in isolated strained human cardiomyocytes and in hearts from patients with advanced heart failure. Further, inhibition of NPPA-AS1 in vitro and in vivo resulted in increased myocardial expression of ANP, increased circulating ANP, increased renal cGMP, and lower blood pressure. The effects of NPPA-AS1 inhibition on NPPA expression in human cardiomyocytes were further marked under cell-strain conditions. Collectively, these results implicate the antisense transcript NPPA-AS1 as part of a physiologic self-regulatory ANP circuit and a viable target for specific ANP augmentation.
Selvi Celik, Mardjaneh Karbalaei Sadegh, Michael Morley, Carolina Roselli, Patrick T. Ellinor, Thomas Cappola, J. Gustav Smith, Olof Gidlöf
Clinical trials of high-dose androgen (HDA) therapy for prostate cancer (PC) have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of HDAs, we applied an unbiased integrative approach using genetic screens and transcriptional profiling of PC cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both HDA and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors that are amenable to HDA therapy.
Michael D. Nyquist, Alexandra Corella, Osama Mohamad, Ilsa Coleman, Arja Kaipainen, Daniel A. Kuppers, Jared M. Lucas, Patrick J. Paddison, Stephen R. Plymate, Peter S. Nelson, Elahe A. Mostaghel
Oxidative stress is a major contributor to chronic lung diseases. Antioxidants such as N-acetylcysteine (NAC) are broadly viewed as protective molecules that prevent the mutagenic effects of reactive oxygen species. Antioxidants may, however, increase the risk of some forms of cancer and accelerate lung cancer progression in murine models. Here, we investigated chronic NAC treatment in aging mice displaying lung oxidative stress and cell senescence due to inactivation of the transcription factor JunD, which is downregulated in diseased human lungs. NAC treatment decreased lung oxidative damage and cell senescence and protected from lung emphysema but concomitantly induced the development of lung adenocarcinoma in 50% of JunD-deficient mice and 10% of aged control mice. This finding constitutes the first evidence to our knowledge of a carcinogenic effect of antioxidant therapy in the lungs of aged mice with chronic lung oxidative stress and warrants the utmost caution when considering the therapeutic use of antioxidants.
Marielle Breau, Amal Houssaini, Larissa Lipskaia, Shariq Abid, Emmanuelle Born, Elisabeth Marcos, Gabor Czibik, Aya Attwe, Delphine Beaulieu, Alberta Palazzo, Jean-Michel Flaman, Brigitte Bourachot, Guillaume Collin, Jeanne Tran Van Nhieu, David Bernard, Fatima Mechta-Grigoriou, Serge Adnot
B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4–/– versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4–/– recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4–/– versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4–/– donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4–/– recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4–/– mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.
Asim Saha, Patricia A. Taylor, Christopher J. Lees, Angela Panoskaltsis-Mortari, Mark J. Osborn, Colby J. Feser, Govindarajan Thangavelu, Wolfgang Melchinger, Yosef Refaeli, Geoffrey R. Hill, David H. Munn, William J. Murphy, Jonathan S. Serody, Ivan Maillard, Katharina Kreymborg, Marcel van den Brink, Chen Dong, Shuyu Huang, Xingxing Zang, James P. Allison, Robert Zeiser, Bruce R. Blazar
Alcohol withdrawal (AW) after chronic alcohol exposure produces a series of symptoms, with AW-associated seizures being among the most serious and dangerous. However, the mechanism underlying AW seizures has yet to be established. In our mouse model, a sudden AW produced 2 waves of seizures: the first wave includes a surge of multiple seizures that occurs within hours to days of AW, and the second wave consists of sustained expression of epileptiform spikes and wave discharges (SWDs) during a protracted period of abstinence. We revealed that the structural and functional adaptations in newborn dentate granule cells (DGCs) in the hippocampus underlie the second wave of seizures but not the first wave. While the general morphology of newborn DGCs remained unchanged, AW increased the dendritic spine density of newborn DGCs, suggesting that AW induced synaptic connectivity of newborn DGCs with excitatory afferent neurons and enhanced excitability of newborn DGCs. Indeed, specific activation and suppression of newborn DGCs by the chemogenetic DREADD method increased and decreased the expression of epileptiform SWDs, respectively, during abstinence. Thus, our study unveiled that the pathological plasticity of hippocampal newborn DGCs underlies AW seizures during a protracted period of abstinence, providing critical insight into hippocampal neural circuits as a foundation to understand and treat AW seizures.
Daehoon Lee, Balu Krishnan, Hai Zhang, Hee Ra Park, Eun Jeoung Ro, Yu-Na Jung, Hoonkyo Suh
Osteolytic bone lesions and hypercalcemia are common, serious complications in adult T cell leukemia/lymphoma (ATL), an aggressive T cell malignancy associated with human T cell leukemia virus type 1 (HTLV-1) infection. The HTLV-1 viral oncogene HBZ has been implicated in ATL tumorigenesis and bone loss. In this study, we evaluated the role of HBZ on ATL-associated bone destruction using HTLV-1 infection and disease progression mouse models. Humanized mice infected with HTLV-1 developed lymphoproliferative disease and continuous, progressive osteolytic bone lesions. HTLV-1 lacking HBZ displayed only modest delays to lymphoproliferative disease but significantly decreased disease-associated bone loss compared with HTLV-1–infected mice. Gene expression array of acute ATL patient samples demonstrated increased expression of RANKL, a critical regulator of osteoclasts. We found that HBZ regulated RANKL in a c-Fos–dependent manner. Treatment of HTLV-1–infected humanized mice with denosumab, a monoclonal antibody against human RANKL, alleviated bone loss. Using patient-derived xenografts from primary human ATL cells to induce lymphoproliferative disease, we also observed profound tumor-induced bone destruction and increased c-Fos and RANKL gene expression. Together, these data show the critical role of HBZ in driving ATL-associated bone loss through RANKL and identify denosumab as a potential treatment to prevent bone complications in ATL patients.
Jingyu Xiang, Daniel A. Rauch, Devra D. Huey, Amanda R. Panfil, Xiaogang Cheng, Alison K. Esser, Xinming Su, John C. Harding, Yalin Xu, Gregory C. Fox, Francesca Fontana, Takayuki Kobayashi, Junyi Su, Hemalatha Sundaramoorthi, Wing Hing Wong, Yizhen Jia, Thomas J. Rosol, Deborah J. Veis, Patrick L. Green, Stefan Niewiesk, Lee Ratner, Katherine N. Weilbaecher
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