Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria–causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage–to–blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.
Erika L. Flannery, Lander Foquet, Vorada Chuenchob, Matthew Fishbaugher, Zachary Billman, Mary Jane Navarro, William Betz, Tayla M. Olsen, Joshua Lee, Nelly Camargo, Thao Nguyen, Carola Schafer, Brandon K. Sack, Elizabeth M. Wilson, Jessica Saunders, John Bial, Brice Campo, Susan A. Charman, Sean C. Murphy, Margaret A. Phillips, Stefan H.I. Kappe, Sebastian A. Mikolajczak
The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.
Wilson I. Gonsalves, Vijay Ramakrishnan, Taro Hitosugi, Toshi Ghosh, Dragan Jevremovic, Tumpa Dutta, Dhananjay Sakrikar, Xuan-Mai Petterson, Linda Wellik, Shaji K. Kumar, K. Sreekumaran Nair
B cells play an important role in type 1 diabetes (T1D) development. However, the role of B cell activation-induced cytidine deaminase (AID) in diabetes development is not clear. We hypothesized that AID is important in the immunopathogenesis of T1D. To test this hypothesis, we generated AID-deficient (AID–/–) NOD mice. We found that AID–/–NOD mice developed accelerated T1D, with worse insulitis and high levels of anti-insulin autoantibody in the circulation. Interestingly, neither maternal IgG transferred through placenta, nor IgA transferred through milk affected the accelerated diabetes development. AID–/–NOD mice showed increased activation and proliferation of B and T cells. We found enhanced T-B cell interactions in AID–/–NOD mice, with increased T-bet and IFN-γ expression in CD4+ T cells in the presence of AID–/– B cells. Moreover, excessive lymphoid expansion was observed in AID–/–NOD mice. Importantly, antigen-specific BDC2.5 CD4+ T cells caused more rapid onset of diabetes when cotransferred with AID–/– B cells than when cotransferred with AID+/+ B cells. Thus, our study provides insights into the role of AID in T1D. Our data also suggest that AID is a negative regulator of immune tolerance and ablation of AID can lead to exacerbated islet autoimmunity and accelerated T1D development.
Qiyuan Tan, Ningwen Tai, Yangyang Li, James Pearson, Sean Pennetti, Zhiguang Zhou, F. Susan Wong, Li Wen
Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-γ, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling.
Danya Liu, I. Raul Badell, Mandy L. Ford
Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K–dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1–/– mice survived longer (1 week) than Ggcx–/– mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1–/–;Vkorc1l1–/– mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1–/– mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1–/– mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.
Julie Lacombe, Mark A. Rishavy, Kathleen L. Berkner, Mathieu Ferron
Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.
Aarti Asnani, Bahoui Zheng, Yan Liu, You Wang, Howard H. Chen, Anita Vohra, An Chi, Ivan Cornella-Taracido, Huijun Wang, Douglas G. Johns, David E. Sosnovik, Randall T. Peterson
BACKGROUND. In type 1 diabetes (T1D), adjuvant treatment with inhibitors of the renin-angiotensin-aldosterone system (RAAS), which dilate the efferent arteriole, is associated with prevention of progressive albuminuria and renal dysfunction. Uncertainty still exists as to why some individuals with long-standing T1D develop diabetic kidney disease (DKD) while others do not (DKD resistors). We hypothesized that those with DKD would be distinguished from DKD resistors by the presence of RAAS activation. METHODS. Renal and systemic hemodynamic function was measured before and after exogenous RAAS stimulation by intravenous infusion of angiotensin II (ANGII) in 75 patients with prolonged T1D durations and in equal numbers of nondiabetic controls. The primary outcome was change in renal vascular resistance (RVR) in response to RAAS stimulation, a measure of endogenous RAAS activation. RESULTS. Those with DKD had less change in RVR following exogenous RAAS stimulation compared with DKD resistors or controls (19%, 29%, 31%, P = 0.008, DKD vs. DKD resistors), reflecting exaggerated endogenous renal RAAS activation. All T1D participants had similar changes in renal efferent arteroilar resistance (9% vs. 13%, P = 0.37) irrespective of DKD status, which reflected less change versus controls (20%, P = 0.03). In contrast, those with DKD exhibited comparatively less change in afferent arteriolar vascular resistance compared with DKD resistors or controls (33%, 48%, 48%, P = 0.031, DKD vs. DKD resistors), indicating higher endogenous RAAS activity. CONCLUSION. In long-standing T1D, the intrarenal RAAS is exaggerated in DKD, which unexpectedly predominates at the afferent rather than the efferent arteriole, stimulating vasoconstriction. FUNDING. JDRF operating grant 17-2013-312.
Julie A. Lovshin, Geneviève Boulet, Yuliya Lytvyn, Leif E. Lovblom, Petter Bjornstad, Mohammed A. Farooqi, Vesta Lai, Leslie Cham, Josephine Tse, Andrej Orszag, Daniel Scarr, Alanna Weisman, Hillary A. Keenan, Michael H. Brent, Narinder Paul, Vera Bril, Bruce A. Perkins, David Z.I. Cherney
Successful tumor eradication by chimeric antigen receptor–expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB–based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB–based CAR and are promising therapeutics for clinical testing.
Sonia Guedan, Avery D. Posey Jr., Carolyn Shaw, Anna Wing, Tong Da, Prachi R. Patel, Shannon E. McGettigan, Victoria Casado-Medrano, Omkar U. Kawalekar, Mireia Uribe-Herranz, Decheng Song, J. Joseph Melenhorst, Simon F. Lacey, John Scholler, Brian Keith, Regina M. Young, Carl H. June
Membrane lipid composition is central to the highly specialized functions of neurological tissues. In the retina, abnormal lipid metabolism causes severe forms of blindness, often through poorly understood neuronal cell death. Here, we demonstrate that deleting the de novo lipogenic enzyme fatty acid synthase (FAS) from the neural retina, but not the vascular retina, results in progressive neurodegeneration and blindness with a temporal pattern resembling rodent models of retinitis pigmentosa. Blindness was not rescued by protection from light-evoked activity; by eating a diet enriched in palmitate, the product of the FAS reaction; or by treatment with the PPARα agonist fenofibrate. Vision loss was due to aberrant synaptic structure, blunted responsiveness to glial-derived neurotrophic factor and ciliary neurotrophic factor, and eventual apoptotic cell loss. This progressive neurodegeneration was associated with decreased membrane cholesterol content, as well as loss of discrete n-3 polyunsaturated fatty acid– and saturated fatty acid–containing phospholipid species within specialized membrane microdomains. Neurotrophic signaling was restored by exogenous cholesterol delivery. These findings implicate de novo lipogenesis in neurotrophin-dependent cell survival by maintaining retinal membrane configuration and lipid composition, and they suggest that ongoing lipogenesis may be required to prevent cell death in many forms of retinopathy.
Rithwick Rajagopal, Sheng Zhang, Xiaochao Wei, Teresa Doggett, Sangeeta Adak, Jennifer Enright, Vaishali Shah, Guoyu Ling, Shiming Chen, Jun Yoshino, Fong-Fu Hsu, Clay F. Semenkovich
Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12–/+/Srb1–/–/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis.
Yvonne Baumer, Qimin Ng, Gregory E. Sanda, Amit K. Dey, Heather L. Teague, Alexander V. Sorokin, Pradeep K. Dagur, Joanna I. Silverman, Charlotte L. Harrington, Justin A. Rodante, Shawn M. Rose, Nevin J. Varghese, Agastya D. Belur, Aditya Goyal, Joel M. Gelfand, Danielle A. Springer, Christopher K.E. Bleck, Crystal L. Thomas, Zu-Xi Yu, Mårten C.G. Winge, Howard S. Kruth, M. Peter Marinkovich, Aditya A. Joshi, Martin P. Playford, Nehal N. Mehta
The mechanisms underlying the development and natural progression of the airway mucus defect in cystic fibrosis (CF) remain largely unclear. New animal models of CF, coupled with imaging using micro-optical coherence tomography, can lead to insights regarding these questions. The Cftr–/– (KO) rat allows for longitudinal examination of the development and progression of airway mucus abnormalities. The KO rat exhibits decreased periciliary depth, hyperacidic pH, and increased mucus solid content percentage; however, the transport rates and viscoelastic properties of the mucus are unaffected until the KO rat ages. Airway submucosal gland hypertrophy develops in the KO rat by 6 months of age. Only then does it induce increased mucus viscosity, collapse of the periciliary layer, and delayed mucociliary transport; stimulation of gland secretion potentiates this evolution. These findings could be reversed by bicarbonate repletion but not pH correction without counterion donation. These studies demonstrate that abnormal surface epithelium in CF does not cause delayed mucus transport in the absence of functional gland secretions. Furthermore, abnormal bicarbonate transport represents a specific target for restoring mucus clearance, independent of effects on periciliary collapse. Thus, mature airway secretions are required to manifest the CF defect primed by airway dehydration and bicarbonate deficiency.
Susan E. Birket, Joy M. Davis, Courtney M. Fernandez, Katherine L. Tuggle, Ashley M. Oden, Kengyeh K. Chu, Guillermo J. Tearney, Michelle V. Fanucchi, Eric J. Sorscher, Steven M. Rowe
Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.
Ruojing Yang, Margaret Wu, Songnian Lin, Ravi P. Nargund, Xinghai Li, Theresa Kelly, Lin Yan, Ge Dai, Ying Qian, Qing Dallas-yang, Paul A. Fischer, Yan Cui, Xiaolan Shen, Pei Huo, Danqing Dennis Feng, Mark D. Erion, David E. Kelley, James Mu
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