BACKGROUND. Subspecies of HDL contain apolipoprotein E (apoE) and/or apoCIII. Both proteins have properties that could affect HDL metabolism. The relation between HDL metabolism and risk of coronary heart disease (CHD) is not well understood. METHODS. Eighteen participants were given a bolus infusion of [D3]L-leucine to label endogenous proteins on HDL. HDL was separated into subspecies containing apoE and/or apoCIII and then into 4 sizes. Metabolic rates for apoA-I in HDL subspecies and sizes were determined by interactive modeling. The concentrations of apoE in HDL that contain or lack apoCIII were measured in a prospective study in Denmark including 1,949 incident CHD cases during 9 years. RESULTS. HDL containing apoE but not apoCIII is disproportionately secreted into the circulation, actively expands while circulating, and is quickly cleared. These are key metabolic steps in reverse cholesterol transport, which may protect against atherosclerosis. ApoCIII on HDL strongly attenuates these metabolic actions of HDL apoE. In the epidemiological study, the relation between HDL apoE concentration and CHD significantly differed depending on whether apoCIII was present. HDL apoE was associated significantly with lower risk of CHD only in the HDL subspecies lacking apoCIII. CONCLUSIONS. ApoE and apoCIII on HDL interact to affect metabolism and CHD. ApoE promotes metabolic steps in reverse cholesterol transport and is associated with lower risk of CHD. ApoCIII, when coexisting with apoE on HDL, abolishes these benefits. Therefore, differences in metabolism of HDL subspecies pertaining to reverse cholesterol transport are reflected in differences in association with CHD. TRIAL REGISTRATION. Clinicaltrials.gov NCT01399632. FUNDING. This work was supported by NIH grant R01HL095964 to FMS and by a grant to the Harvard Clinical and Translational Science Center (8UL1TR0001750) from the National Center for Advancing Translational Science.
Allyson M. Morton, Manja Koch, Carlos O. Mendivil, Jeremy D. Furtado, Anne Tjønneland, Kim Overvad, Liyun Wang, Majken K. Jensen, Frank M. Sacks
BACKGROUND. Programmed death 1 (PD-1) inhibition activates partially exhausted cytotoxic T lymphocytes (peCTLs) and induces tumor regression. We previously showed that the peCTL fraction predicts response to anti–PD-1 monotherapy. Here, we sought to correlate peCTL and regulatory T lymphocyte (Treg) levels with response to combination immunotherapy, and with demographic/disease characteristics, in metastatic melanoma patients. METHODS. Pretreatment melanoma samples underwent multiparameter flow cytometric analysis. Patients were treated with anti–PD-1 monotherapy or combination therapy, and responses determined by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) criteria. peCTL and Treg levels across demographic/disease variables were compared. Low versus high peCTL (≤20% vs. >20%) were defined from a previous study. RESULTS. One hundred and two melanoma patients were identified. The peCTL fraction was higher in responders than nonresponders. Low peCTL correlated with female sex and liver metastasis, but not with lactate dehydrogenase (LDH), tumor stage, or age. While overall response rates (ORRs) to anti–PD-1 monotherapy and combination therapy were similar in high-peCTL patients, low-peCTL patients given combination therapy demonstrated higher ORRs than those who received monotherapy. Treg levels were not associated with these factors nor with response. CONCLUSION. In melanoma, pretreatment peCTL fraction is reduced in women and in patients with liver metastasis. In low-peCTL patients, anti–PD-1 combination therapy is associated with significantly higher ORR than anti–PD-1 monotherapy. Fewer tumor-infiltrating peCTLs may be required to achieve response to combination immunotherapy. TRIAL REGISTRATION. UCSF IRB Protocol 138510 FUNDING. NIH DP2-AR068130, K08-AR062064, AR066821, and Burroughs Wellcome CAMS-1010934 (M.D.R.). Amoroso and Cook Fund, and the Parker Institute for Cancer Immunotherapy (A.I.D.).
Kimberly Loo, Katy K. Tsai, Kelly Mahuron, Jacqueline Liu, Mariela L. Pauli, Priscila M. Sandoval, Adi Nosrati, James Lee, Lawrence Chen, Jimmy Hwang, Lauren S. Levine, Matthew F. Krummel, Alain P. Algazi, Miguel Pampaloni, Michael D Alvarado, Michael D. Rosenblum, Adil I. Daud
BACKGROUND. DC-based tumor vaccines have had limited clinical success thus far. SOCS1, a key inhibitor of inflammatory cytokine signaling, is an immune checkpoint regulator that limits DC immunopotency. METHODS. We generated a genetically modified DC (gmDC) vaccine to perform immunotherapy. The adenovirus (Ad-siSSF) delivers two tumor-associated antigens (TAAs), survivin and MUC1; secretory bacterial flagellin for DC maturation; and an RNA interference moiety to suppress SOCS1. A 2-stage phase I trial was performed for patients with relapsed acute leukemia after allogenic hematopoietic stem cell transplantation: in stage 1, we compared the safety and efficacy between gmDC treatment (23 patients) and standard donor lymphocyte infusion (25 patients); in stage 2, we tested the efficacy of the gmDC vaccine for 12 acute myeloid leukemia (AML) patients with early molecular relapse. RESULTS. gmDCs elicited potent TAA-specific CTL responses in vitro, and the immunostimulatory activity of gmDC vaccination was demonstrated in rhesus monkeys. A stage 1 study established that this combinatory gmDC vaccine is safe in acute leukemia patients and yielded improved survival rate. In stage 2, we observed a complete remission rate of 83% in 12 relapsed AML patients. Overall, no grade 3 or grade 4 graft-versus-host disease incidence was detected in any of the 35 patients enrolled. CONCLUSIONS. This study, with combinatory modifications in DCs, demonstrates the safety and efficacy of SOCS1-silenced DCs in treating relapsed acute leukemia. TRIAL REGISTRATION. ClinicalTrials.gov NCT01956630. FUNDING. National Institute of Health (R01CA90427); the Key New Drug Development and Manufacturing Program of the “Twelfth Five-Year Plan” of China (2011ZX09102-001-29); and Clinical Application Research of Beijing (Z131107002213148).
Danhong Wang, Xue F. Huang, Bangxing Hong, Xiao-Tong Song, Liangding Hu, Min Jiang, Bin Zhang, Hongmei Ning, Yuhang Li, Chen Xu, Xiao Lou, Botao Li, Zhiyong Yu, Jiangwei Hu, Jianlin Chen, Fan Yang, Haiyan Gao, Guoliang Ding, Lianming Liao, Lisa Rollins, Lindsey Jones, Si-Yi Chen, Hu Chen
BACKGROUND. Accumulation of diacylglycerol (DAG) and sphingolipids is thought to promote skeletal muscle insulin resistance by altering cellular signaling specific to their location. However,the subcellular localization of bioactive lipids in human skeletal muscle is largely unknown. METHODS. We evaluated subcellular localization of skeletal muscle DAGs and sphingolipids in lean individuals (n = 15), endurance-trained athletes (n = 16), and obese men and women with (n = 12) and without type 2 diabetes (n = 15). Muscle biopsies were fractionated into sarcolemmal, cytosolic, mitochondrial/ER, and nuclear compartments. Lipids were measured using liquid chromatography tandem mass spectrometry, and insulin sensitivity was measured using hyperinsulinemic-euglycemic clamp. RESULTS. Sarcolemmal 1,2-DAGs were not significantly related to insulin sensitivity. Sarcolemmal ceramides were inversely related to insulin sensitivity, with a significant relationship found for the C18:0 species. Sarcolemmal sphingomyelins were also inversely related to insulin sensitivity, with the strongest relationships found for the C18:1, C18:0, and C18:2 species. In the mitochondrial/ER and nuclear fractions, 1,2-DAGs were positively related to, while ceramides were inversely related to, insulin sensitivity. Cytosolic lipids as well as 1,3-DAG, dihydroceramides, and glucosylceramides in any compartment were not related to insulin sensitivity. All sphingolipids but only specific DAGs administered to isolated mitochondria decreased mitochondrial state 3 respiration. CONCLUSION. These data reveal previously unknown differences in subcellular localization of skeletal muscle DAGs and sphingolipids that relate to whole-body insulin sensitivity and mitochondrial function in humans. These data suggest that whole-cell concentrations of lipids obscure meaningful differences in compartmentalization and suggest that subcellular localization of lipids should be considered when developing therapeutic interventions to treat insulin resistance. FUNDING. National Institutes of Health General Clinical Research Center (RR-00036), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01DK089170), NIDDK (T32 DK07658), and Colorado Nutrition Obesity Research Center (P30DK048520).
Leigh Perreault, Sean A. Newsom, Allison Strauss, Anna Kerege, Darcy E. Kahn, Kathleen A. Harrison, Janet K. Snell-Bergeon, Travis Nemkov, Angelo D’Alessandro, Matthew R. Jackman, Paul S. MacLean, Bryan C. Bergman
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
BACKGROUND. In patients with limited response to conventional therapeutics, repositioning of already approved drugs can bring new, more effective options. Current drug repositioning methods, however, frequently rely on retrospective computational analyses and genetic testing — time consuming methods that delay application of repositioned drugs. Here, we show how proteomic analysis of liquid biopsies successfully guided treatment of neovascular inflammatory vitreoretinopathy (NIV), an inherited autoinflammatory disease with otherwise poor clinical outcomes. METHODS. Vitreous biopsies from NIV patients were profiled by an antibody array for expression of 200 cytokine-signaling proteins. Non-NIV controls were compared with NIV samples from various stages of disease progression. Patterns were identified by 1-way ANOVA, hierarchical clustering, and pathway analysis. Subjects treated with repositioned therapies were followed longitudinally. RESULTS. Proteomic profiles revealed molecular pathways in NIV pathologies and implicated superior and inferior targets for therapy. Anti-VEGF injections resolved vitreous hemorrhages without the need for vitrectomy surgery. Methotrexate injections reversed inflammatory cell reactions without the side effects of corticosteroids. Anti–IL-6 therapy prevented recurrent fibrosis and retinal detachment where all prior antiinflammatory interventions had failed. The cytokine array also showed that TNF-α levels were normal and that corticosteroid-sensitive pathways were absent in fibrotic NIV, helping explain prior failure of these conventional therapeutic approaches. CONCLUSIONS. Personalized proteomics can uncover highly personalized therapies for autoinflammatory disease that can be timed with specific pathologic activities. This precision medicine strategy can also help prevent delivery of ineffective drugs. Importantly, proteomic profiling of liquid biopsies offers an endpoint analysis that can directly guide treatment using available drugs.
Gabriel Velez, Alexander G. Bassuk, Diana Colgan, Stephen H. Tsang, Vinit B. Mahajan
BACKGROUND. Systemic inflammation and muscle wasting are highly prevalent and coexist in patients on maintenance hemodialysis (MHD). We aimed to determine the effects of systemic inflammation on skeletal muscle protein metabolism in MHD patients. METHODS. Whole body and skeletal muscle protein turnover were assessed by stable isotope kinetic studies. We incorporated expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue from integrin β1 gene KO CKD mice models. RESULTS. Among 129 patients with mean (± SD) age 47 ± 12 years, 74% were African American, 73% were male, and 22% had diabetes mellitus. Median high-sensitivity C-reactive protein (hs-CRP) concentration was 13 (interquartile range 0.8, 33) mg/l. There were statistically significant associations between hs-CRP and forearm skeletal muscle protein synthesis, degradation, and net forearm skeletal muscle protein balance (P < 0.001 for all). The associations remained statistically significant after adjustment for clinical and demographic confounders, as well as in sensitivity analysis, excluding patients with diabetes mellitus. In attempting to identify potential mechanisms involved in this correlation, we show increased expressions of E1, E214K, E3αI, E3αII, MuRF-1, and atrogin-1 in skeletal muscle tissue obtained from an animal model of chronic kidney disease. CONCLUSION. These data suggest that systemic inflammation is a strong and independent determinant of skeletal muscle protein homeostasis in MHD patients, providing rationale for further studies using anticytokine therapies in patients with underlying systemic inflammation. FUNDING. This study was in part supported by NIH grants R01 DK45604 and 1K24 DK62849, the Clinical Translational Science Award UL1-TR000445 from the National Center for Advancing Translational Sciences, the Veterans Administration Merit Award I01 CX000414, the SatelliteHealth Normon Coplon Extramural Grant Program, and the FDA grant 000943.
Serpil M. Deger, Adriana M. Hung, Jorge L. Gamboa, Edward D. Siew, Charles D. Ellis, Cindy Booker, Feng Sha, Haiming Li, Aihua Bian, Thomas G. Stewart, Roy Zent, William E. Mitch, Naji N. Abumrad, T. Alp Ikizler
BACKGROUND. HIV-infected individuals, even well controlled with combined antiretroviral therapy (cART), have systemic inflammation and comorbidities. Substance P (SP) is an undecapeptide, which mediates neurotransmission and inflammation through its cognate neurokinin 1 receptor (NK1R). Plasma SP levels are elevated in HIV-infected individuals. The FDA-approved antiemetic aprepitant, an NK1R antagonist, has anti-HIV effects and antiinflammatory actions. We evaluated the safety, pharmacokinetics, and antiinflammatory properties of aprepitant in HIV-positive individuals receiving cART. METHODS. We conducted a phase 1B study of 12 HIV-positive individuals on a ritonavir-containing regimen (HIV viral load less than 40 copies/ml and CD4 > 400 cells/μl). Participants received open-label aprepitant 375 mg per day for 28 days and were followed for an additional 30 days. Changes in plasma levels of proinflammatory markers were assessed using flow cytometry, ELISA, luminex, and SOMAscan assays. RESULTS. The mean peak aprepitant plasma concentration was 30.7 ± 15.3 μg/ml at day 14 and 23.3 ± 12.3 μg/ml at day 28. Aprepitant treatment resulted in decreased plasma SP levels and affected 176 plasma proteins (56 after FDR) and several metabolic pathways, including inflammation and lipid metabolism. No change in soluble CD163 was observed. Aprepitant treatment was associated with a moderate increases in total and HDL cholesterol and affected select hematologic and metabolic markers, which returned to baseline levels 30 days after aprepitant treatment was stopped. There were 12 mild and 10 moderate adverse events (AE). CONCLUSIONS. Aprepitant is safe and well tolerated. The antiinflammatory properties of aprepitant make it a possible adjunctive therapy for comorbid conditions associated with HIV infection. TRIAL REGISTRATION. ClinicalTrials.gov (NCT02154360). FUNDING. This research was funded by NIH UO1 MH090325, P30 MH097488, and PO1 MH105303.
Sergei Spitsin, Pablo Tebas, Jeffrey S. Barrett, Vasiliki Pappa, Deborah Kim, Deanne Taylor, Dwight L. Evans, Steven D. Douglas
BACKGROUND. Our goal was to identify changes in the metabolome in multiple sclerosis (MS) and how vitamin D supplementation alters metabolic profiles in MS patients and healthy controls. METHODS. We applied global untargeted metabolomics to plasma from a cross-sectional cohort of age- and sex-matched MS patients and controls and a second longitudinal cohort of MS patients and healthy controls who received 5,000 IU cholecalciferol daily for 90 days. We applied partial least squares discriminant analysis, weighted correlation network analysis (WGCNA), and pathway analysis to the metabolomics data. Generalized estimating equations models were used to assess change in WGCNA-identified module scores or metabolite pathways with vitamin D supplementation. RESULTS. Utilizing multiple analytical techniques, we identified metabolic alterations in oxidative stress (γ-glutamyl amino acid, glutathione) and xenobiotic metabolism (benzoate, caffeine) in MS patients compared with healthy controls in the first cohort. In the vitamin D supplementation cohort, we identified two sets of metabolites altered differentially between MS patients and healthy controls with vitamin D supplementation. The first included markers of oxidative stress and protein oxidation (P = 0.006), while the second contained lysolipids and fatty acids (P = 0.03). CONCLUSIONS. Using metabolomics, we identified alterations in oxidative stress and xenobiotic metabolism in MS patients and subsequently demonstrated a reduction of oxidative stress markers with vitamin D supplementation in healthy controls but not in MS patients. We demonstrate the utility of metabolomics in identifying aberrant metabolic processes and in monitoring the ability of therapeutic interventions to correct these abnormalities. TRIAL REGISTRATION. ClinicalTrials.gov NCT01667796. FUNDING. This study was supported by NIH grant K23 NS067055, grants from the Race to Erase MS, the National Multiple Sclerosis Society, the American Academy of Neurology, and North American Research Committee on Multiple Sclerosis.
Pavan Bhargava, Kathryn C. Fitzgerald, Peter A. Calabresi, Ellen M. Mowry
It has been suggested that low nephron number contributes to glomerular hypertension and hyperperfusion injury in progressive chronic kidney disease (CKD). The incidence of CKD in Japan is among the highest in the world, but the reasons remain unclear. We estimated total nephron (glomerular) number (NglomTOTAL) as well as numbers of nonsclerosed (NglomNSG) and globally sclerosed glomeruli (NglomGSG), and the mean volume of nonsclerosed glomeruli (VglomNSG) in Japanese normotensive, hypertensive, and CKD subjects and investigated associations between these parameters and estimated glomerular filtration rate (eGFR). Autopsy kidneys from age-matched Japanese men (9 normotensives, 9 hypertensives, 9 CKD) had nephron number and VglomNSG estimated using disector/fractionator stereology. Subject eGFR, single-nephron eGFR (SNeGFR), and the ratio SNeGFR/VglomNSG were calculated. NglomNSG in Japanese with hypertension (392,108 ± 87,605; P < 0.001) and CKD (268,043 ± 106,968; P < 0.001) was less than in normotensives (640,399 ± 160,016). eGFR was directly correlated with NglomNSG (r = 0.70, P < 0.001) and inversely correlated with VglomNSG (r = –0.53, P < 0.01). SNeGFR was higher in hypertensives than normotensives (P = 0.03), but was similar in normotensives and CKD, while the ratio SNeGFR/VglomNSG was similar in normotensives and hypertensives but markedly reduced in CKD. Nephron number in Japanese with hypertension or CKD was low. This results in a higher SNeGFR in hypertensives compared with normotensive and CKD subjects, but lowered SNeGFR/VglomNSG in CKD subjects, suggesting that changes in GFR are accommodated by glomerular hypertrophy rather than glomerular hypertension. These findings suggest glomerular hypertrophy is a dominant factor in maintenance of GFR under conditions of low nephron number.
Go Kanzaki, Victor G. Puelles, Luise A. Cullen-McEwen, Wendy E. Hoy, Yusuke Okabayashi, Nobuo Tsuboi, Akira Shimizu, Kate M. Denton, Michael D. Hughson, Takashi Yokoo, John F. Bertram
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