The underlying pathology of atopic dermatitis (AD) includes impaired skin barrier function, susceptibility to Staphylococcus aureus skin infection, immune dysregulation, and cutaneous dysbiosis. Our recent investigation into the potential role of Gram-negative skin bacteria in AD revealed that isolates of one particular commensal, Roseomonas mucosa, collected from healthy volunteers (HVs) improved outcomes in mouse and cell culture models of AD. In contrast, isolates of R. mucosa from patients with AD worsened outcomes in these models. These preclinical results suggested that interventions targeting the microbiome could provide therapeutic benefit for patients with AD. As a first test of this hypothesis in humans, 10 adult and 5 pediatric patients were enrolled in an open-label phase I/II safety and activity trial (the Beginning Assessment of Cutaneous Treatment Efficacy for Roseomonas in Atopic Dermatitis trial; BACTERiAD I/II). Treatment with R. mucosa was associated with significant decreases in measures of disease severity, topical steroid requirement, and S. aureus burden. There were no adverse events or treatment complications. We additionally evaluated differentiating bacterial metabolites and topical exposures that may contribute to the skin dysbiosis associated with AD and/or influence future microbiome-based treatments. These early results support continued evaluation of R. mucosa therapy with a placebo-controlled trial.
Ian A. Myles, Noah J. Earland, Erik D. Anderson, Ian N. Moore, Mark D. Kieh, Kelli W. Williams, Arhum Saleem, Natalia M. Fontecilla, Pamela A. Welch, Dirk A. Darnell, Lisa A. Barnhart, Ashleigh A. Sun, Gulbu Uzel, Sandip K. Datta
BACKGROUND. Cardiac positron emission testing (PET) is more accurate than single photon emission computed tomography (SPECT) at identifying coronary artery disease (CAD); however, the 2 modalities have not been thoroughly compared in a real-world setting. We conducted a retrospective analysis of 60-day catheterization outcomes and 1-year major adverse cardiovascular events (MACE) after the transition from a SPECT- to a PET-based myocardial perfusion imaging (MPI) program. METHODS. MPI patients at Intermountain Medical Center from January 2011–December 2012 (the SPECT era, n = 6,777) and January 2014–December 2015 (the PET era, n = 7,817) were studied. Outcomes studied were 60-day coronary angiography, high-grade obstructive CAD, left main/severe 3-vessel disease, revascularization, and 1-year MACE-revascularization (MACE-revasc; death, myocardial infarction [MI], or revascularization >60 days). RESULTS. Patients were 64 ± 13 years old; 54% were male and 90% were of European descent; and 57% represented a screening population (no prior MI, revascularization, or CAD). During the PET era, compared with the SPECT era, a higher percentage of patients underwent coronary angiography (13.2% vs. 9.7%, P < 0.0001), had high-grade obstructive CAD (10.5% vs. 6.9%, P < 0.0001), had left main or severe 3-vessel disease (3.0% vs. 2.3%, P = 0.012), and had coronary revascularization (56.7% vs. 47.1%, P = 0.0001). Similar catheterization outcomes were seen when restricted to the screening population. There was no difference in 1-year MACE-revasc (PET [5.8%] vs. SPECT [5.3%], P = 0.31). CONCLUSIONS. The PET-based MPI program resulted in improved identification of patients with high-grade obstructive CAD, as well as a larger percentage of revascularization, thus resulting in fewer patients undergoing coronary angiography without revascularization. FUNDING. This observational study was funded using internal departmental funds.
Stacey Knight, David B. Min, Viet T. Le, Kent G. Meredith, Ritesh Dhar, Santanu Biswas, Kurt R. Jensen, Steven M. Mason, Jon-David Ethington, Donald L. Lappe, Joseph B. Muhlestein, Jeffrey L. Anderson, Kirk U. Knowlton
In patients requiring ventilator support, mechanical ventilation (MV) may induce acute lung injury (ventilator-induced lung injury [VILI]). VILI is associated with substantial morbidity and mortality in mechanically ventilated patients with and without acute respiratory distress syndrome. At the cellular level, VILI induces necrotic cell death. However, the contribution of necroptosis, a programmed form of necrotic cell death regulated by receptor-interacting protein-3 kinase (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL), to the development of VILI remains unexplored. Here, we show that plasma levels of RIPK3, but not MLKL, were higher in patients with MV (i.e., those prone to VILI) than in patients without MV (i.e., those less likely to have VILI) in two large intensive care unit cohorts. In mice, RIPK3 deficiency, but not MLKL deficiency, ameliorated VILI. In both humans and mice, VILI was associated with impaired fatty acid oxidation (FAO), but in mice this association was not observed under conditions of RIPK3 deficiency. These findings suggest that FAO-dependent RIPK3 mediates pathogenesis of acute lung injury.
Ilias I. Siempos, Kevin C. Ma, Mitsuru Imamura, Rebecca M. Baron, Laura E. Fredenburgh, Jin-Won Huh, Jong-Seok Moon, Eli J. Finkelsztein, Daniel S. Jones, Michael Torres Lizardi, Edward J. Schenck, Stefan W. Ryter, Kiichi Nakahira, Augustine M.K. Choi
Using a mouse retroviral model, we have shown that mAb-based immunotherapy can induce life-long endogenous protective immunity (vaccine-like effects). This observation has potentially important consequences for treating life-threatening human viral infections. Here, we investigated the role of neutrophils in this effect. Neutrophils are innate immunity effector cells with well-established microbe-killing activities that are rapidly mobilized upon infection. They are also emerging as orchestrators of innate and adaptive immunities. However, their immunomodulatory activity during antiviral mAb immunotherapies has never been studied. Our data reveal that neutrophils have an essential role in immunotherapy-induced immune protection of infected mice. Unexpectedly, neutrophils have a limited effect in controlling viral propagation upon passive immunotherapy administration, which is mostly mediated by NK cells. Instead, neutrophils operate as essential inducers of a potent host humoral antiviral response. Thus, neutrophils play an unexpected key role in protective immunity induction by antiviral mAbs. Our work opens approaches to improve antiviral immunotherapies, as it suggests that preserving neutrophil functions and counts might be required for achieving mAb-induced protective immunity.
Mar Naranjo-Gomez, Jennifer Lambour, Marc Piechaczyk, Mireia Pelegrin
Metabolic stresses such as dietary energy restriction or physical activity exert beneficial metabolic effects. In the liver, endospanin-1 and endospanin-2 cooperatively modulate calorie restriction–mediated (CR-mediated) liver adaptations by controlling growth hormone sensitivity. Since we found CR to induce endospanin protein expression in skeletal muscle, we investigated their role in this tissue. In vivo and in vitro endospanin-2 triggers ERK phosphorylation in skeletal muscle through an autophagy-dependent pathway. Furthermore, endospanin-2, but not endospanin-1, overexpression decreases muscle mitochondrial ROS production, induces fast-to-slow fiber-type switch, increases skeletal muscle glycogen content, and improves glucose homeostasis, ultimately promoting running endurance capacity. In line, endospanin-2–/– mice display higher lipid peroxidation levels, increased mitochondrial ROS production under mitochondrial stress, decreased ERK phosphorylation, and reduced endurance capacity. In conclusion, our results identify endospanin-2 as a potentially novel player in skeletal muscle metabolism, plasticity, and function.
Steve Lancel, Matthijs K.C. Hesselink, Estelle Woldt, Yves Rouillé, Emilie Dorchies, Stephane Delhaye, Christian Duhem, Quentin Thorel, Alicia Mayeuf-Louchart, Benoit Pourcet, Valérie Montel, Gert Schaart, Nicolas Beton, Florence Picquet, Olivier Briand, Jean Pierre Salles, Hélène Duez, Patrick Schrauwen, Bruno Bastide, Bernard Bailleul, Bart Staels, Yasmine Sebti
Kidney injury is a frequent outcome in patients with disseminated Candida albicans fungal infections. IL-17 receptor (IL-17R) signaling is critical for renal protection against disseminated candidiasis, but the identity and function of IL-17–responsive cells in mediating renal defense remains an active area of debate. Using BM chimeras, we found that IL-17R signaling is required only in nonhematopoietic cells for immunity to systemic C. albicans infection. Since renal tubular epithelial cells (RTEC) are highly responsive to IL-17 in vitro, we hypothesized that RTEC might be the dominant target of IL-17 activity in the infected kidney. We generated mice with a conditional deletion of IL-17 receptor A (Il17ra) in RTEC (Il17raΔRTEC). Strikingly, Il17raΔRTEC mice showed enhanced kidney damage and early mortality following systemic infection, very similar to Il17ra–/– animals. Increased susceptibility to candidiasis in Il17raΔRTEC mice was associated with diminished activation of the renal protective Kallikrein-kinin system (KKS), resulting in reduced apoptosis of kidney-resident cells during hyphal invasion. Moreover, protection was restored by treatment with bradykinin, the major end-product of KKS activation, which was mediated dominantly via bradykinin receptor b1. These data show that IL-17R signaling in RTEC is necessary and likely sufficient for IL-17–mediated renal defense against fatal systemic C. albicans infection.
Kritika Ramani, Chetan V. Jawale, Akash H. Verma, Bianca M. Coleman, Jay K. Kolls, Partha S. Biswas
We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.
Sara D’Angelo, Fernanda I. Staquicini, Fortunato Ferrara, Daniela I. Staquicini, Geetanjali Sharma, Christy A. Tarleton, Huynh Nguyen, Leslie A. Naranjo, Richard L. Sidman, Wadih Arap, Andrew R.M. Bradbury, Renata Pasqualini
Fibrosis is the end result of most inflammatory conditions, but its pathogenesis remains unclear. We demonstrate that, in animals and humans with systemic fibrosis, plasmacytoid DCs (pDCs) are unaffected or are reduced systemically (spleen/peripheral blood), but they increase in the affected organs (lungs/skin/bronchoalveolar lavage). A pivotal role of pDCs was shown by depleting them in vivo, which ameliorated skin and/or lung fibrosis, reduced immune cell infiltration in the affected organs but not in spleen, and reduced the expression of genes and proteins implicated in chemotaxis, inflammation, and fibrosis in the affected organs of animals with bleomycin-induced fibrosis. As with animal findings, the frequency of pDCs in the lungs of patients with systemic sclerosis correlated with the severity of lung disease and with the frequency of CD4+ and IL-4+ T cells in the lung. Finally, treatment with imatinib that has been reported to reduce and/or prevent deterioration of skin and lung fibrosis profoundly reduced pDCs in lungs but not in peripheral blood of patients with systemic sclerosis. These observations suggest a role for pDCs in the pathogenesis of systemic fibrosis and identify the increased trafficking of pDCs to the affected organs as a potential therapeutic target in fibrotic diseases.
Suzanne Kafaja, Isela Valera, Anagha A. Divekar, Rajan Saggar, Fereidoun Abtin, Daniel E. Furst, Dinesh Khanna, Ram Raj Singh
BACKGROUND. Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring. METHODS. We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples. RESULTS. Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38– AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells. CONCLUSION. The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results. FUNDING. National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children’s Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.
Elaine Coustan-Smith, Guangchun Song, Sheila Shurtleff, Allen Eng-Juh Yeoh, Wee Joo Chng, Siew Peng Chen, Jeffrey E. Rubnitz, Ching-Hon Pui, James R. Downing, Dario Campana
Elevated levels of brain natriuretic peptide (BNP) are regarded as an early compensatory response to cardiac myocyte hypertrophy, although exogenously administered BNP shows poor clinical efficacy in heart failure and hypertension. We tested whether phosphodiesterase 2A (PDE2A), which regulates the action of BNP-activated cyclic guanosine monophosphate (cGMP), was directly involved in modulating Ca2+ handling from stellate ganglia (SG) neurons and cardiac norepinephrine (NE) release in rats and humans with an enhanced sympathetic phenotype. SG were also isolated from patients with sympathetic hyperactivity and healthy donor patients. PDE2A activity of the SG was greater in both spontaneously hypertensive rats (SHRs) and patients compared with their respective controls, whereas PDE2A mRNA was only high in SHR SG. BNP significantly reduced the magnitude of the calcium transients and ICaN in normal Wistar Kyoto (WKY) SG neurons, but not in the SHRs. cGMP levels stimulated by BNP were also attenuated in SHR SG neurons. Overexpression of PDE2A in WKY neurons recapitulated the calcium phenotype seen in SHR neurons. Functionally, BNP significantly reduced [3H]-NE release in the WKY rats, but not in the SHRs. Blockade of overexpressed PDE2A with Bay 60-7550 or overexpression of catalytically inactive PDE2A reestablished the modulatory action of BNP in SHR SG neurons. This suggests that PDE2A may be a key target in modulating the action of BNP to reduce sympathetic hyperactivity.
Kun Liu, Dan Li, Guoliang Hao, David McCaffary, Oliver Neely, Lavinia Woodward, Demetris Ioannides, Chieh-Ju Lu, Marcella Brescia, Manuela Zaccolo, Harikrishna Tandri, Olujimi A. Ajijola, Jeffrey L. Ardell, Kalyanam Shivkumar, David J. Paterson
Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2’-O-methyl phosphorothioate oligonucleotides and vivo–phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo–phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.
Eugene J. Wyatt, Alexis R. Demonbreun, Ellis Y. Kim, Megan J. Puckelwartz, Andy H. Vo, Lisa M. Dellefave-Castillo, Quan Q. Gao, Mariz Vainzof, Rita C. M. Pavanello, Mayana Zatz, Elizabeth M. McNally
Sepsis-associated encephalopathy manifesting as delirium is a common problem in critical care medicine. In this study, patients that had delirium due to sepsis had significant cognitive impairments at 12–18 months after hospital discharge when compared with controls and Cambridge Neuropsychological Automated Test Battery–standardized scores in spatial recognition memory, pattern recognition memory, and delayed-matching-to-sample tests but not other cognitive functions. A mouse model of S. pneumoniae pneumonia-induced sepsis, which modeled numerous aspects of the human sepsis-associated multiorgan dysfunction, including encephalopathy, also revealed similar deficits in spatial memory but not new task learning. Both humans and mice had large increases in chemokines for myeloid cell recruitment. Intravital imaging of the brains of septic mice revealed increased neutrophil and CCR2+ inflammatory monocyte recruitment (the latter being far more robust), accompanied by subtle microglial activation. Prevention of CCR2+ inflammatory monocyte recruitment, but not neutrophil recruitment, reduced microglial activation and other signs of neuroinflammation and prevented all signs of cognitive impairment after infection. Therefore, therapeutically targeting CCR2+ inflammatory monocytes at the time of sepsis may provide a novel neuroprotective clinical intervention to prevent the development of persistent cognitive impairments.
Graciela Andonegui, Erin L. Zelinski, Courtney L. Schubert, Derrice Knight, Laura A. Craig, Brent W. Winston, Simon C. Spanswick, Björn Petri, Craig N. Jenne, Janice C. Sutherland, Rita Nguyen, Natalie Jayawardena, Margaret M. Kelly, Christopher J. Doig, Robert J. Sutherland, Paul Kubes
Fibrosis is the common final pathway of virtually all chronic injury to the kidney. While it is well accepted that myofibroblasts are the scar-producing cells in the kidney, their cellular origin is still hotly debated. The relative contribution of proximal tubular epithelium and circulating cells, including mesenchymal stem cells, macrophages, and fibrocytes, to the myofibroblast pool remains highly controversial. Using inducible genetic fate tracing of proximal tubular epithelium, we confirm that the proximal tubule does not contribute to the myofibroblast pool. However, in parabiosis models in which one parabiont is genetically labeled and the other is unlabeled and undergoes kidney fibrosis, we demonstrate that a small fraction of genetically labeled renal myofibroblasts derive from the circulation. Single-cell RNA sequencing confirms this finding but indicates that these cells are circulating monocytes, express few extracellular matrix or other myofibroblast genes, and express many proinflammatory cytokines. We conclude that this small circulating myofibroblast progenitor population contributes to renal fibrosis by paracrine rather than direct mechanisms.
Rafael Kramann, Flavia Machado, Haojia Wu, Tetsuro Kusaba, Konrad Hoeft, Rebekka K. Schneider, Benjamin D. Humphreys
BACKGROUND. There are very few studies investigating metabolic biomarkers to predict acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Metabolic models can provide a framework for analyzing the information-rich omics data sets in this setting. METHODS. Four hundred and fifty-six samples from one hundred and fourteen consecutive patients who underwent HSCT from January 2012 to May 2014 were collected for this study. The changes in serum metabolite levels were investigated using a gas chromatography–mass spectrometry–based metabolomics approach and underwent statistical analysis. RESULTS. Significant metabolic changes were observed on day 7. The stearic acid/palmitic acid (SA/PA) ratio was effective in the diagnosis of grade II–IV aGVHD. Multivariate analysis showed that patients with high SA/PA ratios on day 7 after HSCT were less likely to develop II–IV aGVHD than patients with low SA/PA ratios (odds ratio [OR] = 0.06, 95% CI 0.02–0.18, P < 0.001). After the adjustment for clinical characteristics, the SA/PA ratio had no significant effect on overall survival (hazard ratio [HR] = 1.95, 95% CI 0.92–4.14, P = 0.08), and patients in the high SA/PA ratio group were significantly more likely to relapse than those in the low ratio group (HR = 2.26, 95% CI 1.04–4.91, P = 0.04). CONCLUSION. Our findings suggest that the SA/PA ratio on day 7 after HSCT is an excellent biomarker to predict both aGVHD and relapse. The serum SA/PA ratio measured on day 7 after transplantation may improve risk stratification for aGVHD and relapse after allogeneic stem cell transplantation. FUNDING. National Natural Science Foundation of China (81470346, 81773361), Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Natural Science Foundation (BK20161204), Innovation Capability Development Project of Jiangsu Province (BM2015004), Jiangsu Medical Junior Talent Person award (QNRC2016707), and NIH (AI129582 and NS106170).
Xiaojin Wu, Yiyu Xie, Chang Wang, Yue Han, Xiebing Bao, Shoubao Ma, Ahmet Yilmaz, Bingyu Yang, Yuhan Ji, Jinge Xu, Hong Liu, Suning Chen, Jianying Zhang, Jianhua Yu, Depei Wu