BACKGROUND. Undifferentiated systemic autoinflammatory diseases (USAID) present diagnostic and therapeutic challenges. Chronic interferon (IFN) signaling and cytokine dysregulation may identify diseases with available targeted treatments. METHODS. Sixty-six consecutively-referred USAID patients underwent standardized evaluation of Type-I IFN-response-gene-signature (IRG-S); cytokine profiling, and genetic evaluation by next-generation sequencing. RESULTS. Thirty-six USAID patients (55%) had elevated IRG-S. Neutrophilic panniculitis (40% vs 0%), basal ganglia calcifications (46% vs 0%), interstitial lung disease (47% vs 5%), and myositis (60% vs 10%) were more prevalent in patients with elevated IRG-S. Moderate IRG-S elevation and highly-elevated serum IL-18 distinguished eight patients with pulmonary alveolar proteinosis (PAP) and recurrent macrophage activation syndrome (MAS). Among patients with panniculitis and progressive cytopenias, two patients were compound heterozygous for novel LRBA mutations, four patients harbored novel splice variants in IKBKG/NEMO, and six patients had de novo frameshift mutations in SAMD9L. Of additional 12 patients with elevated IRG-S and CANDLE-, SAVI- or Aicardi-Goutières-Syndrome (AGS)-like phenotypes, five patients carried mutations in either SAMHD1, TREX1, PSMB8 or PSMG2. Two patients had anti-MDA5 autoantibody-positive juvenile dermatomyositis, and seven could not be classified. Patients with LRBA, IKBKG/NEMO and SAMD9L mutations showed a pattern of IRG elevation that suggests prominent NF-κB activation different from the canonical interferonopathies CANDLE, SAVI and AGS. CONCLUSIONS. In patients with elevated IRG-S, we identified characteristic clinical features and 3 additional autoinflammatory diseases: IL-18-mediated PAP and recurrent MAS (IL-18PAP-MAS), NEMO∆5-associated autoinflammatory syndrome (NEMO-NDAS), and SAMD9L-associated autoinflammatory disease (SAMD9L-SAAD). The IRG-S expands the diagnostic armamentarium in evaluating USAIDs and points to different pathways regulating IRG expression.
Adriana A. de Jesus, Yanfeng Hou, Stephen Brooks, Louise Malle, Angelique Biancotto, Yan Huang, Katherine R. Calvo, Bernadette Marrero, Susan Moir, Andrew J. Oler, Zuoming Deng, Gina A. Montealegre Sanchez, Amina Ahmed, Eric Allenspach, Bita Arabshahi, Edward Behrens, Susanne Benseler, Liliana Bezrodnik, Sharon Bout-Tabaku, AnneMarie C. Brescia, Diane Brown, Jon M. Burnham, María Soledad Caldirola, Ruy Carrasco, Alice Y. Chan, Rolando Cimaz, Paul Dancey, Jason Dare, Marietta DeGuzman, Victoria Dimitriades, Ian Ferguson, Polly Ferguson, Laura Finn, Marco Gattorno, Alexei A. Grom, Eric P. Hanson, Philip J. Hashkes, Christian M. Hedrich, Ronit Herzog, Gerd Horneff, Rita Jerath, Elizabeth Kessler, Hanna Kim, Daniel J. Kingsbury, Ronald M. Laxer, Pui Y. Lee, Min Ae Lee-Kirsch, Laura Lewandowski, Suzanne Li, Vibke Lilleby, Vafa Mammadova, Lakshmi N. Moorthy, Gulnara Nasrullayeva, Kathleen M. O’Neil, Karen Onel, Seza Ozen, Nancy Pan, Pascal Pillet, Daniela G.P. Piotto, Marilynn G. Punaro, Andreas Reiff, Adam Reinhardt, Lisa G. Rider, Rafael Rivas-Chacon, Tova Ronis, Angela Rösen-Wolff, Johannes Roth, Natasha Mckerran Ruth, Marite Rygg, Heinrike Schmeling, Grant Schulert, Christiaan Scott, Gisela Seminario, Andrew Shulman, Vidya Sivaraman, Mary Beth Son, Yuriy Stepanovskyy, Elizabeth Stringer, Sara Taber, Maria Teresa Terreri, Cynthia Tifft, Troy Torgerson, Laura Tosi, Annet Van Royen-Kerkhof, Theresa Wampler Muskardin, Scott W. Canna, Raphaela Goldbach-Mansky
BACKGROUND. Cerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics. METHODS. Using multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or –), iRBC sequestration (Seqhi, lo, or 0) and HIV status (HIV+ or –). RESULTS. We identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV– children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ vs –, P = 0.004) and sequestration level (Seqhi > lo, P = 0.010). HIV co-infection significantly increased T cell numbers and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution. CONCLUSION. Within the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease. FUNDING. This research was supported by the Intramural Research Program of the National Institutes of Health.
Brittany A. Riggle, Monica Manglani, Dragan Maric, Kory R. Johnson, Myoung-Hwa Lee, Osorio Lopes Abath Neto, Terrie E. Taylor, Karl B. Seydel, Avindra Nath, Louis H. Miller, Dorian B. McGavern, Susan K. Pierce
BACKGROUND Respiratory syncytial virus (RSV) is an important cause of acute pulmonary disease and one of the last remaining major infections of childhood for which there is no vaccine. CD4+ T cells play a key role in antiviral immunity, but they have been little studied in the human lung.METHODS Healthy adult volunteers were inoculated i.n. with RSV A Memphis 37. CD4+ T cells in blood and the lower airway were analyzed by flow cytometry and immunohistochemistry. Bronchial soluble mediators were measured using quantitative PCR and MesoScale Discovery. Epitope mapping was performed by IFN-γ ELISpot screening, confirmed by in vitro MHC binding.RESULTS Activated CD4+ T cell frequencies in bronchoalveolar lavage correlated strongly with local C-X-C motif chemokine 10 levels. Thirty-nine epitopes were identified, predominantly toward the 3′ end of the viral genome. Five novel MHC II tetramers were made using an immunodominant EFYQSTCSAVSKGYL (F-EFY) epitope restricted to HLA-DR4, -DR9, and -DR11 (combined allelic frequency: 15% in Europeans) and G-DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labeling revealed enrichment of resident memory CD4+ T (Trm) cells in the lower airway; these Trm cells displayed progressive differentiation, downregulation of costimulatory molecules, and elevated CXCR3 expression as infection evolved.CONCLUSIONS Human infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of Trm recognizing novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific CD4+ cells, potentially accelerating the development of effective vaccines.TRIAL REGISTRATION ClinicalTrials.gov NCT02755948.FUNDING Medical Research Council, Wellcome Trust, National Institute for Health Research.
Aleks Guvenel, Agnieszka Jozwik, Stephanie Ascough, Seng Kuong Ung, Suzanna Paterson, Mohini Kalyan, Zoe Gardener, Emma Bergstrom, Satwik Kar, Maximillian S. Habibi, Allan Paras, Jie Zhu, Mirae Park, Jaideep Dhariwal, Mark Almond, Ernie H.C. Wong, Annemarie Sykes, Jerico Del Rosario, Maria-Belen Trujillo-Torralbo, Patrick Mallia, John Sidney, Bjoern Peters, Onn Min Kon, Alessandro Sette, Sebastian L. Johnston, Peter J. Openshaw, Christopher Chiu
Background: DICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist. Methods: Whole exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome wide analyses were performed on 13 tissue samples from familial and non-familial DGCR8-E518K positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTC) and Wilms Tumors. MiRNA profiles of four tissue types were compared, and sequencing of miRNA, pre-miRNA and mRNA was performed in a subset of 9 schwannomas, four of which harbor DGCR8-E518K. Results: We identified c.1552G>A;p.E518K in DGCR8, a microprocessor located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms Tumors and has been identified in two PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTC, MNG, schwannomas and Wilms Tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons. Conclusions: We identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis. Funded by CIHR, Compute Canada, Alex’s Lemonade Stand Foundation, and the Mia Neri Foundation for Childhood Cancer.
Barbara Rivera, Javad Nadaf, Somayyeh Fahiminiya, Maria Apellaniz-Ruiz, Avi Saskin, Anne-Sophie Chong, Sahil Sharma, Rabea Wagener, Timothée Revil, Vincenzo Condello, Zineb Harra, Nancy Hamel, Nelly Sabbaghian, Karl Muchantef, Christian Thomas, Leanne de Kock, Marie-Noëlle Hébert-Blouin, Angelia V. Bassenden, Hannah Rabenstein, Ozgur Mete, Ralf Paschke, Marc P. Pusztaszeri, Werner Paulus, Albert Berghuis, Jiannis Ragoussis, Yuri E. Nikiforov, Reiner Siebert, Steffen Albrecht, Robert Turcotte, Martin Hasselblatt, Marc R. Fabian, William D. Foulkes
Background. An increase in intrahepatic triglyceride (IHTG) is the hallmark feature of nonalcoholic fatty liver disease (NAFLD) and is decreased by weight loss. Hepatic de novo lipogenesis (DNL) contributes to steatosis in people with NAFLD. The physiological factors that stimulate hepatic DNL and the effect of weight loss on hepatic DNL are not clear.Methods. Hepatic DNL, 24-h integrated plasma insulin and glucose concentrations, and both liver and whole-body insulin sensitivity were determined in people who were lean (n = 14), obese with normal IHTG content (Obese, n = 26) and obese with NAFLD (Obese-NAFLD, n = 27). Hepatic DNL was assessed by using the deuterated water method corrected for the potential confounding contribution of adipose tissue DNL. Liver and whole-body insulin sensitivity were assessed by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with glucose tracer infusion. Six subjects in the Obese-NAFLD group were also evaluated before and after 10% diet-induced weight loss.Results. The contribution of hepatic DNL to IHTG-palmitate was 11%, 19% and 38% in the Lean, Obese and Obese-NAFLD groups, respectively. Hepatic DNL was inversely correlated with hepatic and whole-body insulin sensitivity, but directly correlated with 24-h plasma glucose and insulin concentrations. Weight loss decreased IHTG content, in conjunction with a decrease in hepatic DNL and 24-h plasma glucose and insulin concentrations. Conclusions. These data suggest hepatic DNL is an important regulator of IHTG content, and that increases in circulating glucose and insulin stimulate hepatic DNL in people with NAFLD. Weight loss decreases IHTG content, at least in part, by decreasing hepatic DNL.
Gordon I. Smith, Mahalakshmi Shankaran, Mihoko Yoshino, George G. Schweitzer, Maria Chondronikola, Joseph W. Beals, Adewole L. Okunade, Bruce W. Patterson, Edna Nyangau, Tyler Field, Claude B. Sirlin, Saswata Talukdar, Marc K. Hellerstein, Samuel Klein
Background: In retinitis pigmentosa (RP) rod photoreceptors degenerate from one of many mutations after which cones are compromised by oxidative stress. N-acetylcysteine (NAC) reduces oxidative damage and increases cone function/survival in RP models. We tested the safety, tolerability, and visual function effects of oral NAC in RP patients. Methods: Subjects (n = 10 per cohort) received 600 mg (cohort 1), 1200 mg (cohort 2), or 1800 mg (cohort 3) NAC BID for 12 weeks and then TID for 12 weeks. Best-corrected visual acuity (BCVA), macular sensitivity, ellipsoid zone (EZ) width, and aqueous NAC were measured. Linear mixed effects models were used to estimate the rates of changes during the treatment period. Results: There were 9 drug-related gastrointestinal adverse events which resolved spontaneously or with dose reduction (MTD 1800 mg bid). During the 24 week treatment period, mean BCVA significantly improved at 0.4 (95% CI 0.2–0.6, P < 0.001), 0.5 (95% CI 0.3–0.7, P < 0.001) and 0.2 (95% CI 0.02–0.4, P = 0.03) letters/month in cohorts 1, 2 and 3, respectively. There was no significant improvement in mean sensitivity (MS) over time in cohorts 1 and 2, but there was in cohort 3 (0.15 dB/month, 95%CI 0.04–0.26). There was no significant change in mean EZ width in any cohort. Conclusion: Oral NAC is safe and well-tolerated in patients with moderately advanced RP and may improve suboptimally functioning macular cones. A randomized, placebo-controlled trial is needed to determine if oral NAC can provide long term stabilization and/or improvement in visual function in patients with RP.
Peter A. Campochiaro, Mustafa Iftikhar, Gulnar Hafiz, Anam Akhlaq, Grace Tsai, Dagmar Wehling, Lili Lu, G. Michael Wall, Mandeep S. Singh, Xiangrong Kong
Background: Ceramides are sphingolipids that play causative roles in diabetes and heart disease, with their serum levels measured clinically as biomarkers of cardiovascular disease (CVD). Methods: We performed targeted lipidomics on serum samples of individuals with familial coronary artery disease (CAD) (n = 462) and population-based controls (n = 212) to explore the relationship between serum sphingolipids and CAD, employing unbiased machine learning to identify sphingolipid species positively associated with CAD. Results: Nearly every sphingolipid measured (n = 30 of 32) was significantly elevated in subjects with CAD compared with population controls. We generated a novel Sphingolipid Inclusive CAD risk score, termed SIC, that demarcates CAD patients independently and more effectively than conventional clinical CVD biomarkers including LDL-cholesterol and serum triglycerides. This new metric comprises several minor lipids which likely serve as measures of flux through the ceramide biosynthesis pathway, rather than the abundant deleterious ceramide species that are incorporated in other ceramide-based scores. Conclusion: This study validates serum ceramides as candidate biomarkers of cardiovascular disease and suggests that comprehensive sphingolipid panels be considered as measures of CVD.
Annelise M. Poss, J. Alan Maschek, James E. Cox, Benedikt J. Hauner, Paul N. Hopkins, Steven C. Hunt, William L. Holland, Scott A. Summers, Mary C. Playdon
Background: Chronic HCV-infection is characterized by a severe impairment of HCV-specific CD4 T cell help that is driven by chronic antigen stimulation. We aimed to study the fate of HCV-specific CD4 T cells after viral elimination. Methods:HCV-specific CD4 T cell responses were longitudinally analyzed using MHC class II tetramer-technology, multicolor flow cytometry and RNA sequencing in a cohort of chronically HCV-infected patients undergoing therapy with direct-acting antivirals. In addition, HCV-specific neutralizing antibodies and CXCL13 levels were analyzed. Results: We observed that the frequency of HCV-specific CD4 T cells increased within two weeks after initiation of DAA therapy. Multicolor flow cytometry revealed a downregulation of exhaustion and activation markers and an upregulation of memory-associated markers. While cells with a Th1 phenotype were the predominant subset at baseline, cells with phenotypic and transcriptional characteristics of follicular T helper cells increasingly shaped the circulating HCV-specific CD4 T cell repertoire, suggesting antigen-independent survival of this subset. These changes were accompanied by a decline of HCV-specific neutralizing antibodies and the germinal center activity. Conclusion: We identified a population of HCV-specific CD4 T cells with a follicular T helper cell signature that is maintained after therapy-induced elimination of persistent infection and may constitute an important target population for vaccination efforts to prevent re-infection and immunotherapeutic approaches for persistent viral infections.
Maike Smits, Katharina Zoldan, Naveed Ishaque, Zuguang Gu, Katharina Jechow, Dominik Wieland, Christian Conrad, Roland Eils, Catherine Fauvelle, Thomas F. Baumert, Florian Emmerich, Bertram Bengsch, Christoph Neumann-Haefelin, Maike Hofmann, Robert Thimme, Tobias Boettler
Background: Adoptive transfer of donor-derived EBV-specific T-cells (EBV-CTLs) can eradicate EBV associated lymphomas post hematopoietic cell (HCT) or solid organ (SOT) transplants but is not available for most patients. Methods: We developed a 3rd-party, allogeneic, off-the-shelf bank of 330 GMP grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (N=33) or SOT (N=13) with established EBV associated lymphomas, who failed rituximab therapy, with 3rd-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation. Results: The EBV-CTLs did not induce significant toxicities or graft injury. One patient developed grade I skin GVHD requiring topical therapy. Complete and sustained partial remissions were achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, overall survival was 88.9% and 81.8% respectively at 1 year. Although only 1/11 patients (9.1%) with progression of disease (POD) after cycle 1 who received additional EBV-CTLs from the same donor survived, 3 of 5 with POD subsequently treated with EBV-CTLs from a different donor achieved CR or durable PR (60%) and survive > 1 year. Maximal responses were achieved after a median of 2 cycles. Conclusions: Third party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab refractory EBV-associated lymphoma post transplant. Phase II protocols (NCT01498484 and NCT00002663) were approved by the Institutional Review Board at Memorial Sloan Kettering Cancer Center, Food and Drug Administration and National Marrow Donor Program. This work was supported through NIH grants CA23766, NIH R21CA162002, Aubrey Fund, The Claire Tow Foundation, Major Family Foundation, Max Cure Foundation, Richard “Rick” J. EIsemann Pediatric Research Fund, Banbury Foundation, Edith Robertson Foundation, Larry Smead Foundation. In June 2015 Atara Biotherapeutics licensed the EBV-CTL bank and is developing this as ATA-129.
Susan Prockop, Ekaterina Doubrovina, Stephanie Suser, Glenn Heller, Juliet Barker, Parastoo Dahi, Miguel A. Perales, Esperanza Papadopoulos, Craig Sauter, Hugo Castro-Malaspina, Farid Boulad, Kevin J. Curran, Sergio Giralt, Boglarka Gyurkocza, Katharine C. Hsu, Ann Jakubowski, Alan M. Hanash, Nancy A. Kernan, Rachel Kobos, Guenther Koehne, Heather Landau, Doris Ponce, Barbara Spitzer, James W. Young, Gerald Behr, Mark Dunphy, Sofia Haque, Julie Teruya-Feldstein, Maria Arcila, Christine Moung, Susan Hsu, Aisha Hasan, Richard J. O'Reilly
Background: Proteinuria is considered as an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear if all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS) featured by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding. Methods: We used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling and epidemiological methods. Results: We identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene are associated with chronic isolated proteinuria with childhood onset. Since the proteinuria displayed a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and proteinuria-lowering treatments. Yet, renal function was normal in all cases. By contrast, we did not find any biallelic pathogenic CUBN variants in patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 out of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that four C-terminal CUBN variants are associated with albuminuria and moderately increased GFR in meta-analyses of large population-based cohorts. Conclusions: Collectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsies.
Mathilda Bedin, Olivia Boyer, Aude Servais, Yong Li, Laure Villoing-Gaudé, Marie-Josephe Tête, Alexandra Cambier, Julien Hogan, Veronique Baudouin, Saoussen Krid, Albert Bensman, Florie Lammens, Ferielle Louillet, Bruno Ranchin, Cecile Vigneau, Iseline Bouteau, Corinne Isnard-Bagnis, Christoph J. Mache, Tobias Schäfer, Lars Pape, Markus Gödel, Tobias B. Huber, Marcus Benz, Günter Klaus, Matthias Hansen, Kay Latta, Olivier Gribouval, Vincent Morinière, Carole Tournant, Maik Grohmann, Elisa Kuhn, Timo Wagner, Christine Bole-Feysot, Fabienne Jabot-Hanin, Patrick Nitschké, Tarunveer S. Ahluwalia, Anna Köttgen, Christian Brix Folsted Andersen, Carsten Bergmann, Corinne Antignac, Matias Simons
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