BACKGROUND. COVID-19 patients develop pneumonia generally associated to lymphopenia and severe inflammatory response due to uncontrolled cytokine release. These mediators are transcriptionally regulated by the JAK-STAT signaling pathways, which can be disabled by small molecules. METHODS. A group of subjects (n = 20) was treated with baricitinib according to an off-label use of the drug. The study was designed as an observational longitudinal trial and approved by the local ethical committee. The patients were treated with baricitinib 4 mg twice daily for 2 days, followed by 4 mg per day for the remaining 7 days. Changes in the immune phenotype and expression of pSTAT3 in blood cells were evaluated and correlated with serum-derived cytokine levels and antibodies anti-SARS-CoV-2. In a single treated patient, we evaluated also the alteration of myeloid cell functional activity. RESULTS. We provided evidences that baricitinib-treated patients have a marked reduction in serum levels of interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α, a rapid recovery in circulating T and B cell frequencies, and increased antibody production against SARS-CoV-2 spike protein, which were clinically associated with a reduction in oxygen flow need and progressive increase in the P/F. CONCLUSION. Baricitinib prevented the progression towards a severe/extreme form of the viral disease by modulating the patients’ immune landscape and these changes were associated with a safer and favorable clinical outcome of patients with COVID-19 pneumonia. TRIAL REGISTRATION. The ClinicalTrials.gov identifier of this project is protocol NCT04438629. FUNDING. This work was supported by Fondazione Cariverona (ENACT Project) and Fondazione TIM.
Vincenzo Bronte, Stefano Ugel, Elisa Tinazzi, Antonio Vella, Francesco De Sanctis, Stefania Canè, Veronica Batani, Rosalinda Trovato, Alessandra Fiore, Varvara Petrova, Francesca Hofer, Roza Maria Barouni, Chiara Musiu, Simone Caligola, Laura Pinton, Lorena Torroni, Enrico Polati, Katia Donadello, Simonetta Friso, Francesca Pizzolo, Manuela Iezzi, Federica Facciotti, Pier Giuseppe Pelicci, Daniela Righetti, Paolo Bazzoni, Mariaelisa Rampudda, Andrea C. Comel, Walter Mosaner, Claudio Lunardi, Oliviero Olivieri
Background:While mitochondria play an important role in innate immunity, the relationship between mitochondrial dysfunction and inflammation in heart failure (HF) is poorly understood. In this study we aimed to investigate the mechanistic link between mitochondrial dysfunction and inflammatory activation in peripheral blood mononuclear cells (PBMCs), and the potential anti-inflammatory effect of boosting NAD level.Methods:We compared the PBMC mitochondrial respiration of 19 hospitalized Stage D HF patients with 19 healthy participants. We then created an in vitro model of sterile inflammation by treating healthy PBMC with MitoDAMP (Mitochondrial Damage-Associated Molecular Patterns) isolated from human heart tissue. Lastly, we enrolled Stage D HF patients and sampled their blood before and after taking 5-9 days of oral nicotinamide riboside, an NAD precursor.Results:We demonstrated that HF is associated with both reduced respiratory capacity and elevated proinflammatory cytokine gene expressions. In our in vitro model, MitoDAMP-treated PBMCs secreted IL-6 that impaired mitochondrial respiration by reducing Complex I activity. Last, oral NR administration enhanced PBMC respiration and reduced proinflammatory cytokine gene expression in 4 HF subjects.Conclusion:These findings suggest that systemic inflammation in HF patients is causally linked to mitochondrial function of the PBMC. Increasing NAD levels may have the potential to improve mitochondrial respiration and attenuate proinflammatory activation of PBMC in HF. FundingThis study is funded by NIH R21 HL126209 (to RT and KO), NIH R01 HL144937 (to KO and RT) and University of Washington ITHS Catalyst Award (to DDW). Both BZ (18POST33990352) and DDW (18POST34030098) are funded by the AHA Postdoctoral Fellowships.
Bo Zhou, Dennis D. Wang, Yanhua Qiu, Sophia Airhart, Yaxin Liu, April Stempien-Otero, Kevin D. O’Brien, Rong Tian.
Background: The recent failure of checkpoint-blockade therapies for glioblastoma multiforme (GBM) in late-phase clinical trials has directed interest towards adoptive cellular immunotherapies (ACT). In this open-label, first-in-human trial, we have assessed the safety and therapeutic potential of cytomegalovirus (CMV)-specific ACT in an adjuvant setting for patients with primary GBM, with an ultimate goal to prevent or delay recurrence and prolong overall survival. Methods: Twenty-eight patients with primary GBM were recruited to this prospective study, 25 of whom were treated with in vitro-expanded autologous CMV-specific T cells. Participants were monitored for safety, progression-free survival (PFS), overall survival (OS) and immune reconstitution. Results: No participants showed evidence of ACT-related toxicities. Of 25 evaluable participants, ten were alive at the completion of follow-up, while five were disease free. Reconstitution of CMV-specific T-cell immunity was evident and CMV-specific ACT may trigger bystander effect leading to additional T-cell responses to non-viral tumour-associated antigens through epitope spreading. Long-term follow-up of participants treated before recurrence showed significantly improved OS when compared to those who progressed before ACT (median 23 months, range 7–65 vs. median 14 months, range 5–19; p=0.018). Gene expression analysis of the ACT products indicated that a favourable T-cell gene signature was associated with improved long-term survival. Conclusion: Data presented in this study demonstrate that CMV-specific ACT can be safely used as an adjuvant therapy for primary GBM and, if offered before recurrence, this therapy may improve overall survival of GBM patients.Trial registration: anzctr.org.au: ACTRN12615000656538Funding Source:National Health & Medical Research Council (Australia) Trial registration: anzctr.org.au: ACTRN12615000656538 Funding Source: Philanthropic funding &National Health & Medical Research Council (Australia)
Corey Smith, Katie E. Lineburg, J. Paulo Martins, George Ambalathingal, Michelle A. Neller, Beth Morrison, Katherine K. Matthews, Sweera Rehan, Pauline Crooks, Archana Panikkar, Leone Beagley, Laetitia Le Texier, Sriganesh Srihari, David Walker, Rajiv Khanna
Background. Chimeric antigen receptor (CAR) T cell immunotherapy has achieved complete remission and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Methods. We reported the early results of a phase I/II trial in B-cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine induced killer cells (CIK). Results. The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3+ lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were two grade I and a grade II cytokine release syndrome (CRS) cases at the highest dose, in the absence of graft-versus-host disease (GvHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients, receiving the highest doses, achieved CR and CRi at day 28. Five out of 6 patients in CR were also minimal residual disease (MRD)-negative. Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. Conclusion. SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Anti-leukemic activity was achieved without severe toxicities. Trial registration. clinicaltrials.gov NCT03389035.Funding. This study was supported by grants from AIRC; CRUK; FC AECC; Ministero della salute; FRRB.
Chiara F. Magnani, Giuseppe Gaipa, Federico Lussana, Daniela Belotti, Giuseppe Gritti, Sara Napolitano, Giada Matera, Benedetta Cabiati, Chiara Buracchi, Gianmaria Borleri, Grazia Fazio, Silvia Zaninelli, Sarah Tettamanti, Stefania Cesana, Valentina Colombo, Michele Quaroni, Giovanni Cazzaniga, Attilio Rovelli, Ettore Biagi, Stefania Galimberti, Andrea Calabria, Fabrizio Benedicenti, Eugenio Montini, Silvia Ferrari, Martino Introna, Adriana Balduzzi, Maria Grazia Valsecchi, Giuseppe Dastoli, Alessandro Rambaldi, Andrea Biondi
Background: Initial reports from the Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) pandemic described children as being less susceptible to Coronavirus Disease 2019 (COVID-19) than adults. Subsequently, a severe and novel pediatric disorder termed Multisystem Inflammatory Syndrome in Children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology. Methods: We prospectively enrolled hospitalized patients with evidence of SARS-CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data. Twenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8 and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. Cts and burr cells on blood smears also differentiated between patients with severe COVID-19 and those with MIS-C. Conclusion: Pediatric patients with SARS-CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and severe COVID-19.
Caroline Diorio, Sarah E. Henrickson, Laura A. Vella, Kevin O. McNerney, Julie M. Chase, Chakkapong Burudpakdee, Jessica H. Lee, Cristina Jasen, Fran Balamuth, David M. Barrett, Brenda Banwell, Kathrin M. Bernt, Allison M. Blatz, Kathleen Chiotos, Brian T. Fisher, Julie C. Fitzgerald, Jeffrey S. Gerber, Kandace Gollomp, Christopher Gray, Stephan A. Grupp, Rebecca M. Harris, Todd J. Kilbaugh, Audrey R. Odom John, Michele P. Lambert, Emily J. Liebling, Michele Paessler, Whitney Petrosa, Charles A. Phillips, Anne F. Reilly, Neil Romberg, Alix E. Seif, Deborah Sesok-Pizzini, Kathleen Sullivan, Julie Vardaro, Edward M Behrens, David T. Teachey, Hamid Bassiri
Background: Pediatric SARS-CoV-2 infection can be complicated by a dangerous hyperinflammatory condition termed multisystem inflammatory syndrome in children (MIS-C). The clinical and immunologic spectrum of MIS-C and its relationship to other inflammatory conditions of childhood have not been studied in detail. Methods: We retrospectively studied confirmed cases of MIS-C at our institution from March to June 2020. The clinical characteristics, laboratory studies and treatment response were collected. Data were compared with historic cohorts of Kawasaki disease (KD) and macrophage activation syndrome (MAS). Results: Twenty-eight patients fulfilled the case definition of MIS-C. Median age at presentation was 9 years (range 1 month to 17 years); 50% of patients had pre-existing conditions. All patients had laboratory confirmation of SARS-CoV-2 infection. Seventeen patients (61%) required intensive care, including 7 patients (25%) requiring inotrope support. Seven patients (25%) met criteria for complete or incomplete KD and coronary abnormalities were found in 6 cases. Lymphopenia, thrombocytopenia, and elevation in inflammatory markers, D-dimer, B-type natriuretic peptide, IL-6 and IL-10 levels were common but not ubiquitous. Cytopenias distinguished MIS-C from KD and the degree of hyperferritinemia and pattern of cytokine production differed between MIS-C and MAS. Immunomodulatory therapy given to MIS-C patients included IVIG (71%), corticosteroids (61%) and anakinra (18%). Clinical and laboratory improvement were observed in all cases, including 6 cases that did not require immunomodulatory therapy. No mortality was recorded in this cohort. Conclusion: MIS-C encompasses a broad phenotypic spectrum with clinical and laboratory features distinct from Kawasaki disease and macrophage activation syndrome. Funding: This work was supported by the National Institute of Health / National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) K08-AR074562 (PYL), K08-AR AR073339 (LAH), R01-AR065538, R01-AR073201 and P30-AR070253 (PAN); National Institute of Allergy and Infectious Diseases 5T32AI007512-34 (JL, JR, TB, AAN and RWN); Rheumatology Research Foundation Investigator Awards (PYL and LAH) and Medical Education Award (JSH); Boston Children’s Hospital Faculty Career Development Awards (PYL and LAH), the McCance Family Foundation (JWN), and the Samara Jan Turkel Center (JC, RPS, MBS).
Pui Y. Lee, Megan Day-Lewis, Lauren A. Henderson, Kevin Friedman, Jeffrey Lo, Jordan E. Roberts, Mindy S. Lo, Craig D. Platt, Janet Chou, Kacie J. Hoyt, Annette L. Baker, Tina Banzon, Margaret H. Chang, Ezra Cohen, Sarah de Ferranti, Audrey Dionne, Saddiq Habiballah, Olha Halyabar, Jonathan S. Hausmann, Melissa Hazen, Erin Janssen, Esra Meidan, Ryan W. Nelson, Alan A. Nguyen, Robert P. Sundel, Fatma Dedeoglu, Peter A. Nigrovic, Jane W. Newburger, Mary Beth F. Son
BACKGROUND. The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) reduces overall infant mortality. Induction of innate immune memory, also termed trained immunity, contributes towards protection against heterologous infections. Since immune cells display oscillations in numbers and function throughout the day, we investigated the effect of BCG administration time on the induction of trained immunity. METHODS. 18 volunteers were vaccinated with BCG at 6pm and compared with 36 age- and sex-matched volunteers vaccinated between 8-9 am. Peripheral blood mononuclear cells were stimulated with Staphylococcus aureus and Mycobacterium tuberculosis before, as well as two weeks and three months after BCG vaccination. Cytokine production was measured to assess the induction of trained immunity and adaptive responses, respectively. Additionally, the influence of vaccination time on induction of trained immunity was studied in an independent cohort of 302 individuals vaccinated between 8am-12pm with BCG. RESULTS. Compared to evening vaccination, morning vaccination elicited both a stronger trained immunity and adaptive immune phenotype. In a large cohort of 302 volunteers, early morning vaccination resulted in a superior cytokine production capacity compared with later morning. A cellular, rather than soluble, substrate of the circadian effect of BCG vaccination was demonstrated by the enhanced capacity to induce trained immunity in vitro in morning compared to evening isolated monocytes. CONCLUSIONS. BCG vaccination in the morning induces stronger trained immunity and adaptive responses compared to evening vaccination. Future studies should take vaccine administration time into account when studying specific and non-specific effects of vaccines: early morning should be the preferred moment of BCG administration. FUNDING Spinoza grant of the Netherlands Organization for Scientific Research, ERC Advanced Grant (TRAIN-OLD nr. 833247), Danish National Research Foundation (DNRF108).
L. Charlotte J. de Bree, Vera P. Mourits, Valerie A.C.M. Koeken, Simone J.C.F.M. Moorlag, Robine Janssen, Lukas Folkman, Daniele Barreca, Thomas Krausgruber, Victoria Fife-Gernedl, Boris Novakovic, Rob J.W. Arts, Helga Dijkstra, Heidi Lemmers, Christoph Bock, Leo A.B. Joosten, Reinout van Crevel, Christine S. Benn, Mihai G. Netea
Background: Inadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden and accurate point-of-care tests (POCT) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB LAM (FujiLAM) for TB diagnosis in HIV-negative outpatients compared to Alere Determine TB LAM Ag (AlereLAM) and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM). Methods: In this multicentre diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient healthcare centres in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM and EclLAM and the diagnostic accuracy was assessed against microbiological (MRS) and composite reference standards. Results: 372 HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared to the MRS, the sensitivities of AlereLAM, FujiLAM and EclLAM were 10.8% (95% CI 6.3to18.0), 53.2% (43.9to62.1), and 66.7% (57.5to74.7) respectively. The specificities of AlereLAM, FujiLAM and EclLAM were 92.3% (88.5to95.0), 98.9% (96.7to99.6), and 98.1% (95.6to99.2) respectively. Positive Likelihood Ratio of AlereLAM, FujiLAM and EclLAM were 1.4, 46.2, and 34.8 and positive predictive values 37.5%, 95.2%, and 93.7% respectively. Conclusion: Compared to AlereLAM, FujiLAM detected five times more TB patients in HIV-negative participants, has a high positive predictive value and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAMs potential as a biomarker. Additional research is required to assess FujiLAMs performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings.
Tobias Broger, Mark Nicol, George Sigal, Eduardo Gotuzzo, Alexandra J. Zimmer, Shireen Surtie, Tatiana Caceres-Nakiche, Anna Mantsoki, Elena Ivanova Reipold, Rita Székely, Michael Tsionsky, Judith van Heerden, Tatiana Plisova, Kinuyo Chikamatsu, Todd L. Lowary, Abraham Pinter, Satoshi Mitarai, Emmanuel Moreau, Samuel G Schumacher, Claudia M. Denkinger
Background. Induction of innate immune memory, also termed trained immunity, by the anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) contributes to protection against heterologous infections. However, the overall impact of BCG vaccination on the inflammatory status of an individual is not known: while induction of trained immunity may suggest increased inflammation, BCG vaccination has been epidemiologically associated with a reduced incidence of inflammatory and allergic diseases. Methods. We investigated the impact of BCG (BCG-Bulgaria, InterVax) vaccination on systemic inflammation in a cohort of 303 healthy volunteers, as well as the effect of the inflammatory status on the response to vaccination. A targeted proteome platform was used to measure circulating inflammatory proteins before and after BCG vaccination, while ex vivo Mycobacterium tuberculosis and Staphylococcus aureus induced cytokine responses in peripheral blood mononuclear cells were used to assess trained immunity. Results. While BCG vaccination enhanced cytokine responses to restimulation, it reduced systemic inflammation. This effect was validated in three smaller cohorts, and was much stronger in men than in women. In addition, baseline circulating inflammatory markers were associated with ex vivo cytokine responses (trained immunity) after BCG vaccination. Conclusion. The capacity of BCG to enhance microbial responsiveness while dampening systemic inflammation should be further explored for potential therapeutic applications. Funding. This study was funded by a Spinoza grant of the Netherlands Organization for Scientific Research and an ERC Advanced Grant (TRAIN-OLD nr. 833247).
Valerie A. C. M. Koeken, L. Charlotte J. de Bree, Vera P. Mourits, Simone J.C.F.M. Moorlag, Jona Walk, Branko Cirovic, Rob J.W. Arts, Martin Jaeger, Helga Dijkstra, Heidi Lemmers, Leo A.B. Joosten, Christine Stabell Benn, Reinout van Crevel, Mihai Netea
BACKGROUND. Idiopathic CD4 lymphopenia (ICL) is defined by persistent low CD4 counts (<300 cells/µL) in the absence of a causal infection or immune deficiency and can manifest with opportunistic infections. Approximately 30% of ICL patients develop autoimmune disease. The prevalence and breadth of their autoantibodies, however, and their potential contribution to pathogenesis of ICL remain unclear. METHODS. We hybridized 35 and 51 ICL patients’ sera to a 9,000 human proteome array and to a 128 known autoantigens array, respectively. Using a flow-based method, we characterized the presence of anti-lymphocyte Ab in the whole cohort of 72 patients, as well as the Ab functional capability of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), complement deposition, and complement dependent cytotoxicity (CDC). We tested ex vivo the activation of the classical complement pathway on ICL CD4 T cells. RESULTS. All ICL patients had multitude of autoantibodies mostly directed against private (not shared) targets and unrelated quantitatively or qualitatively to the patients’ autoimmune disease status. The targets included lymphocyte intracellular and membrane antigens, confirmed by the detection by flow of IgM and IgG (mostly IgG1 and IgG4) anti-CD4 cell Ab in 50% of the patients with half of these cases triggering lysis of CD4 T cells. We also detected in vivo classical complement activation on CD4 T cells in 14% of the whole cohort. CONCLUSION. Our data demonstrate a high prevalence of autoantibodies in ICL, some of which are specific against CD4 T cells, may contribute to pathogenesis and may represent a potential novel therapeutic target.
Ainhoa Perez-Diez, Chun-Shu Wong, Xiangdong Liu, Harry A. Mystakelis, Jian song, Yong Lu, Virginia Sheikh, Jeffrey S. Bourgeois, Andrea Lisco, Elizabeth Laidlaw, Cornelia D. Cudrici, Chengsong Zhu, Quan-Zhen Li, Alexandra F. Freeman, Peter R. Williamson, Megan V. Anderson, Gregg Roby, John S. Tsang, Richard M. Siegel, Irini Sereti
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