The 2021 Eurpean Alliance of Associations for Rheumatology/American College of Rheumatology points to consider for diagnosis and management of autoinflammatory type i interferonopathies: CANDLE/PRAAS, SAVI and AGS

Objective: Autoinflammatory type I interferonopathies, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature/proteasome-associated autoinflammatory syndrome (CANDLE/PRAAS), stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) and Aicardi-Goutières syndrome (AGS) are rare and clinically complex immunodysregulatory diseases. With emerging knowledge of genetic causes and targeted treatments, a Task Force was charged with the development of \u27points to consider\u27 to improve diagnosis, treatment and long-term monitoring of patients with these rare diseases. Methods: Members of a Task Force consisting of rheumatologists, neurologists, an immunologist, geneticists, patient advocates and an allied healthcare professional formulated research questions for a systematic literature review. Then, based on literature, Delphi questionnaires and consensus methodology, \u27points to consider\u27 to guide patient management were developed. Results: The Task Force devised consensus and evidence-based guidance of 4 overarching principles and 17 points to consider regarding the diagnosis, treatment and long-term monitoring of patients with the autoinflammatory interferonopathies, CANDLE/PRAAS, SAVI and AGS. Conclusion: These points to consider represent state-of-the-art knowledge to guide diagnostic evaluation, treatment and management of patients with CANDLE/PRAAS, SAVI and AGS and aim to standardise and improve care, quality of life and disease outcomes

The 2021 EULAR and ACR points to consider for diagnosis and management of autoinflammatory type I interferonopathies: CANDLE/PRAAS, SAVI and AGS

Objective: Autoinflammatory type I interferonopathies, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature/proteasome-associated autoinflammatory syndrome (CANDLE/PRAAS), stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) and Aicardi-Goutières syndrome (AGS) are rare and clinically complex immunodysregulatory diseases. With emerging knowledge of genetic causes and targeted treatments, a Task Force was charged with the development of 'points to consider' to improve diagnosis, treatment and long-term monitoring of patients with these rare diseases. Methods: Members of a Task Force consisting of rheumatologists, neurologists, an immunologist, geneticists, patient advocates and an allied healthcare professional formulated research questions for a systematic literature review. Then, based on literature, Delphi questionnaires and consensus methodology, 'points to consider' to guide patient management were developed. Results: The Task Force devised consensus and evidence-based guidance of 4 overarching principles and 17 points to consider regarding the diagnosis, treatment and long-term monitoring of patients with the autoinflammatory interferonopathies, CANDLE/PRAAS, SAVI and AGS. Conclusion: These points to consider represent state-of-the-art knowledge to guide diagnostic evaluation, treatment and management of patients with CANDLE/PRAAS, SAVI and AGS and aim to standardise and improve care, quality of life and disease outcomes

The 2021 EULAR and ACR points to consider for diagnosis and management of autoinflammatory type I interferonopathies: CANDLE/PRAAS, SAVI and AGS

Objective: Autoinflammatory type I interferonopathies, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature/proteasome-associated autoinflammatory syndrome (CANDLE/PRAAS), stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) and Aicardi-Goutières syndrome (AGS) are rare and clinically complex immunodysregulatory diseases. With emerging knowledge of genetic causes and targeted treatments, a Task Force was charged with the development of 'points to consider' to improve diagnosis, treatment and long-term monitoring of patients with these rare diseases. Methods: Members of a Task Force consisting of rheumatologists, neurologists, an immunologist, geneticists, patient advocates and an allied healthcare professional formulated research questions for a systematic literature review. Then, based on literature, Delphi questionnaires and consensus methodology, 'points to consider' to guide patient management were developed. Results: The Task Force devised consensus and evidence-based guidance of 4 overarching principles and 17 points to consider regarding the diagnosis, treatment and long-term monitoring of patients with the autoinflammatory interferonopathies, CANDLE/PRAAS, SAVI and AGS. Conclusion: These points to consider represent state-of-the-art knowledge to guide diagnostic evaluation, treatment and management of patients with CANDLE/PRAAS, SAVI and AGS and aim to standardise and improve care, quality of life and disease outcomes

Genetic and phenotypic spectrum associated with IFIH1 gain-of-function

IFIH1 gain-of-function has been reported as a cause of a type I interferonopathy encompassing a spectrum of autoinflammatory phenotypes including Aicardi–Goutières syndrome and Singleton Merten syndrome. Ascertaining patients through a European and North American collaboration, we set out to describe the molecular, clinical and interferon status of a cohort of individuals with pathogenic heterozygous mutations in IFIH1. We identified 74 individuals from 51 families segregating a total of 27 likely pathogenic mutations in IFIH1. Ten adult individuals, 13.5% of all mutation carriers, were clinically asymptomatic (with seven of these aged over 50 years). All mutations were associated with enhanced type I interferon signaling, including six variants (22%) which were predicted as benign according to multiple in silico pathogenicity programs. The identified mutations cluster close to the ATP binding region of the protein. These data confirm variable expression and nonpenetrance as important characteristics of the IFIH1 genotype, a consistent association with enhanced type I interferon signaling, and a common mutational mechanism involving increased RNA binding affinity or decreased efficiency of ATP hydrolysis and filament disassembly rate

Cross-sectional examination of 24-hour movement behaviours among 3-and 4-year-old children in urban and rural settings in low-income, middle-income and high-income countries : the SUNRISE study protocol

Introduction 24-hour movement behaviours (physical activity, sedentary behaviour and sleep) during the early years are associated with health and developmental outcomes, prompting the WHO to develop Global guidelines for physical activity, sedentary behaviour and sleep for children under 5 years of age. Prevalence data on 24-hour movement behaviours is lacking, particularly in low-income and middle-income countries (LMICs). This paper describes the development of the SUNRISE International Study of Movement Behaviours in the Early Years protocol, designed to address this gap. Methods and analysis SUNRISE is the first international cross-sectional study that aims to determine the proportion of 3- and 4-year-old children who meet the WHO Global guidelines. The study will assess if proportions differ by gender, urban/rural location and/or socioeconomic status. Executive function, motor skills and adiposity will be assessed and potential correlates of 24-hour movement behaviours examined. Pilot research from 24 countries (14 LMICs) informed the study design and protocol. Data are collected locally by research staff from partnering institutions who are trained throughout the research process. Piloting of all measures to determine protocol acceptability and feasibility was interrupted by COVID-19 but is nearing completion. At the time of publication 41 countries are participating in the SUNRISE study. Ethics and dissemination The SUNRISE protocol has received ethics approved from the University of Wollongong, Australia, and in each country by the applicable ethics committees. Approval is also sought from any relevant government departments or organisations. The results will inform global efforts to prevent childhood obesity and ensure young children reach their health and developmental potential. Findings on the correlates of movement behaviours can guide future interventions to improve the movement behaviours in culturally specific ways. Study findings will be disseminated via publications, conference presentations and may contribute to the development of local guidelines and public health interventions.Peer reviewe

Virion production is significantly decreased by rapamycin treatment.

<p>BCBL-1 +/− rapamycin 12 nM were induced with CoCl₂, TPA, or VPA. Five days post-induction, virus was concentrated from supernatants. HeLa cells were then infected with concentrated virus in the presence of polybrene. Viral titers were determined by staining HeLa cells for punctate intranuclear LANA dots and analyzing via multispectral imaging flow cytometry. (A) Images show representative nuclear staining with DRAQ5 (Nuc.), LANA antibody stain, and the overlay (Comb.). Upper panel shows three representative HeLa cells infected with inoculum from untreated VPA-induced BCBL-1. Lower panel shows representative HeLas infected with inoculum from rapamycin-treated VPA-induced cells. (B) Graph of viral titers untreated (black bars) or treated with rapamycin (gray bars) for each indicated inducting agent. Representative experiment, n = 2. (C) Inhibition of cell-to-cell virus transmission from spontaneously lytic BCBL-1 was assessed by culture of BCBL-1 in the presence of 12 nM rapamycin for 2 days. Cells were then harvested, placed in fresh media without rapamycin and co-cultured with HeLa cells for 24 h. BCBL-1 cells were removed, HeLa cells washed x 2 to remove non-adherent BCBL-1 cells, and cultured an additional 24 h before harvest. Adherent HeLa cells were trypsinized, fixed and stained for intranuclear LANA dots. LANA⁺ HeLa cells were analyzed by MIFC. Graph shows percentage (mean ± s.d.) of infected cells from triplicate cultures for HeLa cells co-cultured with either rapamycin-treated (gray) or DMSO-treated (black) BCBL-1. ** p<0.01.</p

Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.

<p>BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA⁺ cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl₂ (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA⁺ cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.</p

RTA regulation by rapamycin is mediated at both the mRNA and protein level.

<p>Messenger ORF50 RNA and RTA protein levels were assessed in BCBL-1 pre-treated with rapamycin for 2 hours, and then induced to lytic reactivation using VPA, TPA, or CoCl₂. Samples were collected at 0, 6, 24, and 48 hours post-treatment. (A) Top panel, nuclear extracts were used to determine RTA protein levels at each time point using non-enzymatic, immunoblot quantification and normalization to Ran. Graphs show mean ± s.e.m. of triplicate experiments for DMSO (solid line) and rapamycin-treated (dashed line) samples. Individual experiments were normalized to the maximal protein expression levels in DMSO sample at 48 h post-induction. Bottom panel, total mRNA was also collected for quantitative RT-PCR analysis of ORF50 mRNA from parallel whole lysate samples. Graph shows pooled data from 3 experiments with DMSO (solid line) and rapamycin-treated (dashed line) cultures shown. Means ± s.e.m. (B) BCBL-1 cells treated with rapamycin (dashed lines) or DMSO (solid lines) were induced with either TPA (left panels) or CoCl₂ (right panels). RTA protein levels (top) and mRNA levels (bottom) are shown from representative experiments. (C) For each timepoint and induction, viability was determined for both DMSO (black bar) and rapamycin-treated (gray bar) samples by staining an aliquot from each culture with a live/dead exclusion dye and assessing for dye uptake (cell death) by flow cytometry. Graphs show % viable, mean ± s.e.m. of triplicates.</p

Intracellular Kaposi's Sarcoma-Associated Herpesvirus Load Determines Early Loss of Immune Synapse Components▿

Lifelong infection is a hallmark of all herpesviruses, and their survival depends on countering host immune defenses. The human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of proteins that contribute to immune evasion, including modulator of immune recognition 2 (MIR2), an E3 ubiquitin ligase. Exogenously expressed MIR2 downregulates the surface expression of several immune synapse proteins, including major histocompatibility complex (MHC) class 1, ICAM-1 (CD54), and PECAM (CD31). Although immunofluorescence assays detect this lytic gene in only 1 to 5% of cells within infected cultures, we have found that de novo infection of naive cells leads to the downregulation of these immune synapse components in a major proportion of the population. Investigating the possibility that low levels of MIR2 are responsible for this downregulation in the context of viral infection, we found that MIR2 transduction recapitulated the patterns of surface downregulation following de novo infection and that both MIR2 promoter activation, MIR2 expression level, and immune synapse component downregulation were proportional to the concentration of KSHV added to the culture. Additionally, MIR2-specific small interfering RNA reversed the downregulation effects. Finally, using a sensitive, high-throughput assay to detect levels of the virus in individual cells, we also observed that downregulation of MHC class I and ICAM-1 correlated with intracellular viral load. Together, these results suggest that the effects of MIR2 are gene dosage dependent and that low levels of this viral protein contribute to the widespread downregulation of immune-modulating cell surface proteins during the initial stages of KSHV infection