Background: The epithelial immune regulation is an essential and protective feature of the barrier function of the mucous membranes of the airways. Damage to the epithelial barrier can result in chronic inflammatory diseases, such as chronic rhinosinusitis (CRS) or bronchial asthma. Thymic stromal lymphopoietin (TSLP) is a central regulator in the epithelial barrier function and is associated with type 2 (T2) and non-T2 inflammation. Materials and methods: The immunology of chronic rhinosinusitis with polyposis nasi (CRSwNP) was analyzed in a literature search, and the existing evidence was determined through searches in Medline, Pubmed as well as the national and international study and guideline registers and the Cochrane Library. Human studies or studies on human cells that were published between 2010 and 2020 and in which the immune mechanisms of TSLP in T2 and non-T2 inflammation were examined were considered. Results: TSLP is an epithelial cytokine  and a central regulator of the immune reaction, especially in the case of chronic airway inflammation. Induction of TSLP is implicated in the pathogenesis of many diseases like CRS and triggers a cascade of subsequent inflammatory reactions. Conclusion: Treatment with TSLP-blocking monoclonal antibodies could therefore open up interesting therapeutic options. The long-term safety and effectiveness of TSLP blockade has yet to be investigated.  An intact mucosal barrier is crucial for the maintenance of tissue homeostasis as it protects the organism from infections, environmental toxins, pollutants, and allergens . A disruSome authors even suggest that the increase in epithelial barrier-damaging agents associated with industrialization, urbanization, and the associated “modern life” underlies the rise in allergic, autoimmune, and other chronic mucosal diseases . It seems to be proven that, in addition to the epithelial barrier, an intact mucosal immune system is a prerequisite for the prevention of chronic inflammatory mucosal diseases, and mucosal immune dysfunction is also involved in the development of diseases such as allergic rhinitis and CRS , 2, 3, 4Thymic stromal lymphopoietin (TSLP) is a cytokine primarily expressed by airway epithelium and released in response to environmental factors, triggering various inflammatory processes . TSLP gene have been associated with asthma. TSLP expression is increased in the airways of asthma patients compared to healthy individuals and correlates with disease severity and lung function. Polymorphisms in the Evidence suggests that TSLP is an important factor in the pathophysiology of chronic inflammatory airway diseases, promoting eosinophilic  inflammation, non-eosinophilic inflammation, and airway structural changes through its effects on a variety of adaptive and innate immune cells and epithelial cells . Clinical trials of TSLP blockade with monoclonal antibodies in patients with chronic inflammatory airway disease have been highly successful . CRS is an inflammatory disease of the mucous membranes of the nose and sinuses , 7, 8, 9The inflammation associated with CRS is heterogeneous and has been associated with different inflammatory endotypes. Among the most common endotypes is type 2 (T2) inflammation . As in aAlternatively, non-T2 inflammation may be present  , 35, 36.Immunoglobulin (Ig) E, eosinophils in sputum and blood, interleukin (IL)-4, IL-5, and IL-13 are considered indicative biomarkers. Because there may be evidence of multiple upregulated inflammatory pathways in each individual patient, it may be difficult to identify a single predominant endotype in individual cases , 42, 43.TSLP gene [The immunologic significance of the epithelial cytokine TSLP offers the possibility for a new approach to the treatment of chronic airway inflammation. Epithelial cytokines are commonly referred to as alarmins, which include IL-25 and IL-33. TSLP is released by airway epithelial cells in response to various environmental agents, such as viral and bacterial infections, in response to allergens and chemical irritants, and injury , 45. FunSLP gene , 67, 68.SLP gene , 70, whiSLP gene  and is sSLP gene . TSLP haSLP gene , 74. The role of TSLP in the pathogenesis of chronic inflammatory airway disease has led to the development of anti-TSLP monoclonal antibodies as a therapeutic option for these patients. In asthma, the results of clinical trials of anti-TSLP therapy are very convincing . For the present publication, a selective literature search was performed in Medline, Pubmed, and the national and international trials and guidelines registries and the Cochrane Library, and on the World Wide Web. Human studies or human cell studies published from 2010 to 2020 that investigated the immune mechanisms of TSLP in T2 and non-T2 inflammation were considered. In addition, current publications in literature databases of available German-language journals were analyzed. This literature search considered original and review papers in German or English language. The aim of this review is to summarize the available data on the mechanisms of action of TSLP in CRS across the spectrum of inflammatory endotypes to illustrate the therapeutic potential of novel TSLP-blocking therapies. Deliberately, only human studies were considered here because although the biology of the TSLP pathway appears to be similar in humans and rodents, the ability to use rodent models is limited by the generally poor transferability of rodent models to complex, heterogeneous human diseases , 77. Search terms used were TSLP OR thymic stromal lymphopoietin AND asthma* or AND CRS* or AND CRSwNP*, using the species filter “Humans”. Review articles were also considered. The results of these searches were reviewed for relevance, that is, whether they contained information on sites of TSLP expression, TSLP effector cells, or physiological or clinical effects of TSLP, and were supplemented with other relevant articles known to the authors. Several local effector cells play a role in the development of T2 inflammation. The interaction between airway epithelium and these cells is an important process driving eosinophilic inflammation. TSLP acts directly on immune cells involved in T2 inflammatory processes in CRS. Eosinophilic inflammation contributes significantly to the physiological changes and airway remodeling in chronic inflammatory airway disease. Eosinophils are present in airways altered due to T2 inflammation but also in T2 inflammation of the skin  and are subsequently locally activated , 83, 84.In uncontrolled  or severFew studies have examined the direct effect of TSLP on mature human eosinophils, and cross-sectional comparisons with cells from asthma patients compared with healthy controls are also lacking. Human eosinophils express both TSLPR and IL-7Rα subunits, and their expression is enhanced by tumor necrosis factor (TNF)-α and IL-3 , 89. TSLThis is supported by correlation studies in patients with atopic asthma, in which the level of immunopositive staining for TSLP in bronchial biopsies correlated with airway eosinophilia 24 hours after allergen exposure . In contThere is evidence of a link between allergic respiratory reactions and the mobilization of bone marrow-derived eosinophil progenitor cells (EoPs). Affected tissues support local differentiation, proliferation, maturation, and activation of EoPs that migrate to the site of allergen exposure in the mucosa during allergic airway disease. TSLP has been shown to promote activation, migration, and local differentiation of EoPs in the airways. Cord blood-derived hematopoietic progenitor cells cultured with TSLP at nanomolar concentrations upregulate IL-5Rα expression and then, in combination with IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF), stimulate significant growth of eosinophil/basophil colony-forming units (Eo/Bo-CFUs) . In addition, increased eosinophil activity was detected in bronchial epithelial supernatants from patients with severe eosinophilic asthma compared with patients with mild asthma and healthy controls. This activity was attenuated by a receptor-blocking antibody to TSLP . In picoMigration of progenitor cells into the airways is an important component in triggering local eosinophilic inflammation. Prior exposure to TSLP and IL-33 stimulates progenitor cell migration toward the chemoattractant stromal cell-derived factor (SDF)-1α (CXCL12) . This imILC2s produce substantial amounts of T2 cytokines such as IL-5, IL-13, and IL-9 after activation by alarmin cytokines such as TSLP, IL-25, and IL-33 , 96, 97.Aspergillus or IL-2/IL-33. In contrast, dexamethasone had no effect on airway ILC2s, indicating compartmental differences in steroid resistance of ILC2s [CBX7, MEK2, and TRL2, have been identified in TSLP-stimulated lymphoid cells resistant to dexamethasone treatment [For inflammatory airway diseases, Chen et al.  reportedof ILC2s . This waof ILC2s . Three greatment . TSLP itreatment . Thus, dMast cells play an important role in triggering eosinophilic airway inflammation through IgE-FcεR1 cross-linking, leading to the release of histamine, leukotrienes, and many other cytokines/chemokines. Alarmin cytokines may affect mast cell function. Mast cells express TSLPR and, upon stimulation with TSLP alone or together with IL-1β and TNF-α, produce T2 cytokines and chemokines CXCL8 and CCL1, with no effect on mast cell proliferation or survival , 54, 105Macrophages are leukocytes found throughout the respiratory tract. There are few studies on the effect of TSLP on airway macrophages in humans. Herein, TSLP was shown to increase the expression of the activation marker CD80 in CD14+ monocytes/macrophages, suggesting a role in promoting differentiation into mature macrophages . In addiBasophils play an important role in chronic inflammatory airway disease as a significant source of T2 cytokines, including IL-4, IL-13, and pro-inflammatory mediators such as histamine and leukotrienes. Basophil development, homeostasis, and function are largely regulated by IL-3, but there is increasing evidence that TSLP also affects basophil differentiation. Peripheral blood-derived CD34+ cells incubated with IL-3 and TNF-α prior to incubation have increased sensitivity to TSLP-mediated basophil differentiation . In addiIn blood samples from patients with allergic asthma, significant upregulation of TSLPR on basophils was found after direct stimulation with anti-IgE antibodies, which correlated with total serum IgE . HoweverThus, TSLP is an important mediator of the basophil inflammatory response. Human myeloid dendritic cells express TSLPR , and stiMost studies on the influence of TSLP on lymphocytes referred to the indirect effect of TSLP through dendritic cells on T-cell differentiation , 126; hoThe direct effects of TSLP on Tregs have not been well studied. Tregs express TSLPR, and stimulation with TSLP impairs IL-10 production . ImmunosNon-T2 inflammation in CRS is often mediated by Th17 cells and neutrophils. IL-17A produced by Th17 cells stimulates airway epithelial cells to produce neutrophilia-promoting cytokines such as CXCL8 (IL-8) and GM-CSF  and promTSLP enhances Toll-like receptor (TLR) 3 ligand-induced production of IL-23 by dendritic cells and induces programming of naïve CD4+ T cells into Th17 cells . In addiIn chronic inflammatory airway disease, levels of IL-33 and TSLP, but not IL-25, were significantly elevated compared to control subjects . PreviouIn addition to its effects on specific immune cells, there is now ample evidence that TSLP serves as a mediator between immune cells and epithelial cells in the airways. Dysregulation of the epithelium leads to characteristic airway changes known as airway remodeling, which include reticular basement membrane thickening, goblet cell hyperplasia, subepithelial fibrosis, and ASMC hyperplasia and/or hypertrophy . Numerous reports indicate that TSLP is an important modulator of ASMC activity. Human ASMCs express TSLPR , and stiTSLP can promote airway remodeling in chronic inflammatory airway diseases through activation of fibroblasts  in termsinflammatory airway diseases [The TSLPR complex has been associated with a number of chronic diseases . Two monCSJ117 is a fully human neutralizing Fab fragment (antibody-antigen binding fragment: Fab) belonging to the IgG1/λ isotype subclass. CSJ117 was developed as an inhaled formulation for targeted administration to the lung to bind to TSLP released from airway epithelial cells , 45. GSK2618960 is a humanized IgG1 monoclonal antibody directed against the alpha component  of TSLPR  and is bIn patients with severe eosinophilic airway inflammation , blockinIn a double-blind placebo-controlled phase 1b study to demonstrate the efficacy of tezepelumab in an allergen provocation chamber , tezepelThe second completed study in asthma (PATHWAY) was a large, multicenter, randomized, parallel-group, double-blind, placebo-controlled phase 2 trial . The stuIn addition to the studies with tezepelumab, a multinational proof-of-concept study of CSJ117 in the allergen challenge model was conducted in patients with mild allergic asthma to evaluate safety, tolerability, pharmacokinetics, and pharmacodynamics. 28 participants completed the study, which included daily inhalation of CSJ117 and bronchial allergen challenges at 6 and 12 weeks. The study was completed in 2019; results are pending . Additional clinical trials evaluating the efficacy, mechanisms of action, and long-term safety of tezepelumab are ongoing. Two pivotal phase 3 trials (NAVIGATOR and SOURCE) are being conducted in patients with severe asthma receiving inhaled corticosteroids/long-acting β-agonists with or without oral corticosteroids for maintenance therapy and additional asthma controllers , 160. ThTezepelumab is also tested in a phase III study for CRSwNP. The WAYPOINT study recently started recruiting patients. However, the first study results are not expected until 2024. Two biologics have now been approved in Germany  as an add-on treatment for severe, uncontrolled chronic rhinosinusitis with nasal polyps. Based on positive study data, it is likely that two additional compounds will also receive approval in the future  , 163. AlTSLP plays a central role in chronic airway mucosal inflammation. TSLP is also mostly involved in T2 inflammation, but quite possibly also in non-T2 inflammation, and has multiple effects on a variety of cell types including ILC2s, hematopoietic progenitor cells, eosinophils, basophils, mast cells, monocytes/macrophages, dendritic cells, lymphocytes, neutrophils, smooth muscle cells, and fibroblasts . Treatment options for chronic inflammatory airway diseases have improved significantly in recent years with the introduction of T2-targeting biologics. They act on different targets of T2 inflammation, for example IgE, IL-5, IL-4/IL-13. By activating several downstream inflammatory pathways, TSLP affects airway inflammation more comprehensively than the targets of previously approved biologics, which is why anti-TSLP-targeted biologics can sustainably improve epithelial immune regulation in chronic inflammatory airway disease [TSLP blockade has shown promise in treating both T2-related and non-T2-related  inflammation in asthma when administered over a period of up to 1 year. For patients with non-T2-related inflammation, this would be the first therapeutic option with monoclonal antibodies. As with other biologics, it will be important to find biomarkers that identify patients with a good chance of success with anti-TSLP therapy. Blood eosinophils, serum IgE, and FeNO have been used as biomarkers to analyze treatment with anti-IL-5/IL-5Rα, anti-IgE, and anti-IL-4/IL-13 monoclonal antibodies. While TSLP itself could hypothetically be used as a biomarker, this has not been shown to be effective, primarily because of the difficulty in measuring low concentrations of this cytokine [In addition, TSLP has been shown to be cleaved in nasal polyp tissue by endogenous proteases, resulting in bioactive peptides , 165, maThe clinical relevance of systemic TSLP detection is also unclear, as there are large variations in peripheral blood independent of disease activity in the airways . Attempts to quantify TSLP in patient samples are also complicated by the fact that there are two isoforms of the protein: a long-form TSLP (lfTSLP), in which the protein is full-length, and a form that is approximately half amino acid length (63 amino acids), often referred to as short-form TSLP (sfTSLP) , 169. ItAlthough TSLP is primarily expressed by epithelial cells at barrier surfaces , TSLP can also be produced by a variety of immune cells; therefore, systemic administration of anti-TSLP also has the potential to disrupt other homeostatic functions of TSLP , 174. ThThis is especially true in situations such as the current COVID-19 pandemic, where continuing or reinitiating biologic therapy for CRSwNP is recommended even during SARS-CoV-2 virus infection or in the setting of a planned COVID-19 vaccination . The timBecause of the common T2 endotype, most of the biologics developed for asthma are also suitable for CRSwNP. TSLP expression in asthma patients correlates with both airway obstruction and the severity of the disease. TSLP promotes eosinophilia in the airways. The migration of progenitor cells into the airway is an important component in the process triggering local eosinophilic inflammation. TSLP interacts directly with mast cells to promote eosinophilic inflammation through the production of T2 cytokines. TSLP is also a key mediator of the basophilic inflammatory response. There is also evidence that TSLP can directly modulate human T lymphocytes. The expression of TSLP in bronchial biopsy tissue is limited to fibroblasts. There is evidence that tezepelumab has similar effectiveness in patients with T2-related and those with non-T2 disease. TSLP is involved in both T2 inflammation and non-T2 inflammation and shows effects on a wide variety of cell types. None.L. Klimek reports grants and/or honoraria from Allergopharma, MEDA/Mylan, HAL Allergie, ALK Abelló, LETI Pharma, Stallergenes, Quintiles, Sanofi, ASIT Biotech, Lofarma, Allergy Therapeut., AstraZeneca, GSK, Inmunotk, outside the submitted work; and memberships in the following organizations: AeDA, DGHNO, German Academy of Allergology and Clinical Immunology, ENT-BV GPA, EAACI. J. Hagemann states to have received grants from Novartis Pharma GmbH for lectures. U. Förster-Ruhrmann received honoraria for lectures from Novartis, AstraZeneca, Sanofi, and GSK, outside the present work. Ch. Bergmann, P.V. Tomazic, C. A. Hintschich, T. Huppertz, H.J. Welkoborsky, M. Cuevas, I. Casper, S. Becker, have no conflicts of interest related to this work.


Even though the total fertility rate (TFR) has decreased significantly over the past decades in many countries, it has remained stable in sub-Saharan African (SSA) countries. However, there is variation among the sub-regions and inhabitants of SSA. Therefore, this study aimed to conduct a meta-analysis of demographic and health surveys (DHS) to estimate the pooled level of TFR in SSA and to depict sub-regional and inhabitant differences.www.meaasuredhs.com between 2010 and 2018. The point estimate of the total fertility rate with its corresponding standard error in each sub-Saharan African country was estimated using the DHS.rates R package. Using the point estimate of the TFR with the standard error of each country, the pooled estimate of the TFR was generated by the metan STATA command.The data source for this study was the standard Demographic and Health Survey datasets of 33 sub-Saharan African countries, accessed through The study comprised 1,324,466 live births in total. The pooled estimate of sub-Saharan Africa's overall fertility rate was five children per woman (95% CI: 4.63–5.37). Consequently, the pooled estimate of total fertility for people living in urban and rural areas was 3.90 (95% CI: 3.60–4.21) and 5.82 (95% CI: 5.43–6.21) children per woman, respectively. In sub-group analysis, the pooled estimates of the TFR for the East African, Central African, Southern African, and West African regions, respectively, were 4.74, 5.59, 3.18, and 5.38 children per woman. Total fertility rates were greater in low-income nations (5.45), lower-middle-income countries (4.70), and high-middle-income countries (3.80).SSA has a relatively high total fertility rate. The regions of West and Central Africa have the highest overall fertility rate. The fertility rate is higher in countries with a large rural population and low income. Strategies should be developed to address this public health concern, especially in rural Central and Western Africa. Many women want to have biological offspring , 2. The Expanding access to contraceptives and reducing unmet needs for family planning can help decrease the TFR among reproductive-age group women in SSA . Howeverwww.meaasuredhs.com on all completed population-based surveys conducted under the DHS projects  that did not have a DHS survey report after the 2010/2011 survey year were excluded. Three sub-Saharan countries  were excluded because the dataset was not publicly available .ASFRa is calculated for a reference period of 3 years preceding the survey for seven five-year age groups. Thus, the TFR can be written as follows:The outcome variable of this study was the total fertility rate. The TFR is a hypothetical measure of women's fertility. It could be defined as the number of children born per woman if she experienced current age-specific fertility rates throughout her childbearing years and did not die, according to a current schedule of age-specific fertility rates, and not be subject to mortality. In standard DHS surveys, DHS.rates R package. In each country, along with fertility, the “fert” function estimates standard error (SE), relative standard error (RSE), and confidence interval (CI) for each rate  using the ach rate . The metach rate  was poolOverall, a total of 1,324,466 live births, with a minimum of 14,769 in Comoros and a maximum of 116,888 in Nigeria, were included in this study . The pooThe fertility decline in SSA countries has been relatively steady and occurred later compared to other regions in the world. Its age-specific fertility rate also showed substantial variation among countries , 32, 33.In this study, the overall pooled estimate of the TFR from DHS data in SSA was five per woman. Even though the TFR has fallen significantly in the past decades in many countries, it has remained stable (or “stable population”) at around 6,400 children per 1,000 women in 1990  in SSA cA possible justification for this discrepancy might be the data source that the two studies used. As mentioned previously, there is a considerable decrease in the TFR from time to time in different regions of the world. However, the decrement is steady in SSA. There are several reasons why SSA lacks a marked decline in total fertility, including political instability, poverty, a lack of government commitment to female education, a weak healthcare system, and cultural beliefs that view children as sources of income; in almost all African countries, early marriage is taboo , 36. In In this study, the distributions of total fertility in the sub-region of SSA countries are not similar. Consequently, the pooled estimates of TFR in Eastern Africa, Southern Africa, Central Africa, and Western African countries were 4.74, 3.18, 5.59, and 5.38, respectively. Countries within each sub-region have experienced sociodemographic, socio-political, economic, and cultural events that could influence total fertility variations. In Central Africa and Western African countries, the TFR is higher than the pooled level. This rise could be because >50% of women in these regions experience their first marriage, sexual initiation, and the birth of their first child by the age of 20 . In contBesides, West and Central African countries have low rates of modern contraceptive use, with some of the lowest contraceptive prevalence rates in the world in two sub-regions of Africa ; this coAscertaining fertility levels by residence is crucial for a better understanding of contemporary demographic changes in developing countries. The pooled fertility level was significantly higher in rural inhabitants than in urban inhabitants, at 3.90 and 5.82 per woman, respectively. These findings were also consistent with some studies , 42. BecThis pooled estimate revealed that the TFR in sub-Saharan countries is high, with the highest TFR being reported in central and west African countries. The fertility rate of rural dwellers was higher compared to those living in urban areas. Decreasing total fertility can indirectly lead to a decrease in maternal and child mortality. However, the decrease was steady in SSA countries. Therefore, appropriate efforts should be made to reduce the TFR in sub-Saharan countries, particularly in the Central and Western African sub-regions and among rural inhabitants. The government and health professionals should work toward meeting the reproductive and maternal health needs of the population.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.DT and ST were involved in this study from its inception through the design, acquisition, analysis, interpretation of data, and manuscript drafting. All authors contributed to the conception and design, analysis, and interpretation of the data and reviewed and revised the manuscript for important intellectual content, and also gave final approval for the version to be published and are accountable for all aspects of the study.


Functional neurological disorder (FND) is a common issue in the pediatric population. The concept and our understanding of functional neurological disorders have changed over the past years, and new etiologic models and treatment plans have been explored. Knowledge about FND in the pediatric population, however, is lacking. The aim of this review is to provide an update on pediatric functional neurological disorder. We conducted a literature search of PubMed and SCOPUS databases and reviewed a total of 85 articles to gain insight into the current understanding of FND etiology, diagnosis, treatment, and prognosis in children and adolescents. Functional and high resolution MRI revealed abnormal connectivity and structural changes in patients with functional symptoms. The diagnostic criteria no longer require the presence of a psychological factor and instead focus on a rule-in diagnosis. Treatment of FND includes a clear communication of the diagnosis and the support of a multidisciplinary team. Although FND typically has a poor prognosis, better outcomes appear to have been achieved in children and young adults. We conclude that pediatric functional neurological disorder is a prevalent pathology and that this patient population has additional specific needs compared to the adult population. Functional neurological disorder (FND) is a prevalent pathology in pediatric practice, characterized by neurological symptoms that are not caused by other classic neurological or medical conditions . FND hasChildren with FND present with a broad range of symptoms that can mimic those of neurological diseases, but the most common are seizures and motor-type symptoms  . Motor-tThe aim of this article is to provide a review of FND and summarize the etiology, diagnostic approach, treatment options, and prognosis. There is little information about FND in the pediatric population in the medical literature, further emphasizing the need for higher quality studies to help better understand and provide care for these patients.We present a narrative review on the current concepts of functional neurological disorders in children and young adults. We performed a literature search of PubMed and SCOPUS databases, and reviewed the current understanding of FND etiology, diagnosis, treatment, and prognosis in children and adolescents. Our search consisted of the following terms: conversion disorder, dissociative disorder, and functional neurological disorder. There were no restrictions by article type. The search was limited to articles published in English from 2000 up to September 2022. Our search resulted in 602 articles from which those containing the word “functional” unrelated to functional neurological disorder were removed. The remaining articles were screened by title and abstract, and 108 articles were chosen for a full-text review. For a comprehensive search, all relevant references from the selected articles were included regardless of the publication date. In total, 85 studies were included in the review. As in all narrative reviews, a selection bias has to be taken into account.The etiology of FND is complex and no single causal mechanism has been found. The physical symptoms of patients with FND were previously believed to result from psychological distress, but recent studies show that only about one-third of these patients have a history of trauma . Today, Trauma, stress, and psychiatric disorders are some of the most commonly recognized predisposing factors for the development of FND, but the research findings are inconsistent. A history of trauma, stress or psychiatric disorders has been found in only one-third of the adult population with functional symptoms ,17. A reAlthough anxiety is reported as an important predisposing factor for FND, the evidence is inconsistent. Some studies have reported a higher prevalence of anxiety in these patients  while otThe increased anxiety surrounding the COVID-19 pandemic was expected to aggravate FND symptoms, but no clear correlation was found. In a study conducted on patients with a history of FND, only a minority of patients showed a slight deterioration. Instead, there was an increase in the incidence of FND diagnosis ,27. One Although there is insufficient evidence to support trauma or psychiatric symptoms as the sole cause of FND, there is a higher prevalence of these factors and combined with other predisposing factors, they might increase the risk of developing functional symptoms.The presence of physical trauma and somatic symptoms has been reported for a long time in patients with FND . ChildreNeurological disorders are an important risk factor for FND. A systematic review found a mean frequency of epilepsy of 22% in patients presenting with seizure-type FND and a mean frequency of seizure-type FND of 12% in patients suffering from epilepsy . In almoA prior history of family illness also appears to be associated with FND. One study found a powerful relationship between reported poor health of the parents when the subject was an adolescent and development of symptoms later in life .Given the wide heterogeneity of the predisposing factors found in patients with FND, multiple etiological models that integrate psychological, physical, and neurobiological factors have been published over the last few decades. The constructs that appear to be affected in FND include emotion processing, agency, attention, interoception, and predictive processing . EvidencEmotion processing involves the salience and limbic/paralimbic circuits. Functional studies have shown an increased connectivity between limbic and motor circuits in patients with functional neurological disorder, which might explain why negative emotions have been associated with worsening motor symptoms ,41. PatiIn addition to functional abnormalities, some studies have found structural abnormalities in patients with FND. For example, there is evidence of cortical atrophy in the right motor and premotor areas and the right and left cerebellum in patients with seizure-type FND . FurtherAs the understanding of FND has improved, so has the diagnostic criteria. While the DSM-IV required the exclusion of other diseases and the presence of a psychological factor, the DSM-5 focuses on a positive diagnosis. The DSM-5 diagnostic criteria for FND include: 1) symptoms of altered voluntary motor or sensory function; (2) clinical findings incompatible with neurological or medical conditions; (3) symptoms that cannot be explained by another medical or psychiatric disorder; and (4) symptoms that cause significant distress or impairment in social, occupational, or other important areas of functioning [ symptomsChildren with functional symptoms usually present for the first time to the general practitioner (GP) or pediatric emergency department (ED). These patients can have a wide spectrum of symptoms, such as motor function disturbances, sensory and visual dysfunctions , speech problems , and symptoms resembling epileptic seizures . Motor sSince early diagnosis is linked to a better prognosis , the medFunctional neurological disorder is a diagnosis that can elicit considerable stigma towards patients and their families. Moreover, the idea that these patients are “faking it” or that they are “not really sick” still exists in today’s society. Pediatricians face the challenge of having to explain the disease in a way that can be understood and accepted by both the patient and the family. Delivering the diagnosis is central to treating FND. For example, a study on 54 patients with functional seizures showed that 15 of these patients had a remission in symptoms after receiving their diagnosis . FurtherIn our opinion, when possible, the diagnosis should be given to the patients in the presence of a mental health specialist. Several recommendations have been proposed to help the clinician deliver the diagnosis. Hall-Patch and colleagues developed a crib sheet to ensure that the clinician covers all the points in communicating the diagnosis. These include:1. Recognizing symptoms as genuine;2. Labeling the condition;3. Providing a brief explanation ; 4. Addressing which treatments are effective and which are not;5. Managing expectations  .Treating FND represents a challenge for both the healthcare professional and for the patients–from the delivery of the diagnosis to the selection of the best approach. Communicating the diagnosis in a clear and bias-free manner is the first step in treating these patients ,3,60,61.Cognitive behavioral therapy (CBT) is one of the most widely used interventions for treating functional symptoms and has shown a good response rate in the pediatric population ,69. A prRetraining and Control Therapy is a short-term outpatient intervention that uses cognitive behavioral principles to retrain catastrophic symptom expectations and low sense of control over symptoms. ReACT includes four steps: (1) a clear etiological description based on the Integrated Etiological Summary Model ; (2) an Physiotherapy plays a vital role in managing FND, especially in patients with motor symptoms. A randomized controlled trial on a group of 60 patients with motor FND revealed that physiotherapy shows high acceptability, good treatment effect, and a cost benefit. The follow-up period was 6 months and 72% of the patients in the intervention group showed a good outcome compared with 18% in the control group. A larger proportion of the control group (32%) felt that their symptoms worsened compared with 3% in the intervention group . A systeSeveral studies have researched the efficacy of pharmacotherapy in FND but yielded no clear evidence of any beneficial purpose. A study on motor FND revealed that patients with FND and anxiety or depression might benefit from antidepressants . HoweverFunctional studies have revealed evidence of abnormal connectivity in the central nervous system in patients with FND, which might be a consequence of poor neuroplasticity. Treatment options that aim to modulate this phenomenon are known to be effective in treating major depressive disorder and other trauma-related disorders. Several studies have evaluated the efficacy of transcranial magnetic stimulation (TMS) in adult patients with motor-type FND and found a good response to treatment, but others found only a small reduction in symptom severity. Although TMS appears to be of some benefit in treating motor-type FND, most of the studies on TMS have several limitations: the protocols used show wide heterogeneity; most studies had no placebo control group; and TMS was usually a part of a multidisciplinary approach.Finally, physical activity (PA) is known to reduce depression and anxiety and increase overall quality of life ,76,77,78Functional neurological disorder appears to have a generally unfavorable prognosis. A systematic review evaluated the outcomes of patients with motor FND and revealed that 39% (10–90%) of patient symptoms were the same or worse at follow-up evaluations . CohortsAlthough the diagnosis of FND is usually straightforward, the fear of missing an “organic” disease can make managing these patients difficult. This concern can lead to overuse of medical resources and inappropriate treatment. Over-investigation can cause considerable anxiety to patients and their families about a possibly serious disease. It can also lower confidence in the medical professional and thereby lead to a low compliance to further treatment. Inappropriate use of medical tests can lead to false-positive results and thus, a wrong diagnosis and treatment . As prevWhile the number of studies regarding the etiology and the management of functional neurological disorders has grown over the last several years, there is still a lack of large randomized controlled trials, particularly in the pediatric population. This narrative review provides an overall perspective on the latest findings in the field of functional neurological disorders. In the last decades, many studies have aimed to understand the pathophysiology behind FND. However, the mechanisms leading to functional neurological disorders are still not clearly understood nor is there a clear trigger of symptoms. Instead, etiological models that integrate neurobiological, physical, and psychological factors have been proposed. Owing to the structural changes identified in patients with functional symptoms, the term “non-organic” has become outdated and clinicians should avoid using it. These structural changes can be a sign of poor neuronal plasticity and treatment methods aimed at modulating plasticity, such as transcranial magnetic stimulation, might have a role to play in the management of patients with chronic FND. A better understanding of the pathophysiology and predisposing factors is needed to develop better treatment plans. The diagnosis of functional neurological disorder is usually straightforward and in most cases, no additional investigations are required. However, there is a 4% rate of a false-positive diagnosis and a thorough evaluation during the follow-up period is necessary to identify the patients who may require further investigations. Additional investigations may also help reassure parents of children with FND and raise acceptability as well as treatment compliance. Treatment of the disorder can include a wide spectrum of interventions. A multidisciplinary approach based on cognitive behavioral therapy with additional interventions is typically used. When devising a treatment plan, clinicians should take into consideration the duration of symptoms, psychiatric comorbidities, socioeconomic status, and accessibility to various treatment options. There are, however, no guidelines for the management and follow-up of these patients. Additionally, most of the studies on the efficacy of treatment are in the adult population and do not take into consideration the role of parents in the healing process. This calls for additional RCTs in the pediatric population to evaluate the efficacy of different treatment plans and to better understand the family’s role in the management of this disorder. Functional neurological disorder has an overall poor prognosis; however, children and young adults appear to have a better outcome. Although some factors appear to have a good correlation with better outcomes , the difference in follow-up rates in the current literature (50–100%) provides a substantial bias both in patients with better outcomes as well as in those with poorer ones. Prospective studies with longer follow-up rates and periods are needed to evaluate long-term outcomes, including recurrence of FND and development of other functional symptoms or psychiatric disorders. Additionally, data from these studies could be used to develop follow-ups based on prognostic factors, functionality scales, and treatment response. FND is a common issue in the pediatric population, with additional needs compared to the adult population. First, the family must understand the diagnosis and support the child in the rehabilitation program. Family counselling may also be needed to ensure good compliance. Most of the findings in the literature on etiology, prognostic factors, and treatment plans come from adult studies, which are lacking in information on the particularities of pediatric-onset FND. As the concept of FND diagnosis and treatment evolves over time, in-depth reviews and prospective studies on the pediatric population will be needed to improve our understanding of the particularities of childhood-onset FND and to better manage the children who have this disorder.


Serum uric acid (UA) within appropriate levels is reported to be beneficial in patients with idiopathic short stature (ISS). This study aimed to evaluate the association between serum UA levels and height standard deviation scores (SDS) in patients with ISS during growth hormone (GH) therapy.A longitudinal study (LG Growth Study) of 182 children (mean age: 7.29±2.60 years) with ISS was performed. All participants were in the prepubertal stage and treated with GH, and the data within a treatment period of 30 months were analyzed. In the adjusted Pearson’s correlation, UA was significantly correlated with height SDS after controlling for sex, age, and body mass index (BMI) SDS . In the adjusted multiple regression analyses, the height SDS was significantly associated with UA after controlling for sex, age, and BMI SDS . Within the 30-month treatment period, the UA levels significantly increased as the height SDS increased, and the mean UA levels at baseline and 30 months after treatment were 3.90±0.64 mg/dL and 4.71±0.77 mg/dL, respectively (p=0.007). In conclusion, UA is related to height SDS, and GH treatment leads to a significant increase in UA without hyperuricemia. Elevated UA is considered a favorable outcome of GH therapy, and further studies are needed to determine its role as a monitoring tool.  The number of studies on the association of uric acid (UA) on growth is extremely limited compared with that on the association between UA and metabolic problems. The general UA level in children gradually increases from birth to adolescence and that the fastest increase in UA occurs during puberty , 2. SexuWe used data from a registry study  designed to investigate the efficacy and long-term safety of GH treatment in short Korean children and adolescents with ISS, growth hormone deficiency (GHD), Turner syndrome, small for gestational age (SGA) without catch-up growth, or chronic renal failure. LGS was registered at ClinicalTrials.gov (identifier: NCT01604395). LGS is a multicenter, non-interventional study, and detailed descriptions of its background have been provided in previous publications introducing cohort characteristics and study protocols . WrittenA total of 367 patients with ISS were registered in the LGS between November 2011 and March 2017. The inclusion criteria were:1) height <3rd percentile for sex and age according to the data from the 2017 Korean National Growth Charts for children and adolescents (2). The SDSs for height, weight, and BMI were calculated based on the 2007 Korean National Growth Charts using the LMS method  , creatinine, UA, total protein, albumin, aspartate aminotransferase, alanine aminotransferase, total cholesterol (T-C), triglyceride, IGF-1, and IGF-binding protein 3(IGF-BP3) were performed prior to test drug administration in the GH stimulation test. The pediatric endocrinologists at each center performed the physical examinations to determine the Tanner stage and GH stimulation tests by selecting two of the commonly used methods in clinical practice, such as insulin-induced hypoglycemia, L-dopa, clonidine, and glucagon tests. In all research institutions, height was measured to the nearest 0.1 cm using a Harpenden stadiometer , while body weight was measured to the nearest 0.1 kg using a digital scale. BMI was calculated by dividing the weight by height in meters squared (kg/mriation) . The IGFriation) . Anthrophttps://www.r-project.org). To compare the statistical significance between groups, Fisher’s exact test or chi-square test was used for categorical variables, while the t-test or Mann-Whitney U test was used for continuous variables. Pearson’s correlation tests were performed at baseline to examine the correlation between UA and other anthropometric and biochemical variables, while partial correlation was used after adjusting for sex, age, and BMI SDS. Multiple linear regression analysis was conducted to investigate the independent association between UA and height SDS, after adjusting for sex, age, and BMI SDS at baseline. The clinical variables were expressed as mean ± standard deviation. Statistical significance was set at p<0.05.Statistical analyses were performed using SAS version 9.4  and the R statistical software  and adjusted  correlation analyses. Other variables, including weight, SDS, BUN, total protein, albumin, and T-C, were significantly positively correlated with UA levels in the adjusted correlation analysis (p<0.05).Pearson’s correlation analysis was performed to analyze the relationship between UA levels and anthropometric and biochemical variables. As shown in p=0.023). Height SDS also had a significantly positive association with UA levels in the adjusted multiple linear regression analysis after controlling for sex, age, and BMI SDS . The other variable associated with height SDS in the unadjusted and adjusted multiple linear regression analyses was the weight SDS.Results of the unadjusted and adjusted multiple linear regression analyses of the association between UA and the anthropometric and biochemical variables are shown in p<0.001). The UA levels increased significantly during the treatment period, in line with the increasing trend of these factors (p<0.001). The BMI also steadily increased significantly during the period of GH treatment (p<0.05), but the BMI SDS did not show a statistically significant change.The different variables monitored within the 30-month GH treatment period are listed in Our findings identified an independent correlation between height SDS and serum UA levels in children with ISS, as confirmed by multiple regression analysis after adjusting for age, sex, and BMI SDS. More importantly, the serum UA levels significantly increased with elevation in height SDS during the 30-month GH treatment. No hyperuricemia was observed in the GH-treated patients.The reference range for UA levels in healthy children and adolescents has been reported to have a specific distribution. According to a study of UA reference values for Brazilian children and adolescents, UA slowly increases with age throughout childhood and remains the same in both sexes, and sexual differences in distribution begin at puberty . The resGH treatment results in improvements in body composition and long-term beneficial effects on muscle and adipose tissues , 16. MatHowever, little is known regarding the association of GH therapy with UA metabolism. Dixit et al. reported an increase in UA levels after GH treatment in children with short stature aged 4–17 years . The serThis study, as well as the study by Wang et al. , suggestA major limitation of this study was that data such as dual-energy X-ray absorptiometry related to body composition measurements were not available. Therefore, we could not confirm an association between UA elevation and changes in body composition. Another limitation is that UA can be affected by food intake; however, it was not possible to analyze this because of the nature of this study. Nevertheless, this study had several strengths. In this study, to minimize the factors affecting UA, we included children with no underlying medical conditions such as chronic disease, endocrine disease, brain disease, or chromosomal abnormalities. Furthermore, considering that the characteristics of UA are affected by puberty, only prepubertal patients were included; thus, it is considered a meaningful result compared with other studies that included pubertal patients. The characteristics of multicenter long-term prospective cohort studies may also lend greater significance to the statistical results of this study.In conclusion, serum UA levels are associated with height SDS. The result of a significant elevation in serum UA with an increase in height SDS after GH treatment was an interesting feature found during GH treatment. Further studies are needed to determine whether changes in serum UA levels caused by GH treatment are associated with changes in height and body composition, and to determine the role of UA in monitoring GH treatment.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Review Board of Hallym University Kangdong Sacred Heart Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JSY contributed to the data research and analyses and drafted the initial manuscript; YJS, EBK, and HJL designed the study and interpreted the findings; MJK critically reviewed the manuscript and contributed to the conception of the study; and ITH designed and conceptualized the study and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.We thank all the physicians who provided the data of patients included in the “LG Growth Study”; the authors would also like to thank LG Chem, Ltd., for assisting with the statistical analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RIG-I is essential for host defense against viral pathogens, as it triggers the release of type I interferons upon encounter with viral RNA molecules. In this study, we show that RIG-I is rapidly and efficiently activated by small quantities of incoming viral RNA and that it relies exclusively on the constitutively expressed resident pool of RIG-I receptors for a strong antiviral response. Live-cell imaging of RIG-I following stimulation with viral or synthetic dsRNA reveals that RIG-I signaling occurs without mass aggregation at the mitochondrial membrane. By contrast, interferon-induced RIG-I protein becomes embedded in cytosolic aggregates that are functionally unrelated to signaling. These findings suggest that endogenous RIG-I efficiently recognizes viral RNA and rapidly relays an antiviral signal to MAVS via a transient signaling complex and that cellular aggregates of RIG-I have a function that is distinct from signaling.  Thoresen et al. show that the resident pool of cytoplasmic RIG-I readily detects small quantities of viral RNA, inducing a rapid antiviral response that does not involve massive aggregation at the mitochondria membrane. In contrast, when the RIG-I gene is induced by interferon, RIG-I forms signaling-unrelated cytosolic aggregates. Two critical events are needed to initiate the process of RIG-I signaling upon infection: (1) binding of viral RNA to RIG-I, with concomitant release of the CARDs, and (2) formation of CARD-CARD interaction between RIG-I and MAVS.RIG-I is a cytosolic innate immune receptor that plays a central role in our ability to respond rapidly to infections by RNA viruses.in cellulo have previously shown interferon response to viral infection within 2h,18 and active RIG-I signaling in vivo has been observed within 2 h after injection of RIG-I-specific RNAs.8 Unfortunately, previous studies on subcellular behavior of RIG-I and its responses to RNA activation, including available studies on RIG-I localization, have been conducted at very late time points, ranging from 10–40 h after introduction of viral RNA into cells.21 These late snapshots of RIG-I signaling, together with ex vivo biochemical and biophysical studies performed with transfected or recombinant protein, have led to a “massive oligomer” model for RIG-I-MAVS signal transmission.22 According to this model, RNA-stimulated populations of RIG-I migrate en masse to the mitochondrial network or to the mitochondria-associated membrane (MAM) of the ER,23 where RIG-I becomes enmeshed within a large prion-like MAVS aggregate that is required for signal transduction.13 This subcellular structure is thought to be so large that it must ultimately be cleared by autophagy.24 However, unlike other innate immune signaling complexes, such as the inflammasome25 and myddosome,26 direct visualization of functional RIG-I signaling complexes has been elusive in cells. Indeed, RIG-I signaling complexes have never been characterized or studied in cellulo within the short time frame that is suggested by in vivo studies of RIG-I function.Given the immediate danger posed by viral threats, RIG-I signaling must have the following characteristics: First, it must be highly efficient in sensing the first viral RNAs that have infected the cell. Second, it must be extremely rapid to initiate the cascade of signaling reactions and transcriptional events required for alerting the immune system as quickly as possible. Consistent with the need for speed and efficiency, studies of RIG-I signaling 28 However, naive and unprimed cells must be able to respond to viral infection and trigger a robust immune response or an initial infection would escape detection.29 Therefore, the role of downstream, interferon-induced RIG-I expression remains unclear.Not only does RIG-I trigger the initial interferon response upon infection by RNA viruses, it is itself an ISG. Given this behavior, it has been suggested that interferon-induced RIG-I expression can boost the antiviral response by supplying additional receptors that bind viral RNA and amplify RIG-I signaling, creating a late-stage second wave of interferon induction and a positive feedback loop. Indeed, this “priming” of RIG-I signaling by interferon has been proposed to play a central role in antiviral signaling.in cellulo but that the active RIG-I signal is transmitted to MAVS without visible RIG-I accumulation on the mitochondrial network. We observe two separate phases of RIG-I behavior upon activation by viral RNA ligands. The “early phase” involves a burst of RIG-I signaling and IFN induction that is initiated within an hour and which is not accompanied by increases in RIG-I protein expression. The “late phase” of RIG-I induction involves dramatic increases in RIG-I expression but coincides with a decline in signaling and the production of type I interferon. Rather than accumulating at the mitochondria at long time points after RNA induction, RIG-I forms subcellular bodies at other positions within the cytosol, none of which are involved in RNA-stimulated RIG-I signaling. Importantly, we show that blocking interferon-induced RIG-I protein expression does not disrupt robust RNA-induced RIG-I signaling, thereby demonstrating that the resident cytosolic pool of RIG-I is sufficient for a robust antiviral response. Finally, by carefully quantitating the stoichiometry of stimulatory ligands, we show that the RIG-I response requires only a few cytosolic dsRNA molecules to enter the cell in order to initiate strong interferon induction. Collectively, these findings suggest that the RIG-I signaling pathway is a rapid relay that enables a sensitive antiviral response to be efficiently transmitted by a small number of activated RIG-I receptors.To address these gaps in our understanding and reveal the dynamic behavior of functional RIG-I molecules as they actively engage in signaling, we measured endogenous RIG-I signaling kinetics and efficiency in response to both synthetic and viral pathogen-associated molecular patterns (PAMPs). We then linked these measurements with direct observation of endogenous RIG-I via fluorescence confocal microscopy in order to observe the receptor distribution during active signaling. Here, we demonstrate that not only is RIG-I signaling both rapid and efficient We first sought to comprehensively measure endogenous RIG-I signaling kinetics over 24 h in lung epithelial (A549) cells. Cells were stimulated either via transfection of 1 μg/mL stem-loop RNA-14 (SLR14), a RIG-I-specific RNA ligand containing a single RIG-I-binding site, or with 100 haemagglutination units (HA)/mL Sendai virus (SeV), and the completion of RIG-I signaling was determined via type I interferon mRNA transcription with RT-qPCR. In all cases, regardless of RNA ligand identity or delivery method, overall signaling kinetics were strikingly rapid. SLR14 stimulated IFN-β transcription within 1 h , indicatIn addition to stimulating an early interferon response to intracellular dsRNA, RIG-I is itself an ISG, and this induced population of RIG-I molecules has the potential to influence the course of signaling. To monitor the correlation between kinetics of RIG-I gene expression and the kinetics of signaling, we measured both RIG-I mRNA  and protTo examine whether RIG-I signaling was enhanced by interferon-induced RIG-I expression, we pretreated cells with 100 μg/mL cycloheximide for 2 h prior to SLR14 transfection to block translation and ensure that no additional RIG-I protein was expressed during the 24 h time course. Despite the inhibition of induced RIG-I translation, RIG-I signaling kinetics, as reflected in IFNB mRNA expression, did not differ in either speed or magnitude relative to untreated A549 cells during early time points 1–6 h, . This in−/− A549 cells with SLR14, SeV and OH-SLR14, a stem loop RNA lacking triphosphate, for 6 h demonstrated that RIG-I−/− cells failed to produce any type I interferon  and RIG-Iterferon . This coWe next sought to directly observe changes in RIG-I subcellular distribution following stimulation with RIG-I-specific ligands using fluorescence confocal microscopy. First, we set out to determine whether the fluorescent tags used in this study will perturb RIG-I signaling. Attachment of a mNeonGreen or HaloTag fluorescent tag to the RIG-I amino-terminus via a flexible linker  had no evide infra).Having confirmed that fluorescent labels on both RIG-I and RNA ligand do not disrupt signaling, we sought to visualize RIG-I interactions with, and responses to, a viral RNA mimic in real time with live-cell fluorescent microscopy. Additionally, labeling of the mitochondrial network with potentiometric dyes (MitoTracker-CMXRos and MitoTracker-DeepRed) enabled us to determine whether RIG-I accumulated at the mitochondrial network following RNA activation. Ectopic expression of HaloTag-RIG-I labeled with OregonGreen in HEK293T cells produced robust RIG-I fluorescence which uniformly fills the cytosol in the absence of RNA stimulation (not shown). Following transfection of 1 μg/mL of SLR14-647 for 24 h, the stimulatory RNA concentrated primarily at local cytoplasmic sites that appear as bright puncta  and pUNO-mCherry-IRF3 (yellow) were either mock infected  or infec20Throughout the time courses examined in this study (6–24 h), the distribution of overexpressed RIG-I did not change, regardless of the cell type or stimulatory RNA ligand. Despite a clear uptake of the fluorescently labeled dsRNA and direct evidence of active RIG-I signaling via IRF3, there was no large-scale translocation of RIG-I, in contradiction of previously published studies.To determine whether the lack of apparent subcellular RIG-I redistribution was caused by the inherent limitations of ectopic RIG-I expression or whether it represents an actual property of RIG-I signaling, we used CRISPR-Cas9 to fluorescently tag the endogenous RIG-I amino-terminus with mNeonGreen in A549 cells. In this case, we used the same flexible fluorophore linker as we employed in the transiently expressed construct, and we verified that the fluorescently tagged RIG-I was expressed as effectively as WT using western blotting . In addiIn the absence of dsRNA stimulation , mNG-RIGAt 3 h after transfection of mNG-RIG-I A549 cells with 100 ng/mL SLR14-647, the distribution of mNG-RIG-I (green) remained unchanged, with no significant redistribution to the perinuclear region, as seen by line tracing . In cellTo ensure that the absence of RIG-I redistribution following dsRNA stimulation was not specific to one dsRNA PAMP, we also visualized RIG-I distribution following infection with a high titer of SeV at 3 h post-infection. Although the viral RNA was unlabeled in this case, we were nonetheless able to simultaneously track mNG-RIG-I, mCherry-IRF3 (yellow), and the mitochondrial network (red), enabling us to measure the changes in RIG-I distribution specifically within cells where RIG-I signaling was active. At 3 h after SeV infection, RIG-I did not colocalize with the mitochondria, although in these same cells, IRF3 (yellow) had translocated to the nucleus, thereby indicating that RIG-I signaling was completed . At 3 h To quantitatively analyze any RIG-I-MAVS colocalization, we measured the mean mNG-RIG-I fluorescent signal at the position of the mitochondria and compared it with the mean fluorescent mNG-RIG-I signal in cytosolic regions outside of the mitochondria. The ratio of these values allowed for direct comparison of the mNG-RIG-I cellular distribution between different conditions. Compared with mock-infected cells, stimulation with SLR14-647 at 3 h did not increase RIG-I colocalization with the mitochondria (p = 0.09) and stimulation with SeV at 3 h resulted in less RIG-I colocalization with the mitochondria relative to mock (p < 0.0001) . TherefoIn our immunofluorescence experiments, activation of RIG-I signaling and subsequent IRF3 translocation into the nucleus was not accompanied by a detectable accumulation of mNG-RIG-I at the mitochondrial network 3 h post-stimulation. Therefore, our data suggest that mass-translocation and aggregation are not required during the peak phase of RIG-I signaling.21 and it was therefore of interest to replicate these results.Although RIG-I did not accumulate at the mitochondrial network during the peak phase of RIG-I signaling (3 h), we sought to determine whether this accumulation might instead occur at later times after RIG-I stimulation with viral RNA (vRNA) PAMPs (~24 h), in keeping with results from previous studies conducted at late time points. Prior studies of RIG-I subcellular organization during active signaling had reported RIG-I redistribution 10–40 h after infection,At 12 h post-transfection with 1 μg/mL unlabeled SLR14, mNG-A549 cells continued to actively signal, as seen by the elevated nuclear levels of IRF3 (yellow); however, the distribution of RIG-I remained unchanged . CompariDuring late time points, after cells had been transfected with labeled SLR14, we continued to observe a similar RIG-I subcellular distribution as observed at 3 h. At 24 h post-transfection with 100 ng/mL SLR14-647, the distribution of RIG-I remained uniform, whereas signaling remained active, as seen via IRF3 translocation to the nucleus . As obseTo ensure that the lack of RIG-I colocalization with mitochondria was not limited to these specifically chosen images, we measured the total mNG-RIG-I fluorescent signal at the position of the mitochondria and compared it with the fluorescent mNG-RIG-I signal in cytosolic regions outside of the mitochondria under various conditions, using multiple samples to obtain a good statistical sampling. Compared with mock-infected cells, stimulation with SLR14-647 at 24 h resulted in a small increase in RIG-I colocalization with the mitochondria (p = 0.003), whereas stimulation with SeV at 24 h resulted in less RIG-I colocalization with mitochondria relative to mock (p < 0.0001) .To determine whether the use of mitochondrial labeling was an adequate proxy for the location of MAVS, we then compared the distribution of RIG-I with immunolabeled MAVS in fixed cells in order to determine whether the degree of RIG-I colocalization had changed. Fixed mNG continued to fluoresce, enabling continued visualization of mNG-RIG-I, and mock-treated cells showed a similar diffuse cytosolic distribution of mNG-RIG-I and mCherry-IRF3 as observed in the live-cell images . At 3 h Although endogenous RIG-I did not redistribute toward the mitochondria, some of the RIG-I pool was observed to form puncta that colocalized with transfected RNA throughout the time course of signaling. To determine the kinetics of the appearance of these RIG-I:RNA bodies, we quantified RIG-I puncta at both early and late time points.Although mNG-RIG-I (green) did not colocalize with the mitochondria at either 3 or 24 h post-transfection with 100 ng/mL SLR14-647, RIG-I did appear to form detectable cytosolic bodies in other, non-mitochondrial regions of the cytosol . Further8 At 3 h, OH-SLR14 (red) aggregated in similar puncta within cells, and RIG-I puncta (green) colocalized to similar degree  and observed continued formation of RIG-I aggregations and puncta , indicatGiven that RIG-I aggregation at late times may represent a secondary, non-signaling response to viral infection, we sought to determine if the basal pool of cytoplasmic RIG-I is sufficient to induce formation of these structures. To this end, we used cycloheximide to block downstream, interferon-induced translation of additional RIG-I receptors. Treatment of cells with 100 μg/mL of cycloheximide for 2 h prior to dsRNA stimulation restricted the cellular pool of RIG-I exclusively to the receptors present before viral RNA stimulation, so that by 24 h after transfection with 100 ng/mL SLR14-647, the overall cytosolic level of RIG-I decreased significantly . HoweverGiven the rapid signal produced by RIG-I in response to viral RNA and the absence of observable RIG-I complexes at the mitochondrial network during RIG-I signaling, we wondered whether large amounts of viral RNA are actually required to mount this response, as suggested by the prevailing model for RIG-I signaling. To explore the required RNA stoichiometry using an orthogonal approach, we quantified the number of RIG-I ligands required to stimulate RIG-I signaling.in cellulo in both overexpressed and endogenous imaging experiments. Therefore, despite stimulation with a quantity of vRNA sufficient to saturate the RIG-I signaling response, the total lack of RIG-I redistribution indicates that active RIG-I signaling is again either too minute or too transient to be observed.As an initial approach for determining the response of RIG-I to varying concentrations of viral RNA, we transfected A549 cells with 0.01 to 100 ng/mL SLR14 for 6 h . We obseThe amount of RNA required to stimulate RIG-I signaling was quantitated using SeV defective interfering (DI) RNA . We founIn this study, we directly monitored the kinetic behavior and spatial location of activated RIG-I in living cells. We find that RIG-I responds remarkably rapidly upon stimulation with RNA PAMPs and that the receptor selectively identifies, binds, and transmits a robust transcriptional response in less than an hour. The speed of this response is sufficiently rapid to match the fast kinetics of viral infection and replication, which must be detected immediately to marshal appropriate cellular defensive responses. Signaling processes that occur after 24 h would be too slow to provide an effective antiviral defense, and however, previous studies visualizing the distribution of RIG-I have focused almost exclusively on this late time frame, after which the initial burst of functional signaling has long since passed. It was therefore important to take a fresh look at the RNA ligand requirements and subcellular localization kinetics of the RIG-I receptor during the course of signal induction.30 Like the rapid speed of RIG-I activation, this behavior is consistent with a signaling pathway that must be triggered immediately and efficiently upon viral infection. Our findings similarly align with studies showing that incoming or replication-defective viral RNAs are still capable of initiating a robust RIG-I-specific interferon response.29An examination of RNA ligand stoichiometry revealed that RIG-I signaling requires tiny quantities of RNA PAMPs. We found that the entire process can be triggered by as few as an average of 10 molecules of intracellular viral, in broad agreement with prior measures of RIG-I sensitivity.The fact that antiviral signaling by RIG-I is rapid and highly efficient is consistent with the fact that this process is driven by the resident pool of constitutively expressed RIG-I and that it does not involve additional rounds of RIG-I expression, signal amplification, or engagement of RIG-I as a downstream ISG. Specifically, when cycloheximide is used to block downstream RIG-I translation, one still observes robust RIG-I signaling upon treatment with viral RNA PAMPS, occurring with essentially the same magnitude and kinetics as in the absence of cycloheximide. Again, this makes sense given that the antiviral response must be immediate, and a requirement for additional receptor expression would slow down a critical first-response in the cell.27 we observe that RIG-I is both a trigger of interferon expression and an ISG and that RIG-I protein expression increases after cellular stimulation with RNA PAMPs. However, this rise in protein expression occurs many hours after RNA stimulation, and it correlates with a decline in overall IFN signaling. This finding raises a larger and even more intriguing question: If boosted levels of RIG-I expression do not enhance antiviral signaling, then does the overexpression of RIG-I serve as some other type of antiviral effector? There are several possible models for this, and they are best addressed by monitoring the subcellular localization of RIG-I (vide infra).As reported in previous studies,31 Recent studies have even proposed that ubiquitination strengthens RIG-I signaling by bridging large RIG-I:RNA filaments and thereby increasing the size of signaling complexes.32 This model originated from in vitro studies in which high concentrations of isolated CARD protein domains were shown to aggregate in buffer solution, resulting in a behavior that was taken as proxy for signaling. Because isolated CARDs form aggregated polymers in vitro and because the mutations that disrupted this CARD polymerization in a test tube were also observed to inhibit RIG-I signaling in cells,22 it was extrapolated that large aggregates of full-length RIG-I are necessarily required for signaling in cellulo and that aggregates are markers for RIG-I activation. Furthermore, according to this model, increased RIG-I expression, stimulated by interferon, would be expected to strengthen RIG-I signaling by adding to the size of the RNA:RIG-I mass aggregate. However, published attempts to visualize RIG-I oligomerization, and that of other components of the pathway, directly in cells have failed.33 For example, split-luciferase complementation studies failed to detect any increase in RIG-I complex formation at 4 h post-transfection with poly(I:C).34 Similarly, our observations on the speed and efficiency of RIG-I signaling support a dramatically different mechanistic model for RIG-I signaling, suggesting that it requires only a small handful of active receptors to relay a fully active signal to MAVS and thereby transmit the antiviral signaling response further downstream. Given that conventional models of RIG-I signaling posit the obligate accumulation of RIG-I protein on the mitochondrial membrane, we set out to monitor the dynamics of RIG-I expression and subcellular localization directly.The fact that RIG-I signaling is so fast, requires so few copies of viral RNA, and is fully activated by short dsRNA molecules is inconsistent with conventional models for RIG-I signaling. This is understandable, as earlier models for the pathway were based on experiments conducted long before advances in structural biology, and cellular imaging made it possible to visualize stages of the process. According to the prevailing “mass aggregate” model for RIG-I signaling, viral RNA stimulation causes RIG-I to accumulate, polymerize, and form massive filaments on long regions of double-stranded viral RNA, thereby presenting a large array of free CARDs for interaction with the MAVS adapter protein.35 and SeV36 stimulation of interferon are dependent on MAVS in cellulo and in vivo, the signaling events described in this work are expected to require the RIG-I:MAVS interaction.Using CRISPR-tagged endogenous RIG-I, imaging studies revealed a uniform cytosolic distribution of the receptor, confirming that RIG-I circulates continuously throughout the cytoplasm. Similar results in a broad array of cell lines were observed with tagged, transfected RIG-I. After transfection with RNA PAMPs, RIG-I displays the same uniform cytoplasmic distribution during the most robust stage of signaling, which is inconsistent with the requirement for a massive aggregated signaling complex. During these same early time frames after RNA induction, we confirmed that dsRNA successfully entered the cell, transcription factor IRF3 had translocated to the nucleus and that interferon was expressed, thereby confirming that the RIG-I signaling pathway was fully functional and that we were observing an accurate picture of RIG-I behavior during active signaling. In addition there were no large changes to the subcellular organization of infected cells. Rather, our results suggested that active signaling complexes, such as those involved in a rapid relay from a handful of active receptors are beneath the detection limit for visualization by fluorescence confocal microscopy. It is important to note that the absence of RIG-I colocalization with MAVS does not suggest a MAVS-independent signaling process, which is well supported in the literature. Given that both stem loop RNA38 However, again, this is likely due to differences in the methods employed during earlier studies. Given the increase in total RIG-I ISG expression that occurs after RNA stimulation, RIG-I concentration is likely to be elevated at the mitochondria, just as it is elevated everywhere else in the cell.It is important to note that large, aggregated deposits of RIG-I were never observed at the mitochondrial network, whether measured early or late in the process, in direct contradiction of previous studies suggesting aggregation at either the mitochondria or the perinuclear region.19 These species contained not only RIG-I and influenza A virus (IAV) RNA but also other markers of stress granules, and they also appeared during the latter phases of infection.19 Importantly, the stress granules reported previously were observed only after 12 h post-infection, suggesting that they form too late to be linked with productive antiviral signaling. Indeed, our observation of RIG-I colocalization with both signaling and non-signaling dsRNAs suggests that these puncta of accumulated RIG-I are not related to signaling at all. These RIG-I-containing bodies may simply represent locations where RIG-I accumulates, post-signaling or otherwise, as it awaits degradation. More interesting, however, is the possibility that these RIG-I bodies serve a non-signaling effector function, perhaps by sequestering viral  RNAs in non-membranous subcellular structures where it cannot be replicated or translated.At all time frames after RNA transfection, but especially during later stages (~24 h), cellular puncta containing abundant RIG-I protein accumulate at localized sites in the cell. In certain cases, these RIG-I species co-aggregated with transfected RNA, regardless of whether the RNA was a stimulatory PAMP. Importantly, these RIG-I:RNA bodies, which do not associate with the mitochondrial network, also form after direct treatment with IFN-α (in the absence of transfected RNA), indicating that their formation is independent of RNA identity and is linked to the role of RIG-I as an ISG. These RIG-I RNP bodies are reminiscent of RIG-I-containing “antiviral stress granules” that have been previously reported.In summary, we have determined that RIG-I signaling is quickly initiated from a small number of dsRNAs in the cytosol and that it requires only the constitutively expressed level of RIG-I receptors. Even when large amounts of viral RNA are present, we observe that RIG-I signaling proceeds without aggregation at the mitochondrial network. This suggests that the active RIG-I signal is transmitted transiently, rather than accumulating as a massive oligomeric complex at the MAVS interface. Finally, we directly observe that RIG-I forms large, cytosolic RNP bodies with dsRNA, regardless of the RNA identity or size, suggesting that RIG-I aggregation is connected to the formation of specialized RNP bodies rather than with signaling. We believe that these findings help to distinguish RIG-I signaling from RIG-I oligomerization, showing that these are separate phenomena, thereby enabling future studies to better understand the mechanistic role of RIG-I during the cellular antiviral response.Although this study indicates that certain aspects of the standard RIG-I signaling model require revision, it is not a comprehensive dissection of the entire signaling cascade and all the macromolecular complexes that participate along the pathway. For example, we used endogenously tagged RIG-I to monitor its spatial and temporal behavior during active signaling, but our parallel studies of MAVS and IRF-3 depended on immunolabeling studies of fixed samples, which ultimately limits the conclusions that we can draw about the spatiotemporal dynamics of these important components of the signaling pathway. More thorough live-cell imaging and complementary approaches, such as FRAP (fluorescence recovery after photobleaching), are required for a complete picture of the overall pathway, but these will necessitate a comprehensive endogenous labeling approach that includes tags on MAVS and Riplet. A second limitation is that although our data suggest that massive RIG-I-MAVS oligomers do not contribute to active signaling, our conclusions are restricted by the limited spatial resolution of the methods employed here . Similarly, although endogenous RIG-I-MAVS colocalization was not detectable during active signaling in this study, this may be explained by a relatively transient RIG-I-MAVS interaction. Implementation of endogenous split-reporter systems may ultimately be helpful for investigating such interactions.anna.pyle@yale.edu).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anna Pyle  containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. All cells maintained, and all experiments and imaging carried out, at 37C in 5% CO2.A549 cells, including CRISPR-generated knock-in cell lines and RIG-I43 with either a triphosphate or hydroxyl 5’ guanosine. Synthesized RNAs were deprotected and purified via polyacrylamide gel and analyzed via mass spectrometry for purity. AlexaFluor-647 NHS ester (ThermoFisher) was conjugated to an amino-modified uridine (C6) (ChemGenes) on the loop region (5’ GGAUCGAUCGAUCG UUCG CGAUCGAUCGAUCC 3’).Stem Loop RNAs (5’ GGAUCGAUCGAUCG UUCG CGAUCGAUCGAUCC 3’) were synthesized on a MerMade 12 DNA-RNA synthesizer using previously described methodsSLR14 was added to OptiMEM (ThermoFisher) at 10X indicated concentrations using 20 μL/mL RNAiMAX (Invitrogen), and the RNA/lipid mixture was transfected onto cells at 1/10 total media volume. 2000 HA/mL stocks of Cantell strain Sendai virus (Charles River) were thawed, vortexed, and diluted in OptiMEM to the corresponding doses, then added to cells at 37°C for 60 min before replacement with fresh media.Following RNA transfection/viral infection, total cellular RNA was extracted via column purification (Omega Biotek) and treated with Turbo DNase (ThermoFisher) to remove DNA contamination. cDNA was reverse-transcribed from 500 ng total RNA extract with Superscript III (ThermoFisher) following manufacturer’s protocols using random hexamer primers (ThermoFisher). Following reverse-transcription RNA was degraded with 333 mM NaOH and cDNA purified via ethanol precipitation. RT-qPCR was performed using LightCycler 480 Mastermix (Roche) with the following primers: IFNB  and RIG-I [Fwd: CTGATTGCCACCTCAGTTGC Rev: GTCCCATGTCTGAAGGCGTA] were normalized to ACTB [Fwd: TTCCAGCAGATGTGGATCAG Rev: GGTGTAACGCAACTAAGTCA], and further normalized to Mock-infected wells to determine fold induction of gene expression.2 PVDF membrane, then blocked with TBST + 5% milk before treatment with anti-RIG-I  and anti-GAPDH antibodies overnight at 4C. Following washing, blots were treated with HRP-conjugated secondary antibodies and visualized using a blot imager (Bio-Rad). Blots were background-subtracted and the total band intensity was measured via ImageJ, then the ratio of RIG-I/GAPDH expression calculated from both bands.Protein lysates were extracted from A549 cells treated with 1 μg/mL SLR14 or 100 HA/mL SeV for 1h to 24h, the expression of both RIG-I and GAPDH was measured via western blot, and the difference in RIG-I expression was calculated relative to mock-infected wells. Cells lysed at indicated times with NP-40 buffer containing protease and phosphatase inhibitors (Roche). Equal quantities of lysate were separated on 4-20% SDS-PAGE (Bio-Rad) and transferred onto 0.45 μm5 cells/mL on 18mm acid-washed #1.5 glass coverslips in 1mL media and transfected for the indicated timepoints. HaloTag-RIG-I was labeled with 1 μM HaloTag OregonGreen for 15 min prior to fixation. Mitochondria were labeled with 100 nM MitoTracker-CMXRos or MitoTracker-DeepRed for 30 min prior to fixation. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with PBS + 0.1% Triton X-100 for 10 min, then washed and blocked with PBS + 3% BSA and 5% normal donkey serum for 20 min. Cells were then treated overnight at 4C with RIG-I and/or MAVS antibodies at 1:100 dilution in PBS + 3% BSA, washed five times in PBS + 3% BSA, and then treated with AlexaFluor-conjugated secondary antibodies at 1:500 dilution in PBS + 3% BSA for 30 minutes at RT. Cells were again washed 5 times, then mounted onto glass slides with Fluormount G + DAPI.HEK293T, WT A549 and mNG-RIG-I A549 cells were seeded at 1 x 10RIG-I-targeted sgRNAs  and 3 μg HDR templates were electroporated into A549 cells and expanded for 7-10 days. Cells were then treated with SLR14 at 1 μg/mL for 16h, then FITC+ cells were sorted via FACS and clonally expanded. Successful mNG+ knock-ins were screened via Sanger sequencing and immunoblot. Successful knock-in cells were examined for effective RIG-I signaling via qPCR and increased expression via flow cytometry.mNG-RIG-I A549 cells were stimulated with 1 μg/mL SLR14-647, 100 HA/mL SeV, or mock-stimulated for 3 to 24h, then trypsinized, pelleted and resuspended in PBS + 0.1% BSA, then the FITC fluorescent signal in 10,000 live cells for each condition was measured on a BD FACS LSR Fortessa X20 (Science Hill Flow Cytometry Facility). The mean cellular FITC signal and standard deviation for each condition were calculated via FlowJo.mNG-RIG-I cells were seeded on 35 mm dishes at 100,000 cells/mL in 2 mL F-12k. 48h prior to imaging, cells were transfected with 200 μL plasmid solution (pUNO-mCherry-IRF3 prepared in OptiMEM at 2.5 μg/mL with 40 μL/mL Lipofectamine 2000). Then, at 3-24h prior to imaging, cells were transfected with 100 ng/mL SLR14-647 or infected with 100 HA/mL SeV. Images were collected on an LSM 880 Airyscan confocal microscope (Zeiss) with 63X PlanApo lens. Image processing was carried out in FIJI.For analysis of RIG-I colocalization with mitochondria or MAVS, cells expressing labeled mitochondria or MAVS were imaged and processed in FIJI by enhancing contrast and mean-filtering to generate an accurate mask of the location of mitochondria or MAVS within each cell. The mask was then dilated twice to the region immediately surrounding the mitochondria, and regions within the first dilation excluded from the mask, leaving only cytosolic regions near, but not adjacent to, the mitochondria or MAVS. The mean mNG signal within both masks was then measured and the ratio of the two values (Mito/Outer mito) plotted for each cell. These RIG-I-containing bodies are defined as being at least 1 μm in diameter and at least ten times brighter than the mean RIG-I signal throughout the cytosol.46 and for IFN-β mRNA. To obtain an absolute SeV DI RNA copy number, we normalized to an in vitro transcribed (IVT) fragment of the DI RNA. In validation of our assay, we detected as few as 104 copies of the IVT DI RNA, which corresponds to a limit of detection of ~0.5 DI RNAs per cell. All SeV-infected RNA extracts, but not mock-infected cells, had SeV DI RNA levels above this limit of detection. In brief, a 250 nucleotide SeV DI RNA standard was in vitro transcribed from DNA47 and purified by polyacrylamide gel. Purification of the full-length product was confirmed via BioAnalyzer pico gel (Agilent), and RNA concentration was determined via Qubit RNA Assay (Thermo Fisher). A549 cells were infected with indicated concentrations of SeV, and total RNA was extracted from SeV-infected cells using TRIzol. Following total RNA purification, SeV DI RNA was selectively reverse-transcribed with 50 nM gene-specific primer (AGTCCAAGACTATCTTTATCTATGTCCAC) from both RNA extracts and standard curve reverse-transcribed using MarathonRT. cDNA was purified qPCR was performed using LightCycler 480 Mastermix (Roche) using the following primers: SeV .To better quantitate exactly how many RNA ligands are required to stimulate RIG-I signaling, we titrated cells with SeV and, following total RNA extraction, performed RT-qPCR for the SeV defective interfering (DI) RNA, which is the primary RIG-I ligand in SeV infection of A549 cells,Two-way comparisons were performed via student’s T test and comparisons between more than two conditions via one-way ANOVA in GraphPad Prism.1