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This commentary provides a comprehensive overview of the challenges and opportunities in the field of drug development for rare diseases and especially of gene therapy products for ultra-rare diseases. It discusses the limited market size, reimbursement and scientific complexities that deter pharmaceutical investment in this field. Highlighting the pivotal role of charitable organizations like Fondazione Telethon, it showcases their efforts in funding research and ensuring access to innovative therapies. This commentary also addresses the challenges in therapy distribution, particularly regarding sustainability and global access. It outlines Fondazione Telethon's operational model to try to address these challenges. Finally, it appeals to governments and regulatory bodies to implement policies and incentives aimed at further fostering innovation and accessibility in rare disease drug development and access., Competing Interests: 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., (Copyright © 2024 Vavassori, Russell, Scotti and Benvenuti.)

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival., Methods: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency)., Results: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times., Discussion: This efficiency stemmed from the sole requirement of dissolving ( E )-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants., Conclusion: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes., Competing Interests: 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., (Copyright © 2024 Jorisch-Mühlebach, Pitts, Tinner, Teh, Roelli, Prader, Vavassori and Pachlopnik Schmid.)

Cytotoxic lymphocytes kill target cells through polarized release of the content of cytotoxic granules towards the target cell. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytosis (HLH) that occurs in mice and humans with inborn errors of lymphocyte cytotoxic function. The clinical and preclinical data indicate that the damage seen in severe, virally triggered HLH is due to an overwhelming immune system reaction and not the direct effects of the virus per se . The main HLH-disease mechanism, which links impaired cytotoxicity to excessive release of pro-inflammatory cytokines is a prolongation of the synapse time between the cytotoxic effector cell and the target cell, which prompts the former to secrete larger amounts of cytokines (including interferon gamma) that activate macrophages. We and others have identified novel genetic HLH spectrum disorders. In the present update, we position these newly reported molecular causes, including CD48-haploinsufficiency and ZNFX1-deficiency, within the pathogenic pathways that lead to HLH. These genetic defects have consequences on the cellular level on a gradient model ranging from impaired lymphocyte cytotoxicity to intrinsic activation of macrophages and virally infected cells. Altogether, it is clear that target cells and macrophages may play an independent role and are not passive bystanders in the pathogenesis of HLH. Understanding these processes which lead to immune dysregulation may pave the way to novel ideas for medical intervention in HLH and virally triggered hypercytokinemia., Competing Interests: JPS received honorary from Pharming for participation in a data monitoring committee DMC for Leniolisib. MF received a grant by Jazz Pharmaceuticals. The remaining 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., (Copyright © 2023 Planas, Felber, Vavassori and Pachlopnik Schmid.)

Introduction: Bowel preparation is crucial for colonoscopy completeness and lesions detection. Today, several cleansing products are equally recommended by guidelines, irrespective of patients' characteristics. Identification of preparation-specific risk factors for inadequate bowel preparation may lead to a personalized prescription of cleansing products to refine patients' tolerance and improve endoscopic outcomes., Methods: We prospectively enrolled consecutive outpatients referred for colonoscopy using either a high-volume [HV: 4 l polyethylene glycol (PEG)] or a low-volume (LV: 2 l PEG + bisacodyl) preparation. Day-before regimen or split-dose regimen was used for morning or afternoon colonoscopies, respectively. Univariate and multivariate analyses were conducted to identify risk factors related to inadequate bowel preparation, according to the Boston bowel preparation scale for HV and LV preparations., Results: We enrolled 2040 patients, of which 1815 were included in the final analysis (average age 60.6 years, 50.2% men). Half of them (52%) used LV preparation. Adequate preparation was achieved by 87.6% without differences between the HV and LV groups (89.2% vs. 86.6%; P = 0.098). The use of day-before regimen and incomplete assumption of PEG were independent predictors of poor visibility for either HV or LV preparation. However, different specific risk factors for HV [diabetes: odds ratio (OR), 3.81; 95% confidence interval (CI), 1.91-7.58; low level of instruction: OR, 1.95; 95% CI, 1.11-3.44; and previous abdominal surgery: OR, 2.27; 95% CI, 1.20-4.30] and for LV (heart disease: OR, 2.06; 95% CI, 1.09-3.88; age > 65 years: OR, 1.51; 95% CI, 1.01-2.27) preparations were identified., Conclusion: Day-before preparation and incomplete assumption of the purgative agents affect bowel visibility irrespective of the preparation volume. LV should be preferred to HV preparations in patients with diabetes, low level of instruction, and previous abdominal surgery, whereas an HV preparation should be preferred in patients with heart disease and in older patients., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.

Background: Low-volume preparations for colonoscopy have shown similar efficacy compared to high-volume ones in randomized controlled trials (RCT). However, most RCTs do not provide data about clinical outcomes including lesions detection rate. Moreover, real-life comparisons are lacking., Aim: To compare efficacy (both in terms of adequate bowel preparation and detection of colorectal lesions) and tolerability of a high-volume (HV: 4 L polyethylene glycol, PEG) and a low-volume (LV: 2 L PEG plus bisacodyl) bowel preparation in a real-life setting., Methods: Consecutive outpatients referred for colonoscopy were prospectively enrolled between 1 December 2014 and 31 December 2016. Patients could choose either LV or HV preparation, with a day-before schedule for morning colonoscopies and a split-dose for afternoon procedures. Adequate bowel preparation according to Boston Bowel Preparation Scale (BBPS), clinical outcomes including polyp detection rate (PDR), adenoma detection rate (ADR), advanced adenoma detection rate (AADR), sessile/serrated lesion detection rate (SDR) and cancer detection rate and self-reported tolerability of HV and LV were blindly assessed., Results: Total 2040 patients were enrolled and 1815 (mean age 60.6 years, 50.2% men) finally included. LV was chosen by 52% of patients (50.8% of men, 54.9% of women). Split-dose schedule was more common with HV (44.7% vs 38.2%, P = 0.005). High-definition scopes were used in 33.4% of patients, without difference in the two groups ( P = 0.605). HV and LV preparations showed similar adequate bowel preparation rates (89.2% vs 86.6%, P = 0.098), also considering the two different schedules (HV split-dose 93.8% vs LV split-dose 93.6%, P = 1; HV day-before 85.5% vs LV day-before 82.3%, P = 0.182). Mean global BBPS score was higher for HV preparations (7.1 ± 1.7 vs 6.8 ± 1.6, P < 0.001). After adjustment for sex, age and indications for colonoscopy, HV preparation resulted higher in PDR [Odds ratio (OR) 1.32, 95%CI: 1.07-1.63, P = 0.011] and ADR (OR 1.29, 95%CI 1.02-1.63, P = 0.038) and comparable to LV in AADR (OR 1.51, 95%CI 0.97-2.35, P = 0.069), SDR and cancer detection rate. The use of standard-definition colonoscopes was associated to lower PDR (adjusted OR 1.59, 95%CI: 1.22-2.08, P < 0.001), ADR (adjusted OR 1.71, 95%CI: 1.26-2.30, P < 0.001) and AADR (adjusted OR 1.97, 95%CI: 1.09-3.56, P = 0.025) in patients receiving LV preparation. Mean Visual Analogue Scale tolerability scored equally (7, P = 0.627) but a ≥ 75% dose intake was more frequent with LV (94.6% vs 92.1%, P = 0.003)., Conclusion: In a real-life setting, PEG-based low-volume preparation with bisacodyl showed similar efficacy and tolerability compared to standard HV preparation. However, with higher PDR and ADR, HV should still be considered as the reference standard for clinical trials and the preferred option in screening colonoscopy, especially when colonoscopy is performed with standard resolution imaging., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use., Competing Interests: 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., (Copyright © 2021 Haunerdinger, Moccia, Opitz, Vavassori, Dave and Hauri-Hohl.)

Background: Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019 (COVID-19) have been reported worldwide. Negative polymerase chain reaction (RT-PCR) testing associated with positive serology in most of the cases suggests a postinfectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed., Methods: We report a series of 4 pediatric patients admitted to Geneva University Hospitals with persistent fever and laboratory evidence of inflammation meeting the published definition of MIS-C related to COVID-19, to whom an extensive virological and immunological workup was performed., Results: RT-PCRs on multiple anatomical compartments were negative, whereas anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin A (IgA) and immunoglobulin G (IgG) were strongly positive by enzyme-linked immunosorbent assay and immunofluorescence. Both pseudoneutralization and full virus neutralization assays showed the presence of neutralizing antibodies in all children, confirming a recent infection with SARS-CoV-2. The analyses of cytokine profiles revealed an elevation in all cytokines, as reported in adults with severe COVID-19. Although differing in clinical presentation, some features of MIS-C show phenotypic overlap with hemophagocytic lymphohistiocytosis (HLH). In contrast to patients with primary HLH, our patients showed normal perforin expression and natural killer (NK) cell degranulation. The levels of soluble interleukin (IL)-2 receptor (sIL-2R) correlated with the severity of disease, reflecting recent T-cell activation., Conclusion: Our findings suggest that MIS-C related to COVID-19 is caused by a postinfectious inflammatory syndrome associated with an elevation in all cytokines, and markers of recent T-cell activation (sIL-2R) occurring despite a strong and specific humoral response to SARS-CoV-2. Further functional and genetic analyses are essential to better understand the mechanisms of host-pathogen interactions., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known., Objective: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease., Methods: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids., Results: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes., Conclusion: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease., (Copyright © 2021. Published by Elsevier Inc.)

Background:  Several scientific societies have endorsed non-anesthesiologist sedation (NAS) during gastrointestinal endoscopy, considering it a safe procedure when administered by adequately trained personnel. This study aimed to evaluate the occurrence of adverse events after implementation of the European Society of Gastrointestinal Endoscopy (ESGE) and European Society of Gastroenterology and Endoscopy Nurses and Associates (ESGENA) sedation training program., Methods:  From January 2017 to August 2018, data from all consecutive endoscopic procedures in adults (≥ 18 years) performed at our endoscopy unit were collected using an electronic reporting system., Results:  All staff (physicians and nurses) completed the ESGE-ESGENA sedation course. In total, 12 132 patients underwent endoscopic procedures, 10 755 (88.6 %) of which were performed in a non-anesthesiological setting. Of these, about 20 % used moderate sedation with midazolam + fentanyl and 80 % used deep sedation with additional propofol. No sentinel, 5 (0.05 %) moderate risk, and 18 (0.17 %) minor risk adverse events occurred, all during moderate or deep sedation, and all managed by endoscopy staff without the need for anesthesiologist assistance., Conclusions:  After completing the ESGE-ESGENA sedation training program, the rate of adverse events was very low in our institution. The findings support implementation of the program in all digestive endoscopy units and inclusion in the curriculum for physicians and nurses to ensure safe endoscopic procedures., Competing Interests: The authors declare that they have no conflicts of interest., (Thieme. All rights reserved.)

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Competing Interests: The authors declare that they have no conflict of interest.

We recently demonstrated that removal of one kidney (uninephrectomy [UniNx]) in mice reduced high-fat diet (HFD)-induced adipose tissue inflammation, thereby improving adipose tissue and hepatic insulin sensitivity. Of note, circulating cystatin C (CysC) levels were increased in UniNx compared with sham-operated mice. Importantly, CysC may have anti-inflammatory properties, and circulating CysC levels were reported to positively correlate with obesity in humans and as shown here in HFD-fed mice. However, the causal relationship of such observation remains unclear. HFD feeding of CysC-deficient (CysC knockout [KO]) mice worsened obesity-associated adipose tissue inflammation and dysfunction, as assessed by proinflammatory macrophage accumulation. In addition, mRNA expression of proinflammatory mediators was increased, whereas markers of adipocyte differentiation were decreased. Similar to findings in adipose tissue, expression of proinflammatory cytokines was increased in liver and skeletal muscle of CysC KO mice. In line, HFD-induced hepatic insulin resistance and impairment of glucose tolerance were further aggravated in KO mice. Consistently, chow-fed CysC KO mice were more susceptible to lipopolysaccharide-induced adipose tissue inflammation. In people with obesity, circulating CysC levels correlated negatively with adipose tissue Hif1α as well as IL6 mRNA expression. Moreover, healthy (i.e., insulin-sensitive) subjects with obesity had significantly higher mRNA expression of CysC in white adipose tissue. In conclusion, CysC is upregulated under obesity conditions and thereby counteracts inflammation of peripheral insulin-sensitive tissues and, thus, obesity-associated deterioration of glucose metabolism., (© 2020 by the American Diabetes Association.)

Competing Interests: The authors declare that they have no conflict of interest.