Prospective Newborn Screening for SCID in Germany: A First Analysis by the Pediatric Immunology Working Group (API)

Background: T-cell receptor excision circle (TREC)-based newborn screening (NBS) for severe combined immunodeficiencies (SCID) was introduced in Germany in August 2019. / Methods: Children with abnormal TREC-NBS were referred to a newly established network of Combined Immunodeficiency (CID) Clinics and Centers. The Working Group for Pediatric Immunology (API) and German Society for Newborn Screening (DGNS) performed 6-monthly surveys to assess the TREC-NBS process after 2.5 years. / Results: Among 1.9 million screened newborns, 88 patients with congenital T-cell lymphocytopenia were identified (25 SCID, 17 leaky SCID/Omenn syndrome (OS)/idiopathic T-cell lymphocytopenia, and 46 syndromic disorders). A genetic diagnosis was established in 88%. Twenty-six patients underwent hematopoietic stem cell transplantation (HSCT), 23/26 within 4 months of life. Of these, 25/26 (96%) were alive at last follow-up. Two patients presented with in utero onset OS and died after birth. Five patients with syndromic disorders underwent thymus transplantation. Eight syndromic patients deceased, all from non-immunological complications. TREC-NBS missed one patient, who later presented clinically, and one tracking failure occurred after an inconclusive screening result. / Conclusion: The German TREC-NBS represents the largest European SCID screening at this point. The incidence of SCID/leaky SCID/OS in Germany is approximately 1:54,000, very similar to previous observations from North American and European regions and countries where TREC-NBS was implemented. The newly founded API-CID network facilitates tracking and treatment of identified patients. Short-term HSCT outcome was excellent, but NBS and transplant registries will remain essential to evaluate the long-term outcome and to compare results across the rising numbers of TREC-NBS programs across Europe

A Mild Form of SLC29A3 Disorder: A Frameshift Deletion Leads to the Paradoxical Translation of an Otherwise Noncoding mRNA Splice Variant

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the ‘rescue’ role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic

CD62L (L-selectin) shedding for assessment of perioperative immune sensitivity in patients undergoing cardiac surgery with cardiopulmonary bypass

OBJECTIVE: To investigate the suitability of blood granulocyte and monocyte sensitivity, as measured by the quantity of different agonists required to induce CD62L shedding, for assessment of perioperative immune changes in patients undergoing cardiac surgery with cardiopulmonary bypass. METHODS: Patients scheduled for aortocoronary bypass grafting or for valve surgery were included in this prospective observational study. Blood samples were drawn before anesthesia induction, directly after surgery and 48 hours after anesthesia induction. We determined the concentration of two different inflammatory stimuli--lipoteichoic acid (LTA) and tumor necrosis factor alpha (TNF)--required to induce shedding of 50% of surface CD62L from blood granulocytes and monocytes. In parallel monocyte surface human leukocyte antigen (HLA)-DR, and plasma interleukin (IL)-8, soluble (s)CD62L, soluble (s)Toll-like receptor (TLR)-2 and ADAM17 quantification were used to illustrate perioperative immunomodulation. RESULTS: 25 patients were enrolled. Blood granulocytes and monocytes showed decreased sensitivity to the TLR 2/6 agonist Staphylococcus aureus LTA immediately after surgery (p = 0.001 and p = 0.004 respectively). In contrast, granulocytes (p = 0.01), but not monocytes (p = 0.057) displayed a decreased postoperative sensitivity to TNF. We confirmed the presence of a systemic inflammatory response and a decreased immune sensitivity in the post-surgical period by measuring significant increases in the perioperative plasma concentration of IL-8 (p </= 0.001) and sTLR (p = 0.004), and decreases in monocyte HLA-DR (p<0.001), plasma sCD62L (p </= 0.001). In contrast, ADAM17 plasma levels did not show significant differences over the observation period (p = 0.401). CONCLUSIONS: Monitoring granulocyte and monocyte sensitivity using the "CD62L shedding assay" in the perioperative period in cardiac surgical patients treated with the use of cardiopulmonary bypass reveals common changes in sensitivity to TLR2/6 ligands and to TNF stimulus. Further long-term follow-up studies will address the predictive value of these observations for clinical purposes

Combined immunodeficiency develops with age in immunodeficiency-centromeric instability-facial anomalies syndrome 2 (ICF2)

The autosomal recessive immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) is characterized by immunodeficiency, developmental delay, and facial anomalies. ICF2, caused by biallelic ZBTB24 gene mutations, is acknowledged primarily as an isolated B-cell defect. Here, we extend the phenotype spectrum by describing, in particular, for the first time the development of a combined immune defect throughout the disease course as well as putative autoimmune phenomena such as granulomatous hepatitis and nephritis. We also demonstrate impaired cell-proliferation and increased cell death of immune and non-immune cells as well as data suggesting a chromosome separation defect in addition to the known chromosome condensation defect

Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

BACKGROUND:The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-alpha, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production. CONCLUSIONS/SIGNIFICANCE:The results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-alpha, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88(-/-) mice

Pattern recognition receptors as potential therapeutic targets in inflammatory rheumatic disease

The pattern recognition receptors of the innate immune system are part of the first line of defence against pathogens. However, they also have the ability to respond to danger signals that are frequently elevated during tissue damage and at sites of inflammation. Inadvertent activation of pattern recognition receptors has been proposed to contribute to the pathogenesis of many conditions including inflammatory rheumatic diseases. Prolonged inflammation most often results in pain and damage to tissues. In particular, the Toll-like receptors and nucleotide-binding oligomerisation domain-like receptors that form inflammasomes have been postulated as key contributors to the inflammation observed in rheumatoid arthritis, osteoarthritis, gout and systemic lupus erythematosus. As such, there is increasing interest in targeting these receptors for therapeutic treatment in the clinic. Here the role of pattern recognition receptors in the pathogenesis of these diseases is discussed, with an update on the development of interventions to modulate the activity of these potential therapeutic targets

Role of deficits in pathogen recognition receptors in infection susceptibility

This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), and the Fundação para a Ciência e Tecnologia (FCT) (IF/00735/2014 to A.C. and SFRH/BPD/96176/2013 to C.C.

Is the inflammasome a potential therapeutic target in renal disease?

The inflammasome is a large, multiprotein complex that drives proinflammatory cytokine production in response to infection and tissue injury. Pattern recognition receptors that are either membrane bound or cytoplasmic trigger inflammasome assembly. These receptors sense danger signals including damage-associated molecular patterns and pathogen-associated molecular patterns (DAMPS and PAMPS respectively). The best-characterized inflammasome is the NLRP3 inflammasome. On assembly of the NLRP3 inflammasome, post-translational processing and secretion of pro-inflammatory cytokines IL-1β and IL-18 occurs; in addition, cell death may be mediated via caspase-1. Intrinsic renal cells express components of the inflammasome pathway. This is most prominent in tubular epithelial cells and, to a lesser degree, in glomeruli. Several primary renal diseases and systemic diseases affecting the kidney are associated with NLRP3 inflammasome/IL-1β/IL-18 axis activation. Most of the disorders studied have been acute inflammatory diseases. The disease spectrum includes ureteric obstruction, ischaemia reperfusion injury, glomerulonephritis, sepsis, hypoxia, glycerol-induced renal failure, and crystal nephropathy. In addition to mediating renal disease, the IL-1/ IL-18 axis may also be responsible for development of CKD itself and its related complications, including vascular calcification and sepsis. Experimental models using genetic deletions and/or receptor antagonists/antiserum against the NLRP3 inflammasome pathway have shown decreased severity of disease. As such, the inflammasome is an attractive potential therapeutic target in a variety of renal diseases

Evolutionary Dynamics of Human Toll-Like Receptors and Their Different Contributions to Host Defense

Infectious diseases have been paramount among the threats to health and survival throughout human evolutionary history. Natural selection is therefore expected to act strongly on host defense genes, particularly on innate immunity genes whose products mediate the direct interaction between the host and the microbial environment. In insects and mammals, the Toll-like receptors (TLRs) appear to play a major role in initiating innate immune responses against microbes. In humans, however, it has been speculated that the set of TLRs could be redundant for protective immunity. We investigated how natural selection has acted upon human TLRs, as an approach to assess their level of biological redundancy. We sequenced the ten human TLRs in a panel of 158 individuals from various populations worldwide and found that the intracellular TLRs—activated by nucleic acids and particularly specialized in viral recognition—have evolved under strong purifying selection, indicating their essential non-redundant role in host survival. Conversely, the selective constraints on the TLRs expressed on the cell surface—activated by compounds other than nucleic acids—have been much more relaxed, with higher rates of damaging nonsynonymous and stop mutations tolerated, suggesting their higher redundancy. Finally, we tested whether TLRs have experienced spatially-varying selection in human populations and found that the region encompassing TLR10-TLR1-TLR6 has been the target of recent positive selection among non-Africans. Our findings indicate that the different TLRs differ in their immunological redundancy, reflecting their distinct contributions to host defense. The insights gained in this study foster new hypotheses to be tested in clinical and epidemiological genetics of infectious disease

Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C