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Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries., (© 2024. The Author(s).)

The essential role of B cells is to produce protective immunoglobulins (Ig) that recognize, neutralize, and clear invading pathogens. This results from the integration of signals provided by pathogens or vaccines and the stimulatory microenvironment within sites of immune activation, such as secondary lymphoid tissues, that drive mature B cells to differentiate into memory B cells and antibody (Ab)-secreting plasma cells. In this context, B cells undergo several molecular events including Ig class switching and somatic hypermutation that results in the production of high-affinity Ag-specific Abs of different classes, enabling effective pathogen neutralization and long-lived humoral immunity. However, perturbations to these key signaling pathways underpin immune dyscrasias including immune deficiency and autoimmunity or allergy. Inborn errors of immunity that disrupt critical immune pathways have identified non-redundant requirements for eliciting and maintaining humoral immune memory but concomitantly prevent immune dysregulation. Here, we will discuss our studies on human B cells, and how our investigation of cytokine signaling in B cells have identified fundamental requirements for memory B-cell formation, Ab production as well as regulating Ig class switching in the context of protective versus allergic immune responses., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders., (© 2024. The Author(s).)

The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aim: Coeliac disease (CD) can remain undiagnosed due to absent/atypical symptoms. We evaluated screening for CD in undifferentiated paediatric patients in the emergency department (ED)., Methods: Subjects were all patients presenting to a children's hospital ED during the study period who had blood taken. Plasma remaining after routine care was tested for tissue transglutaminase IgA (tTG IgA) and deamidated gliadin IgG (DGP IgG) antibodies. Patients with positive results were counselled and offered confirmatory testing, then gastroenterology review if warranted., Results: An initial positive result for either DGP IgG or tTG IgA was found in 4.2% (44/1055). There was a normalisation of 76% (19/25) of positive DGP IgG and 44% (4/9) of tTG IgA results on repeat testing, which was not available in 27% (12/44). The prevalence of biopsy-confirmed CD was 0.7% (7/1055), including two new diagnoses and five subjects with known CD. Three likely cases could not be confirmed. All confirmed and likely cases were >10 years old. In children >10 years old, the prevalence of either biopsy-confirmed or likely CD was 3.3% (10/302). A family history of CD, growth concerns, recurrent abdominal pain and lethargy were associated with persistence of positive tests., Conclusion: Opportunistic testing for CD in ED requires further investigation as a CD screening strategy. Our results suggest optimal screening in this setting should be by initially testing for tTG IgA and total IgA in children >10 years old (minimising transiently positive tests). Transiently positive coeliac antibodies may also warrant further investigation as a predictor of future CD., (© 2023 The Authors. Journal of Paediatrics and Child Health published by John Wiley & Sons Australia, Ltd on behalf of Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

B cells are fundamental to host defence against infectious diseases; indeed, the ability of humans to elicit robust antibody responses following exposure to foreign antigens underpins long-lived humoral immunity and serological memory, as well as the success of most currently administered vaccines. However, B cells also have a dark side - they can cause myriad diseases, including autoimmunity, atopy, allergy and malignancy. Thus, it is critical to understand the molecular requirements for generating effective, high-affinity, specific immune responses following natural infection or vaccination, as well as for constraining B-cell function to mitigate B-cell-mediated immune dyscrasias. In this review, we discuss recent developments that have been derived from the identification and detailed analysis of individuals with inborn errors of immunity that disrupt cytokine signalling, resulting in immune dysregulatory conditions. These studies have defined fundamental cytokine/cytokine receptor/signal transducer and activator of transcription (STAT) signalling pathways that are critical for the generation and maintenance of human memory B-cell and plasma cell subsets during host defence, as well as revealed mechanisms of disease pathogenesis causing immune deficiency, autoimmunity and atopy. More importantly, these studies have identified molecules that could be targeted to either enhance humoral immunity in the settings of infection or vaccination, or attenuate humoral immunity that contributes to antibody-mediated autoimmunity or allergy., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases., Competing Interests: Declaration of interests F.H.E. and S.S. are employees and shareholders of BioVinc LLC, who has licensed patents for fluorescent probe compositions used in this work. P.I.C. has grant funding from Amgen. Other authors have no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The dopamine and glutamate hypotheses are well known in psychosis. Recently, the detection of autoantibodies against proteins expressed on the surface of cells in the central nervous system has raised the possibility that specific immune-mediated mechanisms may define a biological subgroup within psychosis, although no cohort of a first episode of psychosis in children has been investigated., Methods: Serum taken during the acute presentation of 43 children with first episode of psychosis and serum from 43 pediatric control subjects was assessed for the presence of immunoglobulin (Ig)G, IgM, or IgA antibodies to dopamine-2 receptor (D2R) and NR1 subunit of the N-methyl-D-aspartate receptor using a flow cytometry live cell-based assay and immunolabeling of murine primary neurons., Results: Using a cutoff of three SD above the control mean, serum antibodies to D2R or NR1 were detected in 8 of 43 psychotic patients but not detected in any of 43 control subjects (p < .001). Positive immunoglobulin binding to D2R was found in 3 of 43 psychosis patients (3 IgG, 1 IgM, 0 IgA) and to N-methyl-D-aspartate receptor in 6 of 43 patients (5 IgG, 1 IgM, 1 IgA). Specificity of antibody was confirmed by immunoaffinity purification and immunoabsorption. Significant differences in antibody binding to live, fixed, and fixed and permeabilized neurons were observed, confirming that only live cells can define surface epitope immunolabeling., Conclusions: This is the first report of serum antibodies to surface D2R and NR1 in pediatric patients with isolated psychosis, which supports the hypothesis that a subgroup of patients may be immune-mediated., (Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Objective: We examined a cohort of adults with aquaporin-4 (AQP4) antibody-negative neuromyelitis optica/neuromyelitis optica spectrum disorder (NMO/NMOSD) for antibodies to myelin oligodendrocyte glycoprotein (MOG)., Methods: We performed a flow cytometry cell-based assay using live human lentivirus-transduced cells expressing full-length surface MOG. Serum was tested in 23 AQP4 antibody-negative NMO/NMOSD patients with bilateral and/or recurrent optic neuritis (BON, n = 11), longitudinally extensive transverse myelitis (LETM, n = 10), and sequential BON and LETM (n = 2), as well as in patients with multiple sclerosis (MS, n = 76) and controls (n = 52)., Results: MOG antibodies were detected in 9/23 AQP4 antibody-negative patients with NMO/NMOSD, compared to 1/76 patients with MS and 0/52 controls (p < 0.001). MOG antibodies were detected in 8/11 patients with BON, 0/10 patients with LETM, and 1/2 patients with sequential BON and LETM. Six of 9 MOG antibody-positive patients had a relapsing course. MOG antibody-positive patients had prominent optic disc swelling and were more likely to have a rapid response to steroid therapy and relapse on steroid cessation than MOG antibody-negative patients (p = 0.034 and p = 0.029, respectively). While 8/9 MOG antibody-positive patients had good follow-up visual acuity, one experienced sustained visual impairment, 3 had retinal nerve fiber layer thinning, and one had residual spinal disability., Conclusions: MOG antibodies have a strong association with BON and may be a useful clinical biomarker. MOG antibody-associated BON is a relapsing disorder that is frequently steroid responsive and often steroid dependent. Failure to recognize the disorder early and institute immunotherapy promptly may be associated with sustained impairment., Classification of Evidence: This study provides Class II evidence that MOG antibodies are associated with AQP4 antibody-negative BON (sensitivity 69%, 95% confidence interval [CI] 42%-87%; specificity 99%, 95% CI 93.7%-99.8%).

Objective: To examine the clinical features of pediatric CNS demyelination associated with positive myelin oligodendrocyte glycoprotein (MOG) antibodies and to examine the functional effects of MOG antibody on oligodendrocyte cytoskeleton., Methods: We measured MOG antibody using a fluorescence-activated cell sorting live cell-based assay in acute sera of 73 children with CNS demyelination (DEM) (median age 8 years, range 1.3-15.3) followed for a median of 4 years. We used MO3.13 cells to examine immunoglobulin (Ig) G effects on oligodendrocyte cytoskeleton using 3D deconvolution imaging., Results: MOG antibodies were found in 31/73 patients with DEM (42%) but in 0/24 controls. At first presentation, MOG antibody-positive patients were more likely to have bilateral than unilateral optic neuritis (ON) (9/10 vs 1/5, respectively, p = 0.03), less likely to have brainstem findings (2/31 vs 16/42, p = 0.005), more likely to have a raised erythrocyte sedimentation rate >20 mm/h (9/19 vs 3/21, p = 0.05), less likely to have intrathecal oligoclonal bands (0/16 vs 5/27, p = 0.18), and less likely to be homozygous or heterozygous for human leukocyte antigen DRB1*1501 (3/18 vs 7/22, p = 0.46). MOG antibody positivity varied according to clinical phenotype, with ON and relapsing ON most likely to be seropositive. Two relapsing MOG antibody-positive patients treated with mycophenolate mofetil remain in remission and have become MOG antibody seronegative. Oligodendrocytes incubated with purified IgG from MOG antibody-positive patients showed a striking loss of organization of the thin filaments and the microtubule cytoskeleton, as evidenced by F-actin and β-tubulin immunolabelings., Conclusions: MOG antibody may define a separate demyelination syndrome, which has therapeutic implications. MOG antibody has functional effects on oligodendrocyte cytoskeleton.

The pathophysiology of psychosis is poorly understood, with both the cognitive and cellular changes of the disease process remaining mysterious. There is a growing body of evidence that points to dysfunction of the immune system in a subgroup of patients with psychosis. Recently, autoantibodies directed against neuronal cell surface targets have been identified in a range of syndromes that feature psychosis. Of interest is the detection of autoantibodies in patients whose presentations are purely psychiatric, such as those suffering from schizophrenia. Autoantibodies have been identified in a minority of patients, suggesting that antibody-associated mechanisms of psychiatric disease likely only account for a subgroup of cases. Recent work has been based on the application of cell-based assays-a paradigm whose strength lies in the expression of putative antigens in their natural conformation on the surface of live cells. The responsiveness of some of these newly described clinical syndromes to immune therapy supports the hypothesis that antibody-associated mechanisms play a role in the pathogenesis of psychotic disease. However, further investigation is required to establish the scope and significance of antibody pathology in psychosis. The identification of a subgroup of patients with antibody-mediated disease would promise more effective approaches to the treatment of these high-morbidity conditions.

Input from unmyelinated and myelinated nociceptors drives somatosympathetic responses to painful stimuli. Here we report that somatosympathetic responses recorded simultaneously in the cervical and splanchnic sympathetic nerves of the urethane-anaesthetized rat are qualitatively different. High intensity electrical stimulation of the sciatic nerve (SN) evoked characteristic biphasic responses in splanchnic nerve activity (N=6), but only monophasic responses in the cervical nerve (N=4). By colliding sympathoexcitatory responses to SN stimulation with precisely triggered baroinhibition evoked by electrical stimulation of the aortic depressor nerve, we found that cervical responses are analogous to the first phase of the splanchnic response, and that the biphasic splanchnic response is due to the arrival of two distinct afferent volleys at the site of sympathetic integration. Extracellular recordings of responses to SN stimulation in barosensitive neurons in the rostral ventrolateral medulla (RVLM; N=16) support these findings; responses were typically biphasic, although the relative magnitudes of the two phases were highly variable, and in some cases the longer-latency volley was completely absent. Our results suggest that sympathetic responses to somatic stimuli, mediated by the RVLM, are non-uniform and are dependent on the target of the particular sympathetic output. The identification of RVLM sympathetic premotor neurons with both biphasic and monophasic responses indicates that the difference in the splanchnic and cervical nerve responses is due to specific channeling of activity evoked by myelinated and unmyelinated nociceptors to the medulla. The results are discussed with regard to the differential control of sympathetic nerve activity.

Photonic crystal fibers (PCF) containing coated holes have recently been demonstrated experimentally, but haven't been studied theoretically and numerically thus far. We extend the multipole formalism to take into account coated cylinders, and demonstrate its accuracy even with metallic coatings. We provide numerical tables for calibration of other numerical methods. Further, we study the guidance properties of several PCF with coated holes: we demonstrate that the confinement mechanisms of PCFs with high index coated holes depend on wavelength, and exhibit plasmonic resonances in metal coated PCFs.