We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

We aimed to investigate the association between polymorphisms located in type I interferon (IFN)-induced genes, genes belonging to the toll-like receptor (TLR) pathway, and genes encoding neurotransmitter receptors and the response to IFN-β treatment in patients with multiple sclerosis (MS).

Variation in genes encoding retinoid acid-inducible gene I (RIG-I)-like receptors (RLRs) has been implicated in the pathogenesis of autoimmune disorders. We investigated if polymorphisms in the IFIH1, RIG-I, LGP2 and VISA genes influence the risk for multiple sclerosis (MS) in a German case-control cohort comprising 716 patients and 706 controls. Evaluation of 18 single nucleotide polymorphisms (SNPs) in the four genes did not reveal significant single-SNP associations with MS risk, but two VISA polymorphisms were modestly associated with age of onset. Further, we provide initial evidence for combinatorial effects of polymorphic variants in the RIG-I, LGP2 and IFIH1 genes on MS risk.

Huntington disease (HD) is an inherited neurodegenerative disease caused by an abnormal expansion of a CAG repeat in the huntingtin HTT (HD) gene. The primary genetic determinant of the age at onset (AO) is the length of the HTT CAG repeat; however, the remaining genetic contribution to the AO of HD has largely not been elucidated. Recent studies showed that impaired functioning of the peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1alpha) contributes to mitochondrial dysfunction and appears to play an important role in HD pathogenesis. Further genetic evidence for involvement of PGC-1alpha in HD pathogenesis was generated by the findings that sequence variations in the PPARGC1A gene encoding PGC-1alpha exert modifying effects on the AO in HD. In this study, we hypothesised that polymorphisms in PGC-1alpha downstream targets might also contribute to the variation in the AO.

The gut microbiome is a critical modulator of host immunity and is linked to the immune response to respiratory viral infections. However, few studies have gone beyond describing broad compositional alterations in severe COVID-19, defined as acute respiratory or other organ failure. We profiled 127 hospitalized patients with COVID-19 (n=79 with severe COVID-19 and 48 with moderate) who collectively provided 241 stool samples from April 2020 to May 2021 to identify links between COVID-19 severity and gut microbial taxa, their biochemical pathways, and stool metabolites. 48 species were associated with severe disease after accounting for antibiotic use, age, sex, and various comorbidities. These included significant in-hospital depletions of Fusicatenibacter saccharivorans and Roseburia hominis, each previously linked to post-acute COVID syndrome or "long COVID", suggesting these microbes may serve as early biomarkers for the eventual development of long COVID. A random forest classifier achieved excellent performance when tasked with predicting whether stool was obtained from patients with severe vs. moderate COVID-19. Dedicated network analyses demonstrated fragile microbial ecology in severe disease, characterized by fracturing of clusters and reduced negative selection. We also observed shifts in predicted stool metabolite pools, implicating perturbed bile acid metabolism in severe disease. Here, we show that the gut microbiome differentiates individuals with a more severe disease course after infection with COVID-19 and offer several tractable and biologically plausible mechanisms through which gut microbial communities may influence COVID-19 disease course. Further studies are needed to validate these observations to better leverage the gut microbiome as a potential biomarker for disease severity and as a target for therapeutic intervention.

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin-derived DCs, tonsil leukocytes, monocyte-derived DCs, CD34-derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell-surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte-surface molecules.

ATP-binding cassette (ABC) transporters prevent the access of pharmacological compounds to the ischemic brain, thereby impeding the efficacy of stroke therapies. ABC transporters can be deactivated by selective inhibitors, which potently increase the brain accumulation of drugs. Concerns have been raised that long-term ABC transporter deactivation may promote neuronal degeneration and, under conditions of ischemic stroke, compromise neurological recovery. To elucidate this issue, we exposed male C57BL/6 mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of the selective ABCB1 inhibitor tariquidar (8 mg/kg/day) or ABCC1 inhibitor MK-571 (10 mg/kg/day), which were administered alone or in combination with each other over up to 28 days, on neurological recovery and brain injury. Mice were sacrificed after 14, 28, or 56 days. The Clark score, RotaRod, tight rope, and open field tests revealed reproducible motor-coordination deficits in mice exposed to intraluminal MCAO, which were not influenced by ABCB1, ABCC1, or combined ABCB1 and ABCC1 deactivation. Brain volume, striatum volume, and corpus callosum thickness were not altered by ABCB1, ABCC1 or ABCB1, and ABCC1 inhibitors. Similarly, neuronal survival and reactive astrogliosis, evaluated by NeuN and GFAP immunohistochemistry in the ischemic striatum, were unchanged. Iba1 immunohistochemistry revealed no changes of the overall density of activated microglia in the ischemic striatum of ABC transporter inhibitor treated mice, but subtle changes of microglial morphology, that is, reduced microglial cell volume by ABCB1 deactivation after 14 and 28 days and reduced microglial ramification by ABCB1, ABCC1 and combined ABCB1 and ABCC1 deactivation after 56 days. Endogenous neurogenesis, assessed by BrdU incorporation analysis, was not influenced by ABCB1, ABCC1 or combined ABCB1 and ABCC1 deactivation. Taken together, this study could not detect any exacerbation of neurological deficits or brain injury after long-term ABC transporter deactivation in this preclinical stroke model.

Localised prostate cancer is commonly treated with external-beam radiotherapy. Moderate hypofractionation has been shown to be non-inferior to conventional fractionation. Ultra-hypofractionated stereotactic body radiotherapy would allow shorter treatment courses but could increase acute toxicity compared with conventionally fractionated or moderately hypofractionated radiotherapy. We report the acute toxicity findings from a randomised trial of standard-of-care conventionally fractionated or moderately hypofractionated radiotherapy versus five-fraction stereotactic body radiotherapy for low-risk to intermediate-risk localised prostate cancer.

c-Jun N-terminal kinase (JNK) is upregulated during multiple sclerosis relapses and at the peak of experimental autoimmune encephalomyelitis (EAE). We aim to investigate the effects of pharmacological pan-JNK inhibition on the course of myelin oligodendrocyte glycoprotein (MOG35-55) EAE disease using in vivo and in vitro experimental models. EAE was induced in female C57BL/6JRj wild type mice using MOG35-55. SP600125 (SP), a reversible adenosine triphosphate competitive pan-JNK inhibitor, was then given orally after disease onset. Positive correlation between SP plasma and brain concentration was observed. Nine, but not three, consecutive days of SP treatment led to a significant dose-dependent decrease of mean cumulative MOG35-55 EAE severity that was associated with increased mRNA expression of interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) in the spinal cord. On a histological level, reduced spinal cord immune cell-infiltration predominantly of CD3+ T cells as well as increased activity of Iba1+ cells were observed in treated animals. In addition, in vitro incubation of murine and human CD3+ T cells with SP resulted in reduced T cell apoptosis and proliferation. In conclusion, our study demonstrates that pharmacological pan-JNK inhibition might be a treatment strategy for autoimmune central nervous system demyelination.

Cre/LoxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. The field of microglial biology has particularly benefited from this technology as microglia have historically been difficult to transduce with virus or electroporation methods for gene delivery. Here, we interrogate four of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency and spontaneous recombination, depending on the Cre line and loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency in microglia, which could be extended to other cell types. There is increasing evidence that microglia are key regulators of neural circuit structure and function. Microglia are also major drivers of a broad range of neurological diseases. Thus, reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field of microglial biology and the development of microglia-based therapeutics.

Volumetric image analysis to detect progressive brain tissue loss in patients with multiple sclerosis (MS) has recently been suggested as a promising marker for "no evidence of disease activity." Software packages for longitudinal whole-brain volume analysis in individual patients are already in clinical use; however, most of these methods have omitted region-based analysis. Here, we suggest a fully automatic analysis pipeline based on the free software packages FSL and FreeSurfer.

Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.

Progressive multifocal leukoencephalopathy (PML) is an opportunistic central nervous system- (CNS-) infection that typically occurs in a subset of immunocompromised individuals. An increasing incidence of PML has recently been reported in patients receiving monoclonal antibody (mAb) therapy for the treatment of autoimmune diseases, particularly those treated with natalizumab, efalizumab and rituximab. Intracellular CD4(+)-ATP-concentration (iATP) functionally reflects cellular immunocompetence and inversely correlates with risk of infections during immunosuppressive therapy. We investigated whether iATP may assist in individualized risk stratification for opportunistic infections during mAb-treatment.

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.

Myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disorder. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal anti-FcRn antibody in murine MOG-IgG-associated experimental autoimmune encephalomyelitis (EAE).

Gut transit time is a key modulator of host-microbiome interactions, yet this is often overlooked, partly because reliable methods are typically expensive or burdensome. The aim of this single-arm, single-blinded intervention study is to assess (1) the relationship between gut transit time and the human gut microbiome, and (2) the utility of the 'blue dye' method as an inexpensive and scalable technique to measure transit time.

Experimental studies indicate shared molecular pathomechanisms in cerebral hypoxia-ischemia and autoimmune neuroinflammation. This has led to clinical studies investigating the effects of immunomodulatory therapies approved in multiple sclerosis on inflammatory damage in stroke. So far, mutual and combined interactions of autoimmune, CNS antigen-specific inflammatory reactions and cerebral ischemia have not been investigated so far.

The detection of new or enlarged white-matter lesions is a vital task in the monitoring of patients undergoing disease-modifying treatment for multiple sclerosis. However, the definition of 'new or enlarged' is not fixed, and it is known that lesion-counting is highly subjective, with high degree of inter- and intra-rater variability. Automated methods for lesion quantification, if accurate enough, hold the potential to make the detection of new and enlarged lesions consistent and repeatable. However, the majority of lesion segmentation algorithms are not evaluated for their ability to separate radiologically progressive from radiologically stable patients, despite this being a pressing clinical use-case. In this paper, we explore the ability of a deep learning segmentation classifier to separate stable from progressive patients by lesion volume and lesion count, and find that neither measure provides a good separation. Instead, we propose a method for identifying lesion changes of high certainty, and establish on an internal dataset of longitudinal multiple sclerosis cases that this method is able to separate progressive from stable time-points with a very high level of discrimination (AUC = 0.999), while changes in lesion volume are much less able to perform this separation (AUC = 0.71). Validation of the method on two external datasets confirms that the method is able to generalize beyond the setting in which it was trained, achieving an accuracies of 75 % and 85 % in separating stable and progressive time-points.