Ultrahigh frequencies of peripherally matured LGI1- and CASPR2-reactive B cells characterize the cerebrospinal fluid in autoimmune encephalitis.

Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with ≥4 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (P < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (P = 0.004 and P < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS.

Insulin-like peptides and ovary ecdysteroidogenic hormone differentially stimulate physiological processes regulating egg formation in the mosquito Aedes aegypti.

Mosquitoes including Aedes aegypti are human disease vectors because females must blood feed to produce and lay eggs. Blood feeding triggers insulin-insulin growth factor signaling (IIS) which regulates several physiological processes required for egg development. A. aegypti encodes 8 insulin-like peptides (ILPs) and one insulin-like receptor (IR) plus ovary ecdysteroidogenic hormone (OEH) that also activates IIS through the OEH receptor (OEHR). In this study, we assessed the expression of A. aegypti ILPs and OEH during a gonadotrophic cycle and produced each that were functionally characterized to further understand their roles in regulating egg formation. All A. aegypti ILPs and OEH were expressed during a gonadotrophic cycle. Five ILPs (1, 3, 4, 7, 8) and OEH were specifically expressed in the head, while antibodies to ILP3 and OEH indicated each was released after blood feeding from ventricular axons that terminate on the anterior midgut. A subset of ILP family members and OEH stimulated nutrient storage in previtellogenic females before blood feeding, whereas most IIS-dependent processes after blood feeding were activated by one or more of the brain-specific ILPs and/or OEH. ILPs and OEH with different biological activities also exhibited differences in IIS as measured by phosphorylation of the IR, phosphoinositide 3-kinase/Akt kinase (AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK). Altogether, our results provide the first results that compare the functional activities of all ILP family members and OEH produced by an insect.

The mosquito Aedes aegypti requires a gut microbiota for normal fecundity, longevity and vector competence.

Mosquitoes shift from detritus-feeding larvae to blood-feeding adults that can vector pathogens to humans and other vertebrates. The sugar and blood meals adults consume are rich in carbohydrates and protein but are deficient in other nutrients including B vitamins. Facultatively hematophagous insects like mosquitoes have been hypothesized to avoid B vitamin deficiencies by carryover of resources from the larval stage. However, prior experimental studies have also used adults with a gut microbiota that could provision B vitamins. Here, we used Aedes aegypti, which is the primary vector of dengue virus (DENV), to ask if carryover effects enable normal function in adults with no microbiota. We show that adults with no gut microbiota produce fewer eggs, live longer with lower metabolic rates, and exhibit reduced DENV vector competence but are rescued by provisioning B vitamins or recolonizing the gut with B vitamin autotrophs. We conclude carryover effects do not enable normal function.

Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis.

Autoantibodies against the extracellular domain of the N-methyl-d-aspartate receptor (NMDAR) NR1 subunit cause a severe and common form of encephalitis. To better understand their generation, we aimed to characterize and identify human germinal centres actively participating in NMDAR-specific autoimmunization by sampling patient blood, CSF, ovarian teratoma tissue and, directly from the putative site of human CNS lymphatic drainage, cervical lymph nodes. From serum, both NR1-IgA and NR1-IgM were detected more frequently in NMDAR-antibody encephalitis patients versus controls (both P < 0.0001). Within patients, ovarian teratoma status was associated with a higher frequency of NR1-IgA positivity in serum (OR = 3.1; P < 0.0001) and CSF (OR = 3.8, P = 0.047), particularly early in disease and before ovarian teratoma resection. Consistent with this immunoglobulin class bias, ovarian teratoma samples showed intratumoral production of both NR1-IgG and NR1-IgA and, by single cell RNA sequencing, contained expanded highly-mutated IgA clones with an ovarian teratoma-restricted B cell population. Multiplex histology suggested tertiary lymphoid architectures in ovarian teratomas with dense B cell foci expressing the germinal centre marker BCL6, CD21+ follicular dendritic cells, and the NR1 subunit, alongside lymphatic vessels and high endothelial vasculature. Cultured teratoma explants and dissociated intratumoral B cells secreted NR1-IgGs in culture. Hence, ovarian teratomas showed structural and functional evidence of NR1-specific germinal centres. On exploring classical secondary lymphoid organs, B cells cultured from cervical lymph nodes of patients with NMDAR-antibody encephalitis produced NR1-IgG in 3/7 cultures, from patients with the highest serum NR1-IgG levels (P < 0.05). By contrast, NR1-IgG secretion was observed neither from cervical lymph nodes in disease controls nor in patients with adequately resected ovarian teratomas. Our multimodal evaluations provide convergent anatomical and functional evidence of NMDAR-autoantibody production from active germinal centres within both intratumoral tertiary lymphoid structures and traditional secondary lymphoid organs, the cervical lymph nodes. Furthermore, we develop a cervical lymph node sampling protocol that can be used to directly explore immune activity in health and disease at this emerging neuroimmune interface.

Detection and significance of neuronal autoantibodies in patients with meningoencephalitis in Vientiane, Lao PDR.

BACKGROUND: The importance of autoimmune encephalitis and its overlap with infectious encephalitides are not well investigated in South-East Asia. METHODS: We report autoantibody testing, using antigen-specific live cell-based assays, in a series of 134 patients (cerebrospinal fluid and sera) and 55 blood donor controls (sera), undergoing lumbar puncture for suspected meningoencephalitis admitted in Vientiane, Lao People's Democratic Republic (PDR). RESULTS: Eight of 134 (6%) patients showed detectable serum neuronal autoantibodies, against the N-methyl-D-aspartate and gamma-aminobutyric acid A receptors (NMDAR and GABAAR), and contactin-associated protein-like 2 (CASPR2). Three of eight patients had accompanying autoantibodies in cerebrospinal fluid (two with NMDAR and one with GABAAR antibodies), and in two of these the clinical syndromes were typical of autoimmune encephalitis. Three of the other five patients had proven central nervous system infections, highlighting a complex overlap between diverse infectious and autoimmune causes of encephalitis. No patients in this cohort were treated with immunotherapy, and the outcomes were poor, with improvement observed in a single patient. CONCLUSIONS: In Lao PDR, autoimmune encephalitis is underdiagnosed and has a poor prognosis. Empiric immunotherapy should be considered after treatable infectious aetiologies are considered unlikely. Awareness and diagnostic testing resources for autoimmune encephalitis should be enhanced in South-East Asia.

Ad libitum consumption of protein- or peptide-sucrose solutions stimulates egg formation by prolonging the vitellogenic phase of oogenesis in anautogenous mosquitoes.

BACKGROUND: Anautogenous mosquitoes commonly consume nectars and other solutions containing sugar but are thought to only produce eggs in discrete gonadotrophic cycles after blood-feeding on a vertebrate host. However, some anautogenous species are known to produce eggs if amino acids in the form of protein are added to a sugar solution. Unclear is how different sources of amino acids in sugar solutions affect the processes that regulate egg formation and whether responses vary among species. In this study, we addressed these questions by focusing on Aedes aegypti and conducting some comparative assays with Aedes albopictus, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus. METHODS: Adult female mosquitoes were fed sugar solutions containing amino acids, peptides or protein. Markers for activation of a gonadotrophic cycle including yolk deposition into oocytes, oviposition, ovary ecdysteroidogenesis, expression of juvenile hormone and 20-hydroxyecdysone-responsive genes, and adult blood-feeding behavior were then measured. RESULTS: The five anautogenous species we studied produced eggs when fed two proteins (bovine serum albumin, hemoglobin) or a mixture of peptides (tryptone) in 10% sucrose but deposited only small amounts of yolk into oocytes when fed amino acids in 10% sucrose. Focusing on Ae. aegypti, cultures were maintained for multiple generations by feeding adult females protein- or tryptone-sugar meals. Ad libitum access to protein- or tryptone-sugar solutions protracted production of ecdysteroids by the ovaries, vitellogenin by the fat body and protease activity by the midgut albeit at levels that were lower than in blood-fed females. Females also exhibited semi-continual oogenesis and repressed host-seeking behavior. CONCLUSIONS: Several anautogenous mosquitoes produce eggs when provided ad libitum access to protein- or peptide-sugar meals, but several aspects of oogenesis also differ from females that blood-feed.

Screening for pathogenic neuronal autoantibodies in serum and CSF of patients with first-episode psychosis.

Patients with autoimmune encephalitides, especially those with antibodies to the N-methyl-D-aspartate receptor (NMDAR), often present with prominent psychosis and respond well to immunotherapies. Although most patients progress to develop various neurological symptoms, it has been hypothesised that a subgroup of patients with first-episode psychosis (FEP) suffer from a forme fruste of autoimmune encephalitis. Without accurate identification, this immunotherapy-responsive subgroup may be denied disease-modifying treatments. Thirty studies addressing aspects of this hypothesis were identified in a systematic review. Amongst other shortcomings, 15/30 reported no control group and only 6/30 determined cerebrospinal fluid (CSF) autoantibodies. To ourselves address these-and other-limitations, we investigated a prospectively ascertained clinically well-characterised cohort of 71 FEP patients without traditional neurological features, and 48 healthy controls. Serum and CSF were tested for autoantibodies against seven neuronal surface autoantigens using live cell-based assays. These identified 3/71 (4%) patient sera with weak binding to either contactin-associated protein-like 2, the NMDAR or glycine receptor versus no binding from 48 control samples (p = 0.28, Fisher's test). The three seropositive individuals showed no CSF autoantibodies and no differences from the autoantibody-negative patients in their clinical phenotypes, or across multiple parameters of peripheral and central inflammation. All individuals were negative for CSF NMDAR antibodies. In conclusion, formes frustes of autoimmune encephalitis are not prevalent among FEP patients admitted to psychiatric care. Our findings do not support screening for neuronal surface autoantibodies in unselected psychotic patients.

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development.

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Whole blood and blood components from vertebrates differentially affect egg formation in three species of anautogenous mosquitoes.

BACKGROUND: Most female mosquitoes are anautogenous and must blood feed on a vertebrate host to produce eggs. Prior studies show that the number of eggs females lay per clutch correlates with the volume of blood ingested and that protein is the most important macronutrient for egg formation. In contrast, how whole blood, blood fractions and specific blood proteins from different vertebrates affect egg formation is less clear. Since egg formation is best understood in Aedes aegypti, we examined how blood and blood components from different vertebrates affect this species and two others: the malaria vector Anopheles gambiae and arbovirus vector Culex quinquefasciatus. METHODS: Adult female mosquitoes were fed blood, blood fractions and purified major blood proteins from different vertebrate hosts. Markers of reproductive response including ovary ecdysteroidogenesis, yolk deposition into oocytes and number of mature eggs produced were measured. RESULTS: Ae. aegypti, An. gambiae and C. quinquefasciatus responded differently to meals of whole blood, plasma or blood cells from human, rat, chicken and turkey hosts. We observed more similarities between the anthropophiles Ae. aegypti and An. gambiae than the ornithophile C. quinquefasciatus. Focusing on Ae. aegypti, the major plasma-derived proteins (serum albumin, fibrinogen and globulins) differentially stimulated egg formation as a function of vertebrate host source. The major blood cell protein, hemoglobin, stimulated yolk deposition when from pigs but not humans, cows or sheep. Serum albumins from different vertebrates also variably affected egg formation. Bovine serum albumin (BSA) stimulated ovary ecdysteroidogenesis, but more weakly induced digestive enzyme activities than whole blood. In contrast, BSA-derived peptides and free amino acids had no stimulatory effects on ecdysteroidogenesis or yolk deposition into oocytes. CONCLUSIONS: Whole blood, blood fractions and specific blood proteins supported egg formation in three species of anautogenous mosquitoes but specific responses varied with the vertebrate source of the blood components tested.

Both living bacteria and eukaryotes in the mosquito gut promote growth of larvae.

We recently reported that larval stage Aedes aegypti and several other species of mosquitoes grow when living bacteria are present in the gut but do not grow when living bacteria are absent. We further reported that living bacteria induce a hypoxia signal in the gut, which activates hypoxia-induced transcription factors and other processes larvae require for growth. In this study we assessed whether other types of organisms induce mosquito larvae to grow and asked if the density of non-living microbes or diet larvae are fed obviate the requirement for living organisms prior results indicated are required for growth. Using culture conditions identical to our own prior studies, we determined that inoculation density of living Escherichia coli positively affected growth rates of Ae. aegypti larvae, whereas non-living E. coli had no effect on growth across the same range of inoculation densities. A living yeast, alga, and insect cell line induced axenic Ae. aegypti first instars to grow, and stimulated similar levels of midgut hypoxia, HIF-α stabilization, and neutral lipid accumulation in the fat body as E. coli. However, the same organisms had no effect on larval growth if heat-killed. In addition, no axenic larvae molted when fed two other diets, when fed diets supplemented with heat-killed microbes or lysed and heat-killed microbes. Experiments conducted with An. gambiae yielded similar findings. Taken together, our results indicate that organisms from different prokaryotic and eukaryotic groups induce mosquito larvae to grow, whereas no conditions were identified that stimulated larvae to grow in the absence of living organisms.

Hypomethylation of CYP2E1 and DUSP22 Promoters Associated With Disease Activity and Erosive Disease Among Rheumatoid Arthritis Patients.

OBJECTIVE: Epigenetic modifications have previously been associated with rheumatoid arthritis (RA). In this study, we aimed to determine whether differential DNA methylation in peripheral blood cell subpopulations is associated with any of 4 clinical outcomes among RA patients. METHODS: Peripheral blood samples were obtained from 63 patients in the University of California, San Francisco RA cohort (all satisfied the American College of Rheumatology classification criteria; 57 were seropositive for rheumatoid factor and/or anti-cyclic citrullinated protein). Fluorescence-activated cell sorting was used to separate the cells into 4 immune cell subpopulations (CD14+ monocytes, CD19+ B cells, CD4+ naive T cells, and CD4+ memory T cells) per individual, and 229 epigenome-wide DNA methylation profiles were generated using Illumina HumanMethylation450 BeadChips. Differentially methylated positions and regions associated with the Clinical Disease Activity Index score, erosive disease, RA Articular Damage score, Sharp score, medication at time of blood draw, smoking status, and disease duration were identified using robust regression models and empirical Bayes variance estimators. RESULTS: Differential methylation of CpG sites associated with clinical outcomes was observed in all 4 cell types. Hypomethylated regions in the CYP2E1 and DUSP22 gene promoters were associated with active and erosive disease, respectively. Pathway analyses suggested that the biologic mechanisms underlying each clinical outcome are cell type-specific. Evidence of independent effects on DNA methylation from smoking, medication use, and disease duration were also identified. CONCLUSION: Methylation signatures specific to RA clinical outcomes may have utility as biomarkers or predictors of exposure, disease progression, and disease severity.

Rheumatoid Arthritis Naive T Cells Share Hypermethylation Sites With Synoviocytes.

OBJECTIVE: To determine whether differentially methylated CpGs in synovium-derived fibroblast-like synoviocytes (FLS) of patients with rheumatoid arthritis (RA) were also differentially methylated in RA peripheral blood (PB) samples. METHODS: For this study, 371 genome-wide DNA methylation profiles were measured using Illumina HumanMethylation450 BeadChips in PB samples from 63 patients with RA and 31 unaffected control subjects, specifically in the cell subsets of CD14+ monocytes, CD19+ B cells, CD4+ memory T cells, and CD4+ naive T cells. RESULTS: Of 5,532 hypermethylated FLS candidate CpGs, 1,056 were hypermethylated in CD4+ naive T cells from RA PB compared to control PB. In analyses of a second set of CpG candidates based on single-nucleotide polymorphisms from a genome-wide association study of RA, 1 significantly hypermethylated CpG in CD4+ memory T cells and 18 significant CpGs (6 hypomethylated, 12 hypermethylated) in CD4+ naive T cells were found. A prediction score based on the hypermethylated FLS candidates had an area under the curve of 0.73 for association with RA case status, which compared favorably to the association of RA with the HLA-DRB1 shared epitope risk allele and with a validated RA genetic risk score. CONCLUSION: FLS-representative DNA methylation signatures derived from the PB may prove to be valuable biomarkers for the risk of RA or for disease status.