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Autistic adults commonly experience anxiety and worry, although knowledge on how worry presents and the content, extent, and experiences among autistic adults is limited. A convergent parallel mixed-methods approach was used to explore the presentation and experiences of worry in autistic and non-autistic adults. Quantitative surveys were used to compare the content and extent of worry in autistic adults to non-autistic adults, with semi-structured interviews also conducted with autistic adults to gain a deeper understanding of the experiences, impacts and content of worry in autistic adults. Findings indicated that autistic adults demonstrated clinically significant levels of worry which were substantially higher than non-autistic adults. Autistic adults described worry as a cycle of negative thoughts impacting their daily life. Findings indicate that autistic adults may worry more than non-autistic adults, impacting on participation in activities of daily living, sleep, and mental health., (© 2023. The Author(s).)

Serum Ab concentrations, selection for higher affinity BCRs, and generation of higher Ab affinities are important elements of immune response optimization and functions of germinal center (GC) reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by mouse GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether glucose uptake or glycolysis increases in GC B cells compared with their naive precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that reduction of the glucose transporter GLUT1 in mice after establishment of a preimmune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and plasma cell outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway, whose activity was important in controlling reactive oxygen species (ROS) and Ab-secreting cell production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose, an efficient precursor to glycosylation, supported robust production of and normal Ab secretion by Ab-secreting cells under glucose-free conditions. Collectively, the findings indicate that GCs depend on normal glucose influx, especially in plasma cell production, but reveal an unexpected metabolic flexibility in hexose requirements., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Cellular metabolism influences immune cell function, with mitochondrial fatty acid β-oxidation and oxidative phosphorylation required for multiple immune cell phenotypes. Carnitine palmitoyltransferase 1a (Cpt1a) is considered the rate-limiting enzyme for mitochondrial metabolism of long-chain fatty acids, and Cpt1a deficiency is associated with infant mortality and infection risk. This study was undertaken to test the hypothesis that impairment in Cpt1a-dependent fatty acid oxidation results in increased susceptibility to infection. Screening the Cpt1a gene for common variants predicted to affect protein function revealed allele rs2229738_T, which was associated with pneumonia risk in a targeted human phenome association study. Pharmacologic inhibition of Cpt1a increases mortality and impairs control of the infection in a murine model of bacterial pneumonia. Susceptibility to pneumonia is associated with blunted neutrophilic responses in mice and humans that result from impaired neutrophil trafficking to the site of infection. Chemotaxis responsible for neutrophil trafficking requires Cpt1a-dependent mitochondrial fatty acid oxidation for amplification of chemoattractant signals. These findings identify Cpt1a as a potential host determinant of infection susceptibility and demonstrate a requirement for mitochondrial fatty acid oxidation in neutrophil biology., (© 2022. The Author(s).)

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation., (Copyright © 2022 The Authors.)

Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways., Competing Interests: Declaration of interests J.C.R. is a founder, scientific advisory board member, and stockholder of Sitryx Therapeutics; a scientific advisory board member and stockholder of Caribou Biosciences; a member of the scientific advisory board of Nirogy Therapeutics; has consulted for Merck, Pfizer, and Mitobridge within the past 3 years; and has received research support from Incyte Corp., Calithera Biosciences, and Tempest Therapeutics. J.D.R. is a co-founder and stockholder in Raze Therapeutics, Toran, Serien Therapeutics, and Farber Partners and an advisor and stockholder in Agios Pharmaceuticals, Kadmon Pharmaceuticals, Bantam Pharmaceuticals, Colorado Research Partners, Rafael Holdings, the Barer Institute, and L.E.A.F. Pharmaceuticals; he has received consulting fees and research funding from Pfizer and Rafael and is the inventor of patents held by Princeton University. A.M.C., P.F., and T.B. are employees of Sitryx Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A key challenge in the development of novel chemotherapeutics is the design of molecules capable of selective toxicity to cancer cells. Antibodies have greater target specificity compared to small molecule drugs, but most are unable to penetrate cells, and predominantly target extracellular antigens. A nuclear-penetrating anti-DNA autoantibody isolated from the MRL/lpr lupus mouse model, 3E10, preferentially localizes to tumors, inhibits DNA repair, and selectively kills cancer cells with defects in DNA repair. A murine divalent single chain variable fragment of 3E10 with mutations for improved DNA binding affinity, 3E10 (D31N) di-scFv, has previously been produced in P. pastoris and yielded promising pre-clinical findings, but is unsuitable for clinical testing. The present study reports the design, expression and testing of a panel of humanized 3E10 (D31N) di-scFvs, some of which contain CDR substitution. These variants were expressed in a modified CHO system and evaluated for their physicochemical attributes and ability to penetrate nuclei to selectively cause DNA damage accumulation in and kill cancer cells with DNA repair defects. Secondary structure was conserved and most variants retained the key characteristics of the murine 3E10 (D31N) di-scFv produced in P. pastoris. Moreover, several variants with CDR substitutions outperformed the murine prototype. In conclusion, we have designed several humanized variants of 3E10 (D31N) di-scFv that have potential for application as monotherapy or conjugates for targeted nuclear drug delivery., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma, and both its surface expression and its biologic significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface GRP78 and leads to disease stabilization when used as single agent in a clinical trial. In this article, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens., Experimental Design: GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of multiple myeloma. Activity of PAT-SM6 was evaluated in combination with anti-multiple myeloma agents lenalidomide, bortezomib, and dexamethasone in vitro Finally, we report on a multiple myeloma patient with relapsed-refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide., Results: Although sGRP78 expression was present at all stages, it increased with disease progression and was even strongly elevated in patients with drug-resistant and extramedullary disease. Pretreatment with dexamethasone as well as dual combination of PAT-SM6/lenalidomide further increased sGRP78 expression and consecutively showed synergistic anti-multiple myeloma effects with PAT-SM6 in proliferation assays. As proof of concept, a 62-year-old male with triple resistant multiple myeloma treated with PAT-SM6, bortezomib, and lenalidomide experienced partial remission of both intra- and extramedullary lesions., Conclusions: PAT-SM6 therapy in combination regimens showed efficacy in relapsed-refractory multiple myeloma. Clin Cancer Res; 22(17); 4341-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a 'steady-state' to a bimodal flow. Initially, a 'shutdown' phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a 'recruitment' phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In a return to the early days of vaccine development during which effective vaccines were produced against viruses, virus-sized vaccine delivery systems have made a comeback. Using modern production technologies these nanoparticles have proved to be very effective at inducing cellular and humoral immune responses. Here, we review a number of vaccine delivery systems based on nanoparticles in the size range of typical viruses. Different strategies for generating these particles, ranging from recombinant virus-like particles to inert nanobeads via ISCOMs and nanoparticle-based DNA vaccine delivery systems, are discussed. In addition, possible mechanisms of immune induction are explored.

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.

Autoimmune gastritis is characterised by lymphocytic infiltration of the gastric submucosa, with loss of parietal and chief cells and achlorhydria. Often, gastritis is expressed clinically as cobalamin deficiency with megaloblastic anaemia, which is generally described as a disease of the elderly. Here, we report on two prepubertal children who developed autoimmune gastritis. One child developed autoimmune gastritis as part of a polyglandular autoimmune disease from a family with polyglandular autoimmune disease type II (PGA type II) and the other as part of a classic "thyro-gastric cluster," which may have been triggered by emotional trauma. Both children presented with normal small bowel biopsies, with abnormal gut permeability, which subsequently resolved. These patients are among the youngest reported to date. The immune systems targetted the gastric parietal cell autoantigens (ATP4A and ATP4B) in both children, similar to the elderly. The study of children with autoimmune gastritis and their families may provide additional insights into the disease's pathogenesis and may also lead to the identification of inheritable factors influencing susceptibility. This report underlines the necessity to screen paediatric patients with organ-specific autoimmune diseases for co-existent conditions. Children with polyglandular autoimmune disease are at particularly high risk.

Vaccination against foot-and-mouth disease virus (FMDV) is a major problem as current vaccines do not allow easy differentiation between infected and vaccinated animals. Furthermore, large scale production of inactivated virus poses significant risks. To address this we investigated the feasibility of using inert nano-beads that target antigen to dendritic cells (DCs) to induce immune responses against FMDV-specific synthetic peptides in sheep. Our results demonstrate that while single peptides induce responses in most sheep, the combination of multiple peptides either conjugated separately to individual nano-beads or conjugated as a mixture induce significant cell-mediated (CM) and humoral immune responses.

Researchers have developed murine lymphopenic, non-lymphopenic, transgenic, spontaneous and infectious agent based models to induce an experimental autoimmune gastritis (EAG) for the study of human organ-specific autoimmune disease. These models result in a chronic inflammatory mononuclear cell infiltrate in the gastric mucosa, destruction of parietal and zymogenic cells with autoantibodies reactive to the gastric parietal cells and the gastric H+/K+ ATPase (ATP4), arguably hallmarks of a human autoimmune gastritis (AIG). In the case of AIG, it is well documented that, in addition to parietal cell antibodies being detected in up to 90% of patients, up to 70% have intrinsic factor antibodies with the later antibodies considered highly specific to patients with pernicious anemia. This is the first report specifically investigating the occurrence of intrinsic factor antibodies, cobalamin deficiency and pernicious anemia in EAG models. We conclude, in contrast to AIG, that, in the three EAG models examined, intrinsic factor is not selected as a critical autoantigen.

The requirements for veterinary vaccines are different to those of human vaccines. Indeed, while more side effects can be tolerated in animals than in humans; there are stricter requirements in terms of cost, ease of delivery (including to wildlife), and a need to develop vaccines in species for which relatively little is known in terms of molecular immunology. By their nature particulate vaccine delivery systems are well suited to address these challenges. Here, we review particulate vaccine delivery systems, ranging from cm-sized long-distance ballistic devices to nano-bead technology for veterinary species and wildlife.

The popularly exploited murine neonatal thymectomy experimental autoimmune gastritis (nTx:EAG) model requires the animal to be in a state of lymphopenia. Here we report on a novel murine immunization (without adjuvant) model that can establish a lasting gastritis. We demonstrate that the immunization model (imm:EAG) results in a bona fide autoimmune disease and define the resulting pathology and serology observed and compare it with that reported for human autoimmune gastritis. Immune cells present in the stomachs of imm:EAG gastritic mice include CD8 T cells, CD11b and Gr1 (granulocytes/monocytes) and B cells. We detected circulating antibodies of immunoglobulin G1 (IgG1) subclass, with some IgG(2a) and IgG(2b) reactive to stomach membranes and the parietal cell proton pump. The class and subclass of autoreactive antibodies resulting from imm:EAG suggest a T helper 1 (Th1)/Th2 immune response. We establish that both self-reactive T and B cells from BALB/cCrSlc imm:EAG gastritic mice have the potential to induce a gastritis in BALB/c syngeneic nu/nu recipients. We conclude that this model is likely to be superior to the currently popularly exploited nTx:EAG and provide insight into and an understanding of the mechanisms of and remedies for autoimmunity in an intact immune system.

The American College of Rheumatology presented a consensus document in 1999 proposing the classification of 19 different syndromes defined by neurological and psychiatric manifestations of systemic lupus erythematosus (SLE). The detection of autoantibodies in patient's serum or cerebrospinal fluid has not been used as diagnostic markers for the proposed neuropsychiatric lupus classifications as their disease associations remain highly contentious. Autoantibodies detected in the serum and/or cerebrospinal fluid, that have been reported to segregate with patients presenting with neuropsychiatric lupus include: (1) anti-neuronal antibodies, (2) brain-lymphocyte cross-reactive antibodies, (3) anti-ribosomal P antibodies, (4) anti-phospholipid antibodies and (5) anti-ganglioside antibodies. Tests for anti-neuronal, anti-brain-lymphocyte cross-reactive and anti-ganglioside antibodies remain highly specialized whereas tests for ribosomal P antibodies and for antiphospholipid antibodies are currently routinely available in most diagnostic laboratories. Anti-ribosomal antibodies segregate with SLE. Antiphospholipid P antibodies are markers for the antiphospholipid syndrome. This syndrome may be associated with another disease, commonly SLE. In this setting, neuropsychiatric manifestations in SLE may arise as a consequence of thrombotic episodes involving the cerebral vasculature. There is a pressing need for antibodies to ribosomal P and to phospholipids to be standardized for routine diagnostic application. We conclude that the search for specific antibody marker(s) that can be applied for the routine laboratory diagnosis for neuropsychiatric lupus remains elusive.

It has previously been reported that neonatal BALB.D2 mice injected with native proton pump antigens without adjuvant develop an irreversible gastritis (Claeys et al, 1997). The ease of inititating gastritis in the neonate stands in contrast with the difficulty in initiating gastritis in adult mice that require repeated immunisation in adjuvant that is reversible following cessation of immunisation (Scarff et al, 1997). In view of these contrasting observations, we set out to ascertain whether we could confirm the observations in neonatal mice as well as further characterise the pathology and the autoantibody response. We found that neonatal gastritis-susceptible BALB/c mice (n=12), immunised with either pig or mouse gastric membranes in the absence of adjuvant, develop gastritis without circulating antibody to parietal cells detected by immunofluorescence, a hallmark of murine and human gastritis (Toh et al, 1997). However, mice immunized with pig gastric membranes (n=6) had circulating antibodies reactive by immunofluorescence to recombinant alpha and/or beta subunit of gastric H+/K+-ATPase expressed by insect cells (Sfalpha and Sfbeta). Four mice from this cohort with antibodies to Sfbeta also had reactivity to gastric H+/K+-ATPase by ELISA, and 3 immunoblotted the beta but not the alpha subunit of the ATPase. In the cohort of mice immunised with mouse gastric membranes (n=6), four produced antibodies reactive by immunofluorescence to Sfalpha, two of which were also reactive to Sfbeta and one developed antibodies detected by ELISA to gastric H+/K+-ATPase. However, no members of this cohort had antibodies reactive by immunoblotting to either the beta or alpha subunit of the ATPase. In all cases gastritic stomachs were characterised by areas deficient in ribosome-rich zymogenic cells and marked reductions in H+/K+-ATPase-positive parietal cells. Metaplasia detected by Maxwell stain, as clusters of mucus-producing cells throughout gastric units, were non-reactive to stomach mucin autoantibody suggesting the mucins comprise other and/or aberrant form(s). Compared to our previous observations in adult mice, our present data confirms that gastric autoimmunity is more readily induced in the neonate than the adult. Our data also affirms that while the neonatal immune system can mount a damaging inflammatory cellular immune response to gastric antigens, it develops an altered antibody response.