LRPAP1 is released from activated microglia and inhibits microglial phagocytosis and amyloid beta aggregation.

Low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1), also known as receptor associated protein (RAP), is an endoplasmic reticulum (ER) chaperone and inhibitor of LDL receptor related protein 1 (LRP1) and related receptors. These receptors have dozens of physiological ligands and cell functions, but it is not known whether cells release LRPAP1 physiologically at levels that regulate these receptors and cell functions. We used mouse BV-2 and human CHME3 microglial cell lines, and found that microglia released nanomolar levels of LRPAP1 when inflammatory activated by lipopolysaccharide or when ER stressed by tunicamycin. LRPAP1 was found on the surface of live activated and non-activated microglia, and anti-LRPAP1 antibodies induced internalization. Addition of 10 nM LRPAP1 inhibited microglial phagocytosis of isolated synapses and cells, and the uptake of Aß. LRPAP1 also inhibited Aß aggregation in vitro. Thus, activated and stressed microglia release LRPAP1 levels that can inhibit phagocytosis, Aß uptake and Aß aggregation. We conclude that LRPAP1 release may regulate microglial functions and Aß pathology, and more generally that extracellular LRPAP1 may be a physiological and pathological regulator of a wide range of cell functions.

Protective and pathogenic functions of innate lymphoid cells in transplantation.

Innate lymphoid cells (ILCs) are a family of lymphocytes with essential roles in tissue homeostasis and immunity. Along with other tissue-resident immune populations, distinct subsets of ILCs have important roles in either promoting or inhibiting immune tolerance in a variety of contexts, including cancer and autoimmunity. In solid organ and hematopoietic stem cell transplantation, both donor and recipient-derived ILCs could contribute to immune tolerance or rejection, yet understanding of protective or pathogenic functions are only beginning to emerge. In addition to roles in directing or regulating immune responses, ILCs interface with parenchymal cells to support tissue homeostasis and even regeneration. Whether specific ILCs are tissue-protective or enhance ischemia reperfusion injury or fibrosis is of particular interest to the field of transplantation, beyond any roles in limiting or promoting allograft rejection or graft-versus host disease. Within this review, we discuss the current understanding of ILCs functions in promoting immune tolerance and tissue repair at homeostasis and in the context of transplantation and highlight where targeting or harnessing ILCs could have applications in novel transplant therapies.

Strategies for optimizing CITE-seq for human islets and other tissues.

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

Clinical Outcomes and Cost Analysis of PreserFlo versus Trabeculectomy for Glaucoma Management in the United Kingdom.

PURPOSE: Clinical evaluation and cost analysis of mitomycin-C-augmented PreserFlo MicroShunt versus trabeculectomy. DESIGN: Retrospective cohort study across 3 teaching hospitals. PARTICIPANTS: A total of 134 consecutive eyes of 129 patients (70 undergoing MicroShunt, 64 trabeculectomy). METHODS: Primary and secondary glaucoma cases with uncontrolled intraocular pressure (IOP) were included. Neovascular glaucoma and surgery combined with cataract extraction were excluded. The cost analysis used results from the clinical study to estimate operative costs (equipment and staff costs) and postoperative costs (follow-up visits, nonglaucoma medications, and postoperative procedures) per eye for PreserFlo and trabeculectomy. MAIN OUTCOME MEASURES: The primary clinical outcome measure was surgical failure (defined as IOP > 21 mmHg or < 20% reduction from baseline, IOP ≤ 5 mmHg, reoperation, or loss of light perception) or qualified and complete success (with or without medication) at 18 months. Secondary measures were IOP, glaucoma medications, visual acuity, mean deviation, time to cessation of steroid drops, complications, surgical time, follow-up visits, postoperative interventions, and reoperations. The cost analysis evaluated costs of PreserFlo compared with trabeculectomy. RESULTS: Baseline characteristics were similar, except for more non-White patients in the trabeculectomy group (51% Black and Asian vs. 32% MicroShunt, P = 0.02) and more cases with prior ab externo glaucoma surgery in the MicroShunt group (19% vs. 3% in the trabeculectomy group, P = 0.004). Overall, 59% of eyes had primary open-angle glaucoma. Mean follow-up was 19.9 months for both groups. At 18 months, surgical failure was 25% for MicroShunt compared with 35% for trabeculectomy (P = 0.18). Failure in MicroShunt cases was due to inadequate IOP reduction (84%) or reoperation for glaucoma (16%). Failure in trabeculectomy cases was due to inadequate IOP reduction (58%), persistent hypotony (29%), or reoperation for glaucoma (13%). Combined blebitis and endophthalmitis rate was 1.4% for MicroShunt and 3.1% for trabeculectomy. Cost analysis showed a savings of £245 to £566 per eye in the MicroShunt group, driven mostly by reduced postoperative procedures and follow-up visits. This is in contrast to prior randomized controlled trial data reporting the incremental cost of $2058 of PreserFlo over trabeculectomy. CONCLUSIONS: Our experience of introducing PreserFlo MicroShunt surgery showed it was safer than trabeculectomy and is a cost-saving and effective option that offers potential to free up highly limited National Health Service resources. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Brain Cells Release Calreticulin That Attracts and Activates Microglia, and Inhibits Amyloid Beta Aggregation and Neurotoxicity.

Calreticulin is a chaperone, normally found in the endoplasmic reticulum, but can be released by macrophages into the extracellular medium. It is also found in cerebrospinal fluid bound to amyloid beta (Aß). We investigated whether brain cells release calreticulin, and whether extracellular calreticulin had any effects on microglia and neurons relevant to neuroinflammation and neurodegeneration. We found that microglia release nanomolar levels of calreticulin when inflammatory-activated with lipopolysaccharide, when endoplasmic reticulum stress was induced by tunicamycin, or when cell death was induced by staurosporine, and that neurons release calreticulin when crushed. Addition of nanomolar levels of extracellular calreticulin was found to chemoattract microglia, and activate microglia to release cytokines TNF-α, IL-6 and IL-1ß, as well as chemokine (C-C motif) ligand 2. Calreticulin blocked Aß fibrillization and modified Aß oligomerization, as measured by thioflavin T fluorescence and transmission electron microscopy. Extracellular calreticulin also altered microglial morphology and proliferation, and prevented Aß-induced neuronal loss in primary neuron-glial cultures. Thus, calreticulin is released by microglia and neurons, and acts: as an alarmin to recruit and activate microglia, as an extracellular chaperone to prevent Aß aggregation, and as a neuroprotectant against Aß neurotoxicity.

Macrophage migration inhibitory factor drives pathology in a mouse model of spondyloarthritis and is associated with human disease.

Spondyloarthritis (SpA), a type 3 immunity-mediated inflammatory arthritis, is a systemic rheumatic disease that primarily affects the joints, spine, gut, skin, and eyes. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, yet MIF's pathological role in SpA is unknown. Here, we observed that the expression of MIF and its receptor CD74 is increased in blood and tissues of curdlan (ß-glucan)­treated SKG mice, a mouse model of SpA. We found that neutrophils substantially expanded and produced MIF in curdlan-treated SKG mice and that human neutrophils from SpA patients secreted higher concentrations of MIF compared to healthy individuals. Although genetic deletion of Mif (Mif−/−) substantially suppressed the severity of SpA features, adoptive transfer of inflammatory neutrophils induced SpA pathology in curdlan-treated Mif−/− SKG mice; in contrast, blocking the function of neutrophils with anti­Gr-1 antibody suppressed the curdlan-induced SpA-like phenotype. We also determined that systemic MIF overexpression was sufficient to induce SpA-like clinical features in SKG mice with enhanced type 3 immunity, whereas SKG mice treated with a MIF antagonist prevented or attenuated curdlan-induced SpA manifestations. Mechanistically, we identified that MIF intensifies type 3 immunity by boosting human and mouse T regulatory cell (Treg) acquisition of a TH17 cell­like phenotype, including the up-regulation of interleukin-17 (IL-17) and IL-22 in vitro. Tregs in blood and synovial fluids from SpA patients have a pathologic TH17 phenotype. These results indicate that MIF is a crucial regulator and a potential therapeutic target in type 3 immunity-mediated arthritis.

Anti-racist interventions to transform ecology, evolution and conservation biology departments.

Racial and ethnic discrimination persist in science, technology, engineering and mathematics fields, including ecology, evolution and conservation biology (EECB) and related disciplines. Marginalization and oppression as a result of institutional and structural racism continue to create barriers to inclusion for Black people, Indigenous people and people of colour (BIPOC), and remnants of historic racist policies and pseudoscientific theories continue to plague these fields. Many academic EECB departments seek concrete ways to improve the climate and implement anti-racist policies in their teaching, training and research activities. We present a toolkit of evidence-based interventions for academic EECB departments to foster anti-racism in three areas: in the classroom; within research laboratories; and department wide. To spark restorative discussion and action in these areas, we summarize EECB's racist and ethnocentric histories, as well as current systemic problems that marginalize non-white groups. Finally, we present ways that EECB departments can collectively address shortcomings in equity and inclusion by implementing anti-racism, and provide a positive model for other departments and disciplines.

Induction of stable human FOXP3+ Tregs by a parasite-derived TGF-ß mimic.

Immune homeostasis in the intestine is tightly controlled by FOXP3+ regulatory T cells (Tregs), defects of which are linked to the development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites boost Treg activity. The parasite Heligmosomoides polygyrus is known to secrete a molecule (Hp-TGM) that mimics the ability of TGF-ß to induce FOXP3 expression in CD4+ T cells. The study aimed to investigate whether Hp-TGM could induce human FOXP3+ Tregs as a potential therapeutic approach for inflammatory diseases. CD4+ T cells from healthy volunteers were expanded in the presence of Hp-TGM or TGF-ß. Treg induction was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CD25 and CTLA-4. Epigenetic changes were detected using ChIP-Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was evaluated by cellular co-culture suppression assays and cytometric bead arrays for secreted cytokines. Hp-TGM efficiently induced FOXP3 expression (> 60%), in addition to CD25 and CTLA-4, and caused epigenetic modification of the FOXP3 locus to a greater extent than TGF-ß. Hp-TGM-induced Tregs had superior suppressive function compared with TGF-ß-induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Furthermore, Hp-TGM induced a Treg-like phenotype in in vivo differentiated Th1 and Th17 cells, indicating its potential to re-program memory cells to enhance immune tolerance. These data indicate Hp-TGM has potential to be used to generate stable human FOXP3+ Tregs to treat IBD and other inflammatory diseases.

The association between social emotional development and symptom presentation in autism spectrum disorder.

Understanding differences in social-emotional behavior can help identify atypical development. This study examined the differences in social-emotional development in children at increased risk of an autism spectrum disorder (ASD) diagnosis (infant siblings of children diagnosed with the disorder). Parents completed the Brief Infant-Toddler Social-Emotional Assessment (BITSEA) to determine its ability to flag children with later-diagnosed ASD in a high-risk (HR) sibling population. Parents of HR (n = 311) and low-risk (LR; no family history of ASD; n = 127) children completed the BITSEA when their children were 18 months old and all children underwent a diagnostic assessment for ASD at age 3 years. All six subscales of the BITSEA (Problems, Competence, ASD Problems, ASD Competence, Total ASD Score, and Red Flags) distinguished between those in the HR group who were diagnosed with ASD (n = 84) compared to non-ASD-diagnosed children (both HR-N and LR). One subscale (BITSEA Competence) differentiated between the HR children not diagnosed with ASD and the LR group. The results suggest that tracking early social-emotional development may have implications for all HR children, as they are at increased risk of ASD but also other developmental or mental health conditions.

Brief Report: Evaluation of the Short Quantitative Checklist for Autism in Toddlers (Q-CHAT-10) as a Brief Screen for Autism Spectrum Disorder in a High-Risk Sibling Cohort.

This study examined the potential of the short form of the Quantitative Checklist for Autism in Toddlers (Q-CHAT-10) to identify autism spectrum disorder (ASD) in a high-risk sibling cohort. High-risk (HR; siblings of children diagnosed with ASD) and low-risk (LR; no family history of ASD) toddlers were assessed prospectively at 18 and 24 months of age using the Q-CHAT-10 and underwent blind diagnostic assessment for ASD at 36 months of age. The results indicated that at 18 and 24 months, total score differentiated between HR toddlers subsequently diagnosed with ASD from other HR and LR toddlers. The sensitivity at both time points was acceptable; however, the specificity was below the level recommended for clinical application.

From resource to female defence: the impact of roosting ecology on a bat's mating strategy.

With their extraordinary species richness and diversity in ecological traits and social systems, bats are a promising taxon for testing socio-ecological hypotheses in order to get new insights into the evolution of animal social systems. Regarding its roosting habits, proboscis bats form an extreme by occupying sites which are usually completely exposed to daylight (e.g. tree trunks, vines or rocks). This is accompanied by morphological and behavioural adaptations to remain cryptic in exposed day roosts. With long-term behavioural observations and genetic parentage analyses of individually marked proboscis bats, we assessed its social dispersion and male mating strategy during day and night. Our results reveal nocturnal male territoriality-a strategy which most closely resembles a resource-defence polygyny that is frequent also in other tropical bats. Its contrasting clumped social dispersion during the day is likely to be the result of strong selection for crypsis in exposed roosts and is accompanied by direct female defence in addition to male territoriality. To the best of our knowledge, such contrasting male mating strategies within a single day-night cycle have not been described in a vertebrate species so far and illustrate a possible evolutionary trajectory from resource-defence to female-defence strategy by small ecologically driven evolutionary steps.

Non-compliance with growth hormone treatment in children is common and impairs linear growth.

BACKGROUND: GH therapy requires daily injections over many years and compliance can be difficult to sustain. As growth hormone (GH) is expensive, non-compliance is likely to lead to suboptimal growth, at considerable cost. Thus, we aimed to assess the compliance rate of children and adolescents with GH treatment in New Zealand. METHODS: This was a national survey of GH compliance, in which all children receiving government-funded GH for a four-month interval were included. Compliance was defined as ≥ 85% adherence (no more than one missed dose a week on average) to prescribed treatment. Compliance was determined based on two parameters: either the number of GH vials requested (GHreq) by the family or the number of empty GH vials returned (GHret). Data are presented as mean ± SEM. FINDINGS: 177 patients were receiving GH in the study period, aged 12.1 ± 0.6 years. The rate of returned vials, but not number of vials requested, was positively associated with HVSDS (p < 0.05), such that patients with good compliance had significantly greater linear growth over the study period (p<0.05). GHret was therefore used for subsequent analyses. 66% of patients were non-compliant, and this outcome was not affected by sex, age or clinical diagnosis. However, Maori ethnicity was associated with a lower rate of compliance. INTERPRETATION: An objective assessment of compliance such as returned vials is much more reliable than compliance based on parental or patient based information. Non-compliance with GH treatment is common, and associated with reduced linear growth. Non-compliance should be considered in all patients with apparently suboptimal response to GH treatment.