A comparison of changes in drug burden index between older inpatients who fell and people who have not fallen: A case-control study.

OBJECTIVE: Older inpatients who fall are often frail, with multiple co-morbidities and polypharmacy. Although the causes of falls are multifactorial, sedating and delirium-inducing drugs increase that risk. The aims were to determine whether people who fell had a change in their sedative and anticholinergic medication burden during an admission compared to people who did not fall. A secondary aim was to determine the factors associated with change in drug burden. METHODS: A retrospective, observational, case-control study of inpatients who fell. Two hundred consecutive people who fell were compared with 200 randomly selected people who had not fallen. Demographics, functional ability, frailty and cognition were recorded. For each patient, their total medications and anticholinergic and sedative burden were calculated on admission and on discharge, using the drug burden index (DBI). RESULTS: People who fell were more dependent and cognitively impaired than people who did not fallen. People who fell had a higher DBI on admission, than people who had not fall (mean: .69 vs .43, respectively, p < .001) and discharge (.66 vs .38, p < .001). For both cohorts, the DBI decreased between admission and discharge (-.03 and -.05), but neither were clinically significant. Higher total medications and a higher number DBI medications on admission were both associated with greater DBI changes (p = .003 and <.001, respectively). However, the presence (or absence) of cognitive impairment, dependency, frailty and single vs multiple falls were not significantly associated with DBI changes. CONCLUSIONS: In older people, DBI medications and falls are both common and have serious consequences, yet this study was unable to demonstrate any clinically relevant reduction in average DBI either in people who fell or people who had not fallen during a hospital admission.

Fusion of Rabies Virus Glycoprotein or gh625 to Iduronate-2-Sulfatase for the Treatment of Mucopolysaccharidosis Type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously delivered IDS is unable to cross the blood-brain barrier (BBB). Hematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (rabies virus glycoprotein [RVG] and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via hematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared with LV.IDS.ApoEII and LV.IDS in MPS II mice at 6 months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG- and LV.IDS.gh625-treated mice than in LV.IDS.ApoEII- and LV.IDS-treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis, and lysosomal swelling were partially normalized in MPS II mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalized by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared with control tissue from LV.IDS- and LV.IDS.ApoEII-transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPS II and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPS II disease than IDS alone.

Establishment of the Effectiveness of Early Versus Late Stem Cell Gene Therapy in Mucopolysaccharidosis II for Treating Central Versus Peripheral Disease.

Mucopolysaccharidosis type II (MPSII) is a rare pediatric X-linked lysosomal storage disease, caused by heterogeneous mutations in the iduronate-2-sulfatase (IDS) gene, which result in accumulation of heparan sulfate (HS) and dermatan sulfate within cells. This leads to severe skeletal abnormalities, hepatosplenomegaly, and cognitive deterioration. The progressive nature of the disease is a huge obstacle to achieve full neurological correction. Although current therapies can only treat somatic symptoms, a lentivirus-based hematopoietic stem cell gene therapy (HSCGT) approach has recently achieved improved central nervous system (CNS) neuropathology in the MPSII mouse model following transplant at 2 months of age. In this study, we evaluate neuropathology progression in 2-, 4- and 9-month-old MPSII mice, and using the same HSCGT strategy, we investigated somatic and neurological disease attenuation following treatment at 4 months of age. Our results showed gradual accumulation of HS between 2 and 4 months of age, but full manifestation of microgliosis/astrogliosis as early as 2 months. Late HSCGT fully reversed the somatic symptoms, thus achieving the same degree of peripheral correction as early therapy. However, late treatment resulted in slightly decreased efficacy in the CNS, with poorer brain enzymatic activity, together with reduced normalization of HS oversulfation. Overall, our findings confirm significant lysosomal burden and neuropathology in 2-month-old MPSII mice. Peripheral disease is readily reversible by LV.IDS-HSCGT regardless of age of transplant, suggesting a viable treatment for somatic disease. However, in the brain, higher IDS enzyme levels are achievable with early HSCGT treatment, and later transplant seems to be less effective, supporting the view that the earlier patients are diagnosed and treated, the better the therapy outcome.

Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC.

BACKGROUND: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9. METHODS: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread. RESULTS: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere. CONCLUSIONS: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.

Study protocol: PreOperative Brain Irradiation in Glioblastoma (POBIG) - A phase I trial.

Background: Glioblastoma is a high-grade aggressive neoplasm whose outcomes have not changed in decades. In the current treatment pathway, tumour growth continues and remains untreated for several weeks post-diagnosis. Intensified upfront therapy could target otherwise untreated tumour cells and improve the treatment outcome. POBIG will evaluate the safety and feasibility of single-fraction preoperative radiotherapy for newly diagnosed glioblastoma, assessed by the maximum tolerated dose (MTD) and maximum tolerated irradiation volume (MTIV). Methods: POBIG is an open-label, dual-centre phase I dose and volume escalation trial that has received ethical approval. Patients with a new radiological diagnosis of glioblastoma will be screened for eligibility. This is deemed sufficient due to the high accuracy of imaging and to avoid treatment delay. Eligible patients will receive a single fraction of preoperative radiotherapy ranging from 6 to 14 Gy followed by their standard of care treatment comprising maximal safe resection and postoperative chemoradiotherapy (60 Gy/30 fr) with concurrent and adjuvant temozolomide). Preoperative radiotherapy will be directed to the part of the tumour that is highest risk for remaining as postoperative residual disease (hot spot). Part of the tumour will remain unirradiated (cold spot) and sampled separately for diagnostic purposes. Dose/volume escalation will be guided by a Continual Reassessment Method (CRM) model. Translational opportunities will be afforded through comparison of irradiated and unirradiated primary glioblastoma tissue. Discussion: POBIG will help establish the role of radiotherapy in preoperative modalities for glioblastoma. Trial registration: NCT03582514 (clinicaltrials.gov).

Increased Circulating Chemokines and Macrophage Recruitment in Growing Vestibular Schwannomas.

BACKGROUND: There is evidence that macrophage infiltration in the tumor microenvironment promotes vestibular schwannoma (VS) growth. Efficacy of bevacizumab in NF2-associated VS demonstrates the value of therapies targeting the microvascular tumor microenvironment, and tumor-associated macrophages (TAMs) may represent another druggable target. OBJECTIVE: To characterize the relationship between growth, TAM infiltration, and circulating monocyte chemokines in a large cohort of patients with VS. METHODS: Immunostaining for Iba1 (macrophages), CD31 (endothelium), and fibrinogen (permeability) was performed on 101 growing and 19 static sporadic VS. The concentrations of monocyte-specific chemokines were measured in the plasma of 50 patients with growing VS and 25 patients with static VS. RESULTS: The Iba1 + cell count was significantly higher in growing as compared with static VS (592 vs 226/×20 HPF, P =<0.001). Similarly, the CD31 + % surface area was higher in growing VS (2.19% vs 1.32%, P = .01). There was a positive correlation between TAM infiltration and VS growth rate, which persisted after controlling for the effect of tumor volume (aR2 = 0.263, P =<0.001). The plasma concentrations of several monocytic chemokines were higher in patients with growing rather than static VS. CONCLUSION: There is a strong positive correlation between TAM infiltration and volumetric growth of VS, and this relationship is independent of tumor size. There is a colinear relationship between TAM infiltration and tumor vascularity, implying that inflammation and angiogenesis are interlinked in VS. Chemokines known to induce monocyte chemotaxis are found in higher concentrations in patients with growing VS, suggestive of a potential pathophysiological mechanism.

Tuft Cells: Context- and Tissue-Specific Programming for a Conserved Cell Lineage.

Tuft cells are found in tissues with distinct stem cell compartments, tissue architecture, and luminal exposures but converge on a shared transcriptional program, including expression of taste transduction signaling pathways. Here, we summarize seminal and recent findings on tuft cells, focusing on major categories of function-instigation of type 2 cytokine responses, orchestration of antimicrobial responses, and emerging roles in tissue repair-and describe tuft cell-derived molecules used to affect these functional programs. We review what is known about the development of tuft cells from epithelial progenitors under homeostatic conditions and during disease. Finally, we discuss evidence that immature, or nascent, tuft cells with potential for diverse functions are driven toward dominant effector programs by tissue- or perturbation-specific contextual cues, which may result in heterogeneous mature tuft cell phenotypes both within and between tissues.

Bile acid-sensitive tuft cells regulate biliary neutrophil influx.

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell-deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid-sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions.

Beyond Antoni: A Surgeon's Guide to the Vestibular Schwannoma Microenvironment.

Introduction Vestibular schwannomas (VS) are histologically benign tumors arising from cranial nerve VIII. Far from a homogenous proliferation of Schwann cells, mounting evidence has highlighted the complex nature of the inflammatory microenvironment in these tumors. Methods A review of the literature pertaining to inflammation, inflammatory molecular pathways, and immune-related therapeutic targets in VS was performed. Relevant studies published up to June 2020 were identified based on a literature search in the PubMed and MEDLINE databases and the findings were synthesized into a concise narrative review of the topic. Results The VS microenvironment is characterized by a dense infiltrate of inflammatory cells, particularly macrophages. Significantly higher levels of immune cell infiltration are observed in growing versus static tumors, and there is a demonstrable interplay between inflammation and angiogenesis in growing VS. While further mechanistic studies are required to ascertain the exact role of inflammation in angiogenesis, tumor growth, and Schwann cell control, we are beginning to understand the key molecular pathways driving this inflammatory microenvironment, and how these processes can be monitored and targeted in vivo . Conclusion Observational research has revealed a complex and heterogeneous tumor microenvironment in VS. The functional landscape and roles of macrophages and other immune cells in the VS inflammatory infiltrate are, however, yet to be established. The antiangiogenic drug bevacizumab has shown the efficacy of targeted molecular therapies in VS and there is hope that agents targeting another major component of the VS microenvironment, inflammation, will also find a place in their future management.

The spatial phenotype of genotypically distinct meningiomas demonstrate potential implications of the embryology of the meninges.

Meningiomas are the most common primary brain tumor and their incidence and prevalence is increasing. This review summarizes current evidence regarding the embryogenesis of the human meninges in the context of meningioma pathogenesis and anatomical distribution. Though not mutually exclusive, chromosomal instability and pathogenic variants affecting the long arm of chromosome 22 (22q) result in meningiomas in neural-crest cell-derived meninges, while variants affecting Hedgehog signaling, PI3K signaling, TRAF7, KLF4, and POLR2A result in meningiomas in the mesodermal-derived meninges of the midline and paramedian anterior, central, and ventral posterior skull base. Current evidence regarding the common pathways for genetic pathogenesis and the anatomical distribution of meningiomas is presented alongside existing understanding of the embryological origins for the meninges prior to proposing next steps for this work.

The inflammatory microenvironment in vestibular schwannoma.

Vestibular schwannomas are tumors arising from the vestibulocochlear nerve at the cerebellopontine angle. Their proximity to eloquent brainstem structures means that the pathology itself and the treatment thereof can be associated with significant morbidity. The vast majority of these tumors are sporadic, with the remainder arising as a result of the genetic syndrome Neurofibromatosis Type 2 or, more rarely, LZTR1-related schwannomatosis. The natural history of these tumors is extremely variable, with some tumors not displaying any evidence of growth, others demonstrating early, persistent growth and a small number growing following an extended period of indolence. Emerging evidence now suggests that far from representing Schwann cell proliferation only, the tumor microenvironment is complex, with inflammation proposed to play a key role in their growth. In this review, we provide an overview of this new evidence, including the role played by immune cell infiltration, the underlying molecular pathways involved, and biomarkers for detecting this inflammation in vivo. Given the limitations of current treatments, there is a pressing need for novel therapies to aid in the management of this condition, and we conclude by proposing areas for future research that could lead to the development of therapies targeted toward inflammation in vestibular schwannoma.

The microenvironment in sporadic and neurofibromatosis type II-related vestibular schwannoma: the same tumor or different? A comparative imaging and neuropathology study.

OBJECTIVE: Inflammation and angiogenesis may play a role in the growth of sporadic and neurofibromatosis type 2 (NF2)-related vestibular schwannoma (VS). The similarities in microvascular and inflammatory microenvironment have not been investigated. The authors sought to compare the tumor microenvironment (TME) in sporadic and NF2-related VSs using a combined imaging and tissue analysis approach. METHODS: Diffusion MRI and high-temporal-resolution dynamic contrast-enhanced (DCE) MRI data sets were prospectively acquired in 20 NF2-related and 24 size-matched sporadic VSs. Diffusion metrics (mean diffusivity, fractional anisotropy) and DCE-MRI-derived microvascular biomarkers (transfer constant [Ktrans], fractional plasma volume, tissue extravascular-extracellular space [ve], longitudinal relaxation rate, tumoral blood flow) were compared across both VS groups, and regression analysis was used to evaluate the effect of tumor size, pretreatment tumor growth rate, and tumor NF2 status (sporadic vs NF2-related) on each imaging parameter. Tissues from 17 imaged sporadic VSs and a separate cohort of 12 NF2-related VSs were examined with immunohistochemistry markers for vessels (CD31), vessel permeability (fibrinogen), and macrophage density (Iba1). The expression of vascular endothelial growth factor (VEGF) and VEGF receptor 1 was evaluated using immunohistochemistry, Western blotting, and double immunofluorescence. RESULTS: Imaging data demonstrated that DCE-MRI-derived microvascular characteristics were similar in sporadic and NF2-related VSs. Ktrans (p < 0.001), ve (p ≤ 0.004), and tumoral free water content (p ≤ 0.003) increased with increasing tumor size and pretreatment tumor growth rate. Regression analysis demonstrated that with the exception of mean diffusivity (p < 0.001), NF2 status had no statistically significant effect on any of the imaging parameters or the observed relationship between the imaging parameters and tumor size (p > 0.05). Tissue analysis confirmed the imaging metrics among resected sporadic VSs and demonstrated that across all VSs studied, there was a close association between vascularity and Iba1+ macrophage density (r = 0.55, p = 0.002). VEGF was expressed by Iba1+ macrophages. CONCLUSIONS: The authors present the first in vivo comparative study of microvascular and inflammatory characteristics in sporadic and NF2-related VSs. The imaging and tissue analysis results indicate that inflammation is a key contributor to TME and should be viewed as a therapeutic target in both VS groups.

Haematopoietic stem cell gene therapy with IL-1Ra rescues cognitive loss in mucopolysaccharidosis IIIA.

Mucopolysaccharidosis IIIA is a neuronopathic lysosomal storage disease, characterised by heparan sulphate and other substrates accumulating in the brain. Patients develop behavioural disturbances and cognitive decline, a possible consequence of neuroinflammation and abnormal substrate accumulation. Interleukin (IL)-1ß and interleukin-1 receptor antagonist (IL-1Ra) expression were significantly increased in both murine models and human MPSIII patients. We identified pathogenic mechanisms of inflammasome activation, including that disease-specific 2-O-sulphated heparan sulphate was essential for priming an IL-1ß response via the Toll-like receptor 4 complex. However, mucopolysaccharidosis IIIA primary and secondary storage substrates, such as amyloid beta, were both required to activate the NLRP3 inflammasome and initiate IL-1ß secretion. IL-1 blockade in mucopolysaccharidosis IIIA mice using IL-1 receptor type 1 knockout or haematopoietic stem cell gene therapy over-expressing IL-1Ra reduced gliosis and completely prevented behavioural phenotypes. In conclusion, we demonstrate that IL-1 drives neuroinflammation, behavioural abnormality and cognitive decline in mucopolysaccharidosis IIIA, highlighting haematopoietic stem cell gene therapy treatment with IL-1Ra as a potential neuronopathic lysosomal disease treatment.

Regulation of immune responses by tuft cells.

Tuft cells are rare, secretory epithelial cells that generated scant immunological interest until contemporaneous reports in 2016 linked tuft cells with type 2 immunity in the small intestine. Tuft cells have the capacity to produce an unusual spectrum of biological effector molecules, including IL-25, eicosanoids implicated in allergy (such as cysteinyl leukotrienes and prostaglandin D2) and the neurotransmitter acetylcholine. In most cases, the extracellular signals controlling tuft cell effector function are unknown, but signal transduction is thought to proceed via canonical, G protein-coupled receptor-dependent pathways involving components of the signalling pathway used by type II taste bud cells to sense sweet, bitter and umami compounds. Tuft cells are ideally positioned as chemosensory sentinels that can detect and relay information from diverse luminal substances via what appear to be stereotyped outputs to initiate both positive and aversive responses through populations of immune and neuronal cells. Despite recent insights, numerous questions remain regarding tuft cell lineage, diversity and effector mechanisms and how tuft cells interface with the immunological niche in the tissues where they reside.

An Improved Adeno-Associated Virus Vector for Neurological Correction of the Mouse Model of Mucopolysaccharidosis IIIA.

Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored. Following preclinical evaluation in MPSIIIA mice, an adeno-associated virus vector of serotype rh10 designed to deliver SGSH and sulfatase modifying factor 1 (SAF301) was trialed in four MPSIIIA patients, showing good tolerance and absence of adverse events with some improvements in neurocognitive measures. This study aimed to improve SAF301 further by removing sulfatase modifying factor 1 (SUMF1) and assessing if expression of this gene is needed to increase the SGSH enzyme activity (SAF301b). Second, the murine phosphoglycerate kinase (PGK) promotor was exchanged with a chicken beta actin/CMV composite (CAG) promotor (SAF302) to see if SGSH expression levels could be boosted further. The three different vectors were administered to MPSIIIA mice via intracranial injection, and SGSH expression levels were compared 4 weeks post treatment. Removal of SUMF1 resulted in marginal reductions in enzyme activity. However, promotor exchange significantly increased the amount of SGSH expressed in the brain, leading to superior therapeutic correction with SAF302. Biodistribution of SAF302 was further assessed using green fluorescent protein (GFP), indicating that vector spread was limited to the area around the injection tract. Further modification of the injection strategy to a single depth with higher injection volume increased vector distribution, leading to more widespread GFP distribution and sustained expression, suggesting this approach should be adopted in future trials.

Strategies for the Induction of Immune Tolerance to Enzyme Replacement Therapy in Mucopolysaccharidosis Type I.

Enzyme replacement therapy with laronidase is an established treatment for Mucopolysaccharidosis type I (MPS I), but its efficacy may be limited by the development of anti-drug antibodies, which inhibit cellular uptake of the enzyme. In a related disorder, infantile Pompe disease, immune tolerance induction with low-dose, short-course methotrexate appears to reduce antibody formation. We investigated a similar regimen using oral methotrexate in three MPS I patients. All patients developed anti-laronidase immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies, and they had clinically relevant levels of cellular uptake inhibition. We then explored several immune tolerance induction strategies in MPS I mice: (1) methotrexate, (2) combination of non-depleting anti-CD4 and anti-CD8 monoclonal antibodies, (3) methotrexate with anti-CD4 and anti-CD8 monoclonals, (4) anti-CD4 monoclonal, and (5) anti-CD8 monoclonal. Treated mice received 10 weekly laronidase injections, and laronidase was delivered with adjuvant on day 49 to further challenge the immune system. Most regimens were only partially effective at reducing antibody responses, but two courses of non-depleting anti-CD4 monoclonal antibody (mAb) ablated immune responses to laronidase in seven of eight MPS I mice (87.5%), even after adjuvant stimulation. Immune tolerance induction with methotrexate does not appear to be effective in MPS I patients, but use of non-depleting anti-CD4 monoclonal is a promising strategy.

Non-myeloablative busulfan chimeric mouse models are less pro-inflammatory than head-shielded irradiation for studying immune cell interactions in brain tumours.

BACKGROUND: Chimeric mouse models generated via adoptive bone marrow transfer are the foundation for immune cell tracking in neuroinflammation. Chimeras that exhibit low chimerism levels, blood-brain barrier disruption and pro-inflammatory effects prior to the progression of the pathological phenotype, make it difficult to distinguish the role of immune cells in neuroinflammatory conditions. Head-shielded irradiation overcomes many of the issues described and replaces the recipient bone marrow system with donor haematopoietic cells expressing a reporter gene or different pan-leukocyte antigen, whilst leaving the blood-brain barrier intact. However, our previous work with full body irradiation suggests that this may generate a pro-inflammatory peripheral environment which could impact on the brain's immune microenvironment. Our aim was to compare non-myeloablative busulfan conditioning against head-shielded irradiation bone marrow chimeras prior to implantation of glioblastoma, a malignant brain tumour with a pro-inflammatory phenotype. METHODS: Recipient wild-type/CD45.1 mice received non-myeloablative busulfan conditioning (25 mg/kg), full intensity head-shielded irradiation, full intensity busulfan conditioning (125 mg/kg) prior to transplant with whole bone marrow from CD45.2 donors and were compared against untransplanted controls. Half the mice from each group were orthotopically implanted with syngeneic GL-261 glioblastoma cells. We assessed peripheral blood, bone marrow and spleen chimerism, multi-organ pro-inflammatory cytokine profiles at 12 weeks and brain chimerism and immune cell infiltration by whole brain flow cytometry before and after implantation of glioblastoma at 12 and 14 weeks respectively. RESULTS: Both non-myeloablative conditioning and head-shielded irradiation achieve equivalent blood and spleen chimerism of approximately 80%, although bone marrow engraftment is higher in the head-shielded irradiation group and highest in the fully conditioned group. Head-shielded irradiation stimulated pro-inflammatory cytokines in the blood and spleen but not in the brain, suggesting a systemic response to irradiation, whilst non-myeloablative conditioning showed no cytokine elevation. Non-myeloablative conditioning achieved higher donor chimerism in the brain after glioblastoma implantation than head-shielded irradiation with an altered immune cell profile. CONCLUSION: Our data suggest that non-myeloablative conditioning generates a more homeostatic peripheral inflammatory environment than head-shielded irradiation to allow a more consistent evaluation of immune cells in glioblastoma and can be used to investigate the roles of peripheral immune cells and bone marrow-derived subsets in other neurological diseases.

Brain-targeted stem cell gene therapy corrects mucopolysaccharidosis type II via multiple mechanisms.

The pediatric lysosomal storage disorder mucopolysaccharidosis type II is caused by mutations in IDS, resulting in accumulation of heparan and dermatan sulfate, causing severe neurodegeneration, skeletal disease, and cardiorespiratory disease. Most patients manifest with cognitive symptoms, which cannot be treated with enzyme replacement therapy, as native IDS does not cross the blood-brain barrier. We tested a brain-targeted hematopoietic stem cell gene therapy approach using lentiviral IDS fused to ApoEII (IDS.ApoEII) compared to a lentivirus expressing normal IDS or a normal bone marrow transplant. In mucopolysaccharidosis II mice, all treatments corrected peripheral disease, but only IDS.ApoEII mediated complete normalization of brain pathology and behavior, providing significantly enhanced correction compared to IDS. A normal bone marrow transplant achieved no brain correction. Whilst corrected macrophages traffic to the brain, secreting IDS/IDS.ApoEII enzyme for cross-correction, IDS.ApoEII was additionally more active in plasma and was taken up and transcytosed across brain endothelia significantly better than IDS via both heparan sulfate/ApoE-dependent receptors and mannose-6-phosphate receptors. Brain-targeted hematopoietic stem cell gene therapy provides a promising therapy for MPS II patients.

A Metabolite-Triggered Tuft Cell-ILC2 Circuit Drives Small Intestinal Remodeling.

The small intestinal tuft cell-ILC2 circuit mediates epithelial responses to intestinal helminths and protists by tuft cell chemosensory-like sensing and IL-25-mediated activation of lamina propria ILC2s. Small intestine ILC2s constitutively express the IL-25 receptor, which is negatively regulated by A20 (Tnfaip3). A20 deficiency in ILC2s spontaneously triggers the circuit and, unexpectedly, promotes adaptive small-intestinal lengthening and remodeling. Circuit activation occurs upon weaning and is enabled by dietary polysaccharides that render mice permissive for Tritrichomonas colonization, resulting in luminal accumulation of acetate and succinate, metabolites of the protist hydrogenosome. Tuft cells express GPR91, the succinate receptor, and dietary succinate, but not acetate, activates ILC2s via a tuft-, TRPM5-, and IL-25-dependent pathway. Also induced by parasitic helminths, circuit activation and small intestinal remodeling impairs infestation by new helminths, consistent with the phenomenon of concomitant immunity. We describe a metabolic sensing circuit that may have evolved to facilitate mutualistic responses to luminal pathosymbionts.