Equine grass sickness (EGS or equine dysautonomia) is a predominantly fatal multi-system neuropathy affecting grazing horses, likely caused by a neurotoxic phospholipase A2 (nPLA2) derived from a plant or microorganism. We studied neuronal tissue gene and protein expression patterns in EGS to elucidate the possible mechanisms of neurotoxicity and neurodegeneration. Tissue from the cranial cervical ganglion of eight EGS horses and six controls was examined histologically and used for transcriptomic analysis. These transcriptomic data were compared with previously published EGS-related proteomic datasets from different horses. Results were visualized using the network analysis tool BioLayout and Ingenuity Pathway Analysis. The cranial cervical ganglia from all affected horses showed pathology typical of EGS. They also showed distinct gene and protein expression profiles that were different from the controls. The EGS signature consisted ofreduced expression of genes and proteins involved in neurological function (including ion-channel and synaptic-function genes and genes encoding mitochondrial proteins) and increased expression of genes and proteins indicative of cellular stress, cell death and inflammation. This signature likely reflects more generalized neurodegeneration. This study thus improves our understanding of the molecular changes likely to be associated with a neurotoxic neurodegenerative process.

Sulfate is a vital nutrient for healthy brain development. More than 90 sulfate-related genes are highly conserved across mammalian species, with 16 of these genes being clinically reportable for adverse brain conditions. To determine the potential involvement of additional sulfate-related genes in human neuropathology, this study curated the spatial and temporal expression patterns of all known sulfate biology genes in the human fetal brain from 8 to 37 post conception weeks (pcw) using data from the BrainSpan database and performed network analysis to cluster sulfate-related genes with genes involved in neurodevelopmental processes. A total of 64 sulfate-related genes were abundantly or moderately expressed in 11 brain regions throughout gestation. Steady state expression was observed for some of these genes from 8 to 37 pcw, including genes that encode sulfotransferases (CHST12, CHST7), sulfatases (ARSA, SULF2, TPST1, TPST2), sulfatase modifying enzyme (SUMF2), key enzymes in amino acid metabolism (CDO1, CTH), sulfate transporter (SLC26A11), as well as genes involved in neurodevelopmental processes (ACTG1, TUBA1A, MAPT, UBE3A, DCHS1, WWOX). Between 21-24 weeks, there were numerous clusters of sulfate biology genes with neurodevelopmental genes involved in neuronal migration (FAT4) and synaptogenesis (CBLN2, WNT4, MAPT, FOXP2). At 8-13 and 17-21 pcw, fifteen sulfate genes (ARSF, CHST1, CHST2, CHST13, GAL3ST3, GOT1, IDS, HS3ST2, HS3ST4, HS6ST3, SLC26A8, STS, SULT1A1, SULT4A1, UST) were expressed in the hippocampus and clustered with genes involved in neurogenesis, differentiation and synaptogenesis (MAPT, DNER, NEUROD1). Overall, this study identified 48 sulfate-related genes with moderate/abundant expression in the fetal brain that are coexpressed with genes for neurodevelopmental processes but are not considered in clinical settings. These findings provide information for future studies into the physiological roles of sulfate-related genes that are expressed in the fetal brain.

Crohn's disease is a chronic, transmural inflammatory disease of the human gut. Changes in the fecal microbial composition and dysbiosis are consistent features in studies of Crohn's disease patients, but whether dysbiosis is a cause or consequence of inflammation remains unresolved. Genetic susceptibility plays a role in the development of Crohn's disease and has been linked to genes involved in recognition of intestinal bacteria by the mononuclear phagocyte system. The earliest visible lesions in Crohn's disease are aphthous ulcers, overlying Peyer's patches and lymphoid follicles. To identify mechanisms underlying the earliest stages of disease we compared gene expression in aphthous ulcers, Peyer's patches, inflamed and endoscopically normal mucosa from patients and controls using total RNA‐seq. The resulting data were subjected to network analysis to identify coregulated gene expression signatures of cell types and processes. These results were compared to single‐cell RNA‐seq analysis of intestinal macrophages in normal and diseased mucosa. The analysis of aphthous ulcers revealed signatures of epithelial stress and antimicrobial defense, plasma cell activation and immunoglobulin production, monocyte recruitment, inflammatory gene expression and induction of interferon‐γ. These signatures were not present in the normal appearing mucosa adjacent to aphthous ulcers, which were similar to healthy control mucosa. Given the role of Peyer's patches and lymphoid follicles in sampling the luminal contents, these findings suggest the initial lesion in Crohn's disease arises from the uptake of bacteria and the activation of multiple host defense pathways rather than the breakdown of epithelial barrier integrity and widespread bacterial translocation.

Adaptation to existence outside the womb is a key event in the life of a mammal. The absence of macrophages in rats with a homozygous mutation in the colony-stimulating factor 1 receptor (Csf1r) gene (Csf1rko) severely compromises pre-weaning somatic growth and maturation of organ function. Transfer of wild-type bone marrow cells (BMT) at weaning rescues tissue macrophage populations permitting normal development and long-term survival. To dissect the phenotype and function of macrophages in postnatal development, we generated transcriptomic profiles of all major organs of wild-type and Csf1rko rats at weaning and selected organs following rescue by BMT. The transcriptomic profiles revealed subtle effects of macrophage deficiency on development of all major organs. Network analysis revealed a common signature of CSF1R-dependent resident tissue macrophages that includes the components of complement C1Q (C1qa/b/c genes). Circulating C1Q was almost undetectable in Csf1rko rats and rapidly restored to normal levels following BMT. Tissue-specific macrophage signatures were also identified, notably including sinus macrophage populations in the lymph nodes. Their loss in Csf1rko rats was confirmed by immunohistochemical localisation of CD209B (SIGNR1). By 6-12 weeks, Csf1rko rats succumb to emphysema-like pathology associated with the selective loss of interstitial macrophages and granulocytosis. This pathology was reversed by BMT. Along with physiological rescue, BMT precisely regenerated the abundance and expression profiles of resident macrophages. The exception was the brain, where BM-derived microglia-like cells had a distinct expression profile compared to resident microglia. In addition, the transferred BM failed to restore blood monocyte or CSF1R-positive bone marrow progenitors. These studies provide a model for the pathology and treatment of CSF1R mutations in humans and the innate immune deficiency associated with prematurity.

More than 300 genomic loci have been associated with increased susceptibility to inflammatory bowel disease (IBD) through genome-wide association studies. A major challenge in the translation of genome-wide association studies to mechanistic insights lies in connecting noncoding variants to function. For example, single-nucleotide variants (SNVs) in the vicinity of the gene encoding the transcription factor ETS2 on human chromosome 21 are associated with the risk of developing IBD in Europeans. The peak of SNV association lies within a distal enhancer that may regulate ETS2 transcription. The interpretation of this and many other SNV associations with IBD depends on a model linking variation in transcriptional regulation to the likelihood of developing chronic intestinal inflammation. One model for the ETS2 locus is that overexpression in monocytes is causally associated with the risk allele, which in turn leads to a hyperinflammatory state. Here we summarize evidence for an alternative mechanism focused on negative regulators of monocyte-macrophage activation. We argue that IBD susceptibility arises from dysregulation of monocyte adaptation in the intestinal milieu to form resident intestinal macrophages that are anergic to inflammatory stimuli. This process depends on signals initiated by macrophage colony-stimulating factor (CSF1) binding to its receptor (CSF1R). Within this framework, ETS2 is a myeloid-specific transcription factor, expressed in pluripotent and committed progenitors and monocytes, and is down-regulated by CSF1, in common with many genes associated with IBD susceptibility, including NOD2. ETS2 is also both a downstream target and a mediator of the CSF1/CSF1R signaling pathway. Therapeutic targeting of ETS2 and its upstream regulators has the potential to prevent CSF1-dependent monocyte differentiation toward a prorepair resident macrophage phenotype and consequently exacerbate intestinal inflammation.

AbstractGerm-line deletion of a conserved enhancer (the Fms intrinsic regulatory element, FIRE) in the mouseCsf1rlocus causes congenital absence of microglia. Homozygous FIRE deletion on a C57BL/6J background leads to perinatal lethality and hydrocephalus (HC) in surviving pups. We developed a congenic C57BL/6J line with defined regions of non-C57BL/6J genomic DNA, increased postnatal viability and reduced incidence of HC. Both perinatal lethality and HC were eliminated in F2 mice following outcross of the congenic line to CBA/J or BALBc/J backgrounds. To assess the impacts of microglial deficiency in postnatal neurodevelopment we analyzed deep total RNA-seq data from multiple brain regions of wild-type andCsf1rΔFIRE/ΔFIREmice. Aside from the loss of microglial-specific transcripts, we found no significant alterations in relative abundance of any cell-type or region-specific transcriptomic signature. Transcripts associated with endosome/lysosome function, which are enriched in microglia, were not affected, suggesting compensatory expression by other cell types. On the C57BL/6J x CBA/J F2 background, congenital absence of microglia did not affect motor activity, behavior or myelination up to 7 months of age but was associated with astrocytosis and calcification in the thalamus. In the congenic C57BL/6JCsf1rΔFIRE/ΔFIREmouse line, intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning led to complete repopulation of the brain with microglia-like cells without giving rise to monocytic intermediates. Our results suggest novel strategies for treatment of microglial deficiency.

168 Background: Waldenstrom macroglobulinemia (WM) is a rare haematological malignancy that has seen an emergence of novel targeted therapies. With a range of clinical features impacting quality of life (QoL), there is a need for disease-specific patient-reported outcomes (PRO), outside of existing measures with limited validity in WM. The aim of this work was to enrich understanding of the patient experience and identify potential WM-specific metrics, through wearable-captured physiological metrics and electronic-PROs (ePROs). Methods: Informed consent was provided by 79 patients with WM for the analysis of data from an FDA-cleared wearable smartwatch capturing activity, sleep, and heart rate, and a mobile app for patients to input daily ePROs. These ePROs included treatments taken, EQ-5D-5L, and disease symptoms (fatigue, weakness, numbness/tingling, breathlessness, difficulty concentrating/confusion, vision, rash, night sweats, dizziness/light-headedness, constipation, loss of appetite, diarrhoea, and vomiting). Data was integrated within a digital platform, and anonymised extracts analysed as a cohort and by treatment. Results: Patients had a mean±SD (range) age of 65±10 (43-88) years. 53% were female. As of the most recent self-reported treatment entry, 22% (17/79) reported Bruton tyrosine kinase inhibitor (BTKi) use. Completion rates over a 142-day snapshot for daily PROs was 60%, and 85% across wearable activity and sleep metrics (devices synced=58/79). A cohort mean EQ-5D-5L Score of 0.760±0.181 and Health State (0-100) of 74±18 was reported. The highest symptom severity scores (1-5) were ‘fatigue’ (1.9/5), ‘weakness’ (1.7/5), and ‘numbness/tingling’ (1.5/5), all other symptoms reporting a mean score of 1.4 or less. Increasing activity levels significantly correlated with decreasing symptom severity and increasing QoL scores (p<0.05). Lower symptom severity also correlated with higher total sleep durations, contrasting higher ‘difficulty concentrating/confusion’, ‘dizziness/light-headedness’, and ‘usual activity impairment’ scores with increasing levels of deep sleep. Breathing disturbance intensities (36.8 vs. 17.6, p=0.008) and nightly wakeup counts (1.9 vs. 1.6, p=0.038), tracked by wearables, were significantly higher in patients reporting BTKi treatment compared to the remaining cohort. Conclusions: Our data support the enrichment of knowledge around baseline physiological and QoL metrics for patients with WM, demonstrating the feasibility of a digital ecosystem of wearable-captured and ePRO metrics at a high data completeness level. Further work will ensure the generation of more population-representative outputs, through the expansion of the patient cohort, and a greater depth of insight into the impact of health events, disease complications, and therapeutic intervention on these remotely-tracked metrics.

Amino acid substitutions in the kinase domain of the human CSF1R protein are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Previous analysis demonstrated that heterozygous mutation (Csf1rE631K/+) had a dominant inhibitory effect on CSF1R signaling in vitro and in vivo but did not recapitulate human disease pathology. We speculated that leukoencephalopathy in humans requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles. Here we examine the Csf1rE631K/+ mutation impact on microglial phenotype, postnatal brain development, age-related changes in gene expression and on prion disease and experimental autoimmune encephalitis (EAE), two pathologies in which microgliosis is a prominent feature. The Csf1rE631K/+ mutation reduced microglial abundance and the expression of microglial-associated transcripts relative to wild-type controls at 12 and 43 weeks of age. There was no selective effect on homeostatic markers e. g. P2ry12, or age-related changes in gene expression in striatum and hippocampus. An epistatic interaction was demonstrated between Csf1rE631K/+ and Cx3cr1EGFP/+ genotypes leading to dysregulated microglial and neuronal gene expression in hippocampus and striatum. Heterozygous Csf1rE631K mutation reduced the microgliosis associated with both diseases. There was no significant impact on disease severity or progression in prion disease. In EAE, inflammation-associated transcripts in the hippocampus and striatum were suppressed in parallel with microglia-specific transcripts. The results support a dominant inhibitory model of CSF1R-related leukoencephalopathy and likely contributions of an environmental trigger and/or genetic background to neuropathology.

There is no effective treatment preventing the progression of neurodegenerative diseases such as prion and Alzheimer’s diseases. These fatal diseases of the central nervous system, involve progressive accumulation of a misfolded protein long before overt clinical signs of disease. Removal of prion protein early in the pathological process appears to halt the progression however, it is not known whether intervention at later disease stages could be effective. We investigated the potential for intervention throughout the course of prion disease, by developing a mouse model in which Prnp expression can be manipulated in a tissue specific and time dependent manner. Depleting Prnp from neuronal populations in CNS throughout the preclinical phase substantially prolonged incubation. The pathology was dramatically altered to a pattern of astrocytic associated prion deposition. However once overt clinical symptoms of disease were apparent Prnp depletion did not alter disease progression. This study establishes a wide window for intervention, and suggests timely treatment could delay the onset of clinical disease potentially well beyond the lifetime of an individual. ### Competing Interest Statement The authors have declared no competing interest.

Adaptation to existence outside the womb is a key event in the life of a mammal. The absence of macrophages in rats with a homozygous mutation in the Csf1r gene ( Csf1rko ) severely compromises pre-weaning somatic growth and maturation of organ function. Transfer of wild-type bone marrow cells (BMT) at weaning rescues tissue macrophage populations permitting normal development and long-term survival. To dissect the phenotype and function of macrophages in postnatal development, we generated transcriptomic profiles of all major organs of wild-type and Csf1rko rats at weaning and selected organs following rescue by BMT. The transcriptomic profiles revealed subtle effects of macrophage deficiency on development of all major organs. Network analysis revealed a common signature of CSF1R-dependent resident tissue macrophages that includes the components of complement C1Q ( C1qa/b/c genes). Circulating C1Q was almost undetectable in Csf1rko rats and rapidly restored to normal levels following BMT. Tissue-specific macrophage signatures were also identified, notably including sinus macrophage populations in the lymph nodes. Their loss in Csf1rko rats was confirmed by immunohistochemical localisation of CD209B (SIGNR1). By 6-12 weeks, Csf1rko rats succumb to emphysema-like pathology associated with the selective loss of interstitial macrophages and granulocytosis. This pathology was prevented by BMT. Along with physiological rescue, BMT precisely regenerated the abundance and expression profiles of resident macrophages. The exception was the brain, where BM-derived microglia-like cells had a distinct expression profile compared to resident microglia. In addition, the transferred BM failed to restore blood monocyte or CSF1R-positive bone marrow progenitors. Considering the integrated data we provide insight into the inter-related systemic consequences of developmental delay in bone, liver and pituitary and potential contributions to somatic growth deficiency in Csf1rko rats.   ### Competing Interest Statement The authors have declared no competing interest.

The differentiation of resident intestinal macrophages from blood monocytes depends upon signals from the macrophage colony-stimulating factor receptor (CSF1R). Analysis of genome-wide association studies (GWAS) indicates that dysregulation of macrophage differentiation and response to microorganisms contributes to susceptibility to chronic inflammatory bowel disease (IBD). Here, we analyzed transcriptomic variation in monocyte-derived macrophages (MDM) from affected and unaffected sib pairs/trios from 22 IBD families and 6 healthy controls. Transcriptional network analysis of the data revealed no overall or inter-sib distinction between affected and unaffected individuals in basal gene expression or the temporal response to lipopolysaccharide (LPS). However, the basal or LPS-inducible expression of individual genes varied independently by as much as 100-fold between subjects. Extreme independent variation in the expression of pairs of HLA-associated transcripts (HLA-B/C, HLA-A/F and HLA-DRB1/DRB5) in macrophages was associated with HLA genotype. Correlation analysis indicated the downstream impacts of variation in the immediate early response to LPS. For example, variation in early expression of IL1B was significantly associated with local SNV genotype and with subsequent peak expression of target genes including IL23A, CXCL1, CXCL3, CXCL8 and NLRP3. Similarly, variation in early IFNB1 expression was correlated with subsequent expression of IFN target genes. Our results support the view that gene-specific dysregulation in macrophage adaptation to the intestinal milieu is associated with genetic susceptibility to IBD.

Glutaric aciduria type II (GAII) is a heterogeneous genetic disorder affecting mitochondrial fatty acid, amino acid and choline oxidation. Clinical manifestations vary across the lifespan and onset may occur at any time from the early neonatal period to advanced adulthood. Historically, some patients, in particular those with late onset disease, have experienced significant benefit from riboflavin supplementation. GAII has been considered an autosomal recessive condition caused by pathogenic variants in the gene encoding electron-transfer flavoprotein ubiquinone-oxidoreductase (ETFDH) or in the genes encoding electron-transfer flavoprotein subunits A and B (ETFA and ETFB respectively). Variants in genes involved in riboflavin metabolism have also been reported. However, in some patients, molecular analysis has failed to reveal diagnostic molecular results. In this study, we report the outcome of molecular analysis in 28 Australian patients across the lifespan, 10 paediatric and 18 adult, who had a diagnosis of glutaric aciduria type II based on both clinical and biochemical parameters. Whole genome sequencing was performed on 26 of the patients and two neonatal onset patients had targeted sequencing of candidate genes. The two patients who had targeted sequencing had biallelic pathogenic variants (in ETFA and ETFDH). None of the 26 patients whose whole genome was sequenced had biallelic variants in any of the primary candidate genes. Interestingly, nine of these patients (34.6%) had a monoallelic pathogenic or likely pathogenic variant in a single primary candidate gene and one patient (3.9%) had a monoallelic pathogenic or likely pathogenic variant in two separate genes within the same pathway. The frequencies of the damaging variants within ETFDH and FAD transporter gene SLC25A32 were significantly higher than expected when compared to the corresponding allele frequencies in the general population. The remaining 16 patients (61.5%) had no pathogenic or likely pathogenic variants in the candidate genes. Ten (56%) of the 18 adult patients were taking the selective serotonin reuptake inhibitor antidepressant sertraline, which has been shown to produce a GAII phenotype, and another two adults (11%) were taking a serotonin-norepinephrine reuptake inhibitor antidepressant, venlafaxine or duloxetine, which have a mechanism of action overlapping that of sertraline. Riboflavin deficiency can also mimic both the clinical and biochemical phenotype of GAII. Several patients on these antidepressants showed an initial response to riboflavin but then that response waned. These results suggest that the GAII phenotype can result from a complex interaction between monoallelic variants and the cellular environment. Whole genome or targeted gene panel analysis may not provide a clear molecular diagnosis.

CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.

Fibrillin-1 is a major component of the extracellular microfibrils, where it interacts with other extracellular matrix proteins to provide elasticity to connective tissues, and regulates the bioavailability of TGFβ family members. A peptide consisting of the C-terminal 140 amino acids of fibrillin-1 has recently been identified as a glucogenic hormone, secreted from adipose tissue during fasting and targeting the liver to release glucose. This fragment, called asprosin, also signals in the hypothalamus to stimulate appetite. Asprosin levels are correlated with many of the pathologies indicative of metabolic syndrome, including insulin resistance and obesity. Previous studies and reviews have addressed the therapeutic potential of asprosin as a target in obesity, diabetes and related conditions without considering mechanisms underlying the relationship between generation of asprosin and expression of the much larger fibrillin-1 protein. Profibrillin-1 undergoes obligatory cleavage at the cell surface as part of its assembly into microfibrils, producing the asprosin peptide as well as mature fibrillin-1. Patterns of FBN1 mRNA expression are inconsistent with the necessity for regulated release of asprosin. The asprosin peptide may be protected from degradation in adipose tissue. We present evidence for an alternative possibility, that asprosin mRNA is generated independently from an internal promoter within the 3' end of the FBN1 gene, which would allow for regulation independent of fibrillin-synthesis and is more economical of cellular resources. The discovery of asprosin opened exciting possibilities for treatment of metabolic syndrome related conditions, but there is much to be understood before such therapies could be introduced into the clinic.

The fibrillinopathies represent a group of diseases in which the 10-12 nm extracellular microfibrils are disrupted by genetic variants in one of the genes encoding fibrillin molecules, large glycoproteins of the extracellular matrix. The best-known fibrillinopathy is Marfan syndrome, an autosomal dominant condition affecting the cardiovascular, ocular, skeletal, and other systems, with a prevalence of around 1 in 3,000 across all ethnic groups. It is caused by variants of the FBN1 gene, encoding fibrillin-1, which interacts with elastin to provide strength and elasticity to connective tissues. A number of mouse models have been created in an attempt to replicate the human phenotype, although all have limitations. There are also natural bovine models and engineered models in pig and rabbit. Variants in FBN2 encoding fibrillin-2 cause congenital contractural arachnodactyly and mouse models for this condition have also been produced. In most animals, including birds, reptiles, and amphibians, there is a third fibrillin, fibrillin-3 (FBN3 gene) for which the creation of models has been difficult as the gene is degenerate and nonfunctional in mice and rats. Other eukaryotes such as the nematode C. elegans and zebrafish D. rerio have a gene with some homology to fibrillins and models have been used to discover more about the function of this family of proteins. This review looks at the phenotype, inheritance, and relevance of the various animal models for the different fibrillinopathies.

Background Voxelotor is a haemoglobin oxygen-affinity modulator that leads to inhibited red blood cell sickling, has demonstrated reduced markers of haemolysis and anaemia improvement, and is one of a small number of novel disease-modifiers approved in the US for sickle cell disease (SCD) treatment. It has marketing authorisation in the UK and is currently undergoing reimbursement appraisal. The modelled impact of voxelotor on health-related quality of life (HRQoL)remains a key issue in current trial evidence. Therefore, the need for real-world data and understanding of patient-reported outcomes and experiences following treatment remains critical. Aims This work sought to characterise the side effects, HRQoL, and wearable-captured sleep and activity metrics of SCD patients in the UK, before and after treatment initiation. Methods Feedback from 19 SCD patients who had received voxelotor was captured through an online survey (Aug 2022-Mar 2023). 15 of these patients were also consented and enrolled within an artificial intelligence (AI) platform of over 600 SCD patients that combined genomic, medical record, real-time wearable biometric, and patient-reported outcomes data into a single platform. This included a mobile phone app for day-to-day QoL questionnaire (EQ-5D-5L) entry and an FDA-cleared smartwatch to capture sleep quality and physical activity. Convenience sampling selected data for a 17-patient (89%) subcohort with available pre- and post-voxelotor initiation EQ-5D-5L scores, either through the platform or follow-up survey snapshots. For each patient, this was analysed up to the date on which they completed the survey. EQ-5D-5L was also analysed in comparison to platform patients who had hydroxyurea (HU) in the past (n=19) or were receiving HU at the time of analysis (n=60), in addition to those with no recorded HU use (n=116). Additional analysis compared pre- and post-initiation activity levels (n=6), 4 of whom also had sleep quality data. Results The 19 surveyed patients had received voxelotor for a mean ± SD (range) of 194 ± 415 (17-1,826) days. 11 (58%) had also received HU and 1 (5%) received crizanlizumab. The mean age was 33 ± 13 (14-52) years, 12 (63%) were male, and 16 (84%) had HbSS. Side effects were reported in 8 (42%) patients, mostly diarrhea (6, 32%), stomach pains (2, 11%), and nausea/vomiting (2, 11%). Comparison of the 17-patient pre- to post-voxelotor cohort showed a statistically significant (p<0.001) increase in pooled EQ-5D-5L scores, from 0.558 ± 0.314 to 0.761 ± 0.262 (Figure 1). Post-initiation EQ-5D-5L for these 17 patients was significantly higher than for those receiving HU at 0.731 ± 0.232 (p=0.023) or who had previously received HU at 0.704 ± 0.256 (p=0.040) (Figure 2). Analysis of pre- and post-initiation wearables data saw increased deep sleep in 50% (2/4) and decreased nightly wakeups in 50% (2/4) of patients. Pooled pre- and post-initiation data showed a significant difference in deep sleep (43% vs. 47%, p=0.011). The mean number of steps and distance increased for 50% (3/6) of patients. While soft activity (Metabolic Equivalent of Task (METs): <3, e.g., sleeping, sitting quietly, slow walk) decreased for 67% (4/6) of patients, moderate activity (MET: 3-6, e.g., fast walk, cleaning, gardening) increased in 67% (4/6) and intense activity (MET: >6, e.g., running, hiking) increased in 60% (3/5). Conclusions Our data present a different lens to the voxelotor treatment experience of patients in the UK, with a side effect profile similar to that of other published studies. We demonstrated that EQ-5D-5L increased by 0.203 pre- to post-initiation, and was 0.030 and 0.057 higher than those receiving HU and those who had previously received HU, respectively, in the voxelotor-treated group. These are in line with the ranges of minimally important differences (0.030-0.098) reported for the EQ-5D-5L in other chronic diseases. As such and despite the limitations of small sample size and variability in pre- to post-initiation changes, a statistically significant improvement in HRQoL was observed after initiating voxelotor, as well as in comparison to HU treatment pathways. Similarly, pre- and post-initiation wearables data demonstrated early positive trends in voxelotor's impacts on sleep and activity using an FDA-cleared device, highlighting a need for further research into these metrics and the impact of SCD-modifying treatment.

Homozygous null mutation of the Csf1r gene (Csf1rko) in rats leads to the loss of most tissue macrophage populations and pleiotropic impacts on postnatal growth and organ maturation, leading to early mortality. The phenotype can be reversed by intraperitoneal transfer of WT BM cells (BMT) at weaning. Here, we used a Csf1r-mApple transgenic reporter to track the fate of donor-derived cells. Following BMT into Csf1rko recipients, mApple+ve cells restored IBA1+ tissue macrophage populations in every tissue. However, monocytes, neutrophils, and B cells in the BM, blood, and lymphoid tissues remained of recipient (mApple-ve ) origin. An mApple+ve cell population expanded in the peritoneal cavity and invaded locally in the mesentery, fat pads, omentum, and diaphragm. One week after BMT, distal organs contained foci of mApple+ve , IBA1-ve immature progenitors that appeared to proliferate, migrate, and differentiate locally. We conclude that rat BM contains progenitor cells that are able to restore, replace, and maintain all tissue macrophage populations in a Csf1rko rat directly without contributing to the BM progenitor or blood monocyte populations.

Background Sickle Cell Disease (SCD) is a genetic disorder caused by an HBB gene mutation and resulting structural variant haemoglobin (HbS) that polymerises upon deoxygenation, leading to a sickled morphology of red blood cells ( Chakravorty et al., Arch Dis Child 2015). This causes acute pain episodes, known as vaso-occlusive crises (VOC), that can result in severe end-organ damage in the long-term and a shortened lifespan ( Jang et al., J Transl Med 2021). Responsible for an estimated 95% of SCD hospitalisations and as a key predictor of death ( Darbari et al., Eur J Haematol 2020, Ballas et al., Am J Hematol 2005), it is critical to find new ways to drive early detection of VOCs in order to support preventative interventions. Aims This work aimed to develop a machine learning (ML) algorithm capable of predicting the potential onset of a VOC, using physiological data captured longitudinally by a wearable smartwatch, as well as patient-reported outcomes (PROs) entered via a specialised mobile phone application. Methods Following informed patient consent, participants gained access to a mobile phone application, or “digital patient wallet”, encompassing their health data and access to a PRO entry portal ( Summers et al., HemaSphere 2023). This allowed for daily recording of PROs including EQ-5D-5L, pain, mood, and fatigue scores, alongside self-reported VOCs. Participants were also provided with a Withings ScanWatch to be worn day and night, capturing physical activity, sleep quality, and heart rate data in an automated manner. Medical record data including healthcare utilisation, pathology, and demographic data was obtained through participant completion of a Subject Access Request form. Data for a snapshot cohort of 186 patients over a 1-month period (May-June 2023) were analysed through an ensemble of ML models: gradient boosting machine, neural network, and k-means clustering. All collated metrics were fed into the model, with the exclusion of hospitalisation as an 'a posteriori' variable, to ensure predictions were based on appropriate variables and not strengthened by self-confirming factors. The resulting predicted likelihood of a VOC for each participant was calculated on each day and given as a scale from 0-100%, with a likelihood of 75% or higher deemed to be a predicted VOC. This was then assessed against the true incidence of the participants' self-reported VOCs. Results The mean age was 36 (SD 11.6) years, and most patients were female (70%). The HbSS genotype was most common (75%), followed by HbSC (16%), and HbS Beta + Thalassemia (5%). 82,804 datapoints were collated for all participants across this period, encompassing 90 different variables. 69 instances of a VOC were self-reported by patients during the period of analysis, 58 (84%) of which were accurately predicted by the algorithm. 2,366 instances of no VOC were self-recorded, giving a negative predicted value of 83% (1,958/2,366 predicted). Further breakdown by the predicted likelihood score is displayed in Figure 2, highlighting the spread of individually calculated VOC likelihood scores per day against the incidence of patient-reported VOCs. Summary/Conclusions This work provides an important translation of daily trackable metrics through a remote, digital ecosystem, to the real-world prediction of potential VOC events in SCD. The purpose of this algorithm is, at its core, to act as a pre-emptive warning system from which proactive behavioural changes or interventions can be enacted. As such, the prediction of additional potential risk scores above the 75% threshold reflects an approach of ‘over-predicting’ considered to be a preferable strategy for the algorithm, given the severe nature of VOCs and the health impacts of missing a potential event. Our ongoing aim is to further develop the algorithm and its contributing metrics against medical data, in addition to patient self-reported events, to further optimise the predictor. This includes the curation of core metrics requiring minimal patient input, in order to reduce missing datapoints across those that feed into the algorithm and thereby reduce unpredicted VOCs. Ultimately, we hope to provide an early alert system that will help patients to identify their VOCs sooner, and thus action the next steps that may prevent possible hospital admissions.