DNL343 is an investigational CNS penetrant eukaryotic initiation factor 2B activator that prevents and reverses the effects of neurodegeneration caused by the integrated stress response.

The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. DNL343 is an investigational CNS-penetrant small-molecule ISR inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. DNL343 reduced CNS ISR activity and neurodegeneration in a dose-dependent manner in two established in vivo models - the optic nerve crush injury and an eIF2B loss of function (LOF) mutant - demonstrating neuroprotection in both and preventing motor dysfunction in the LOF mutant mouse. Treatment with DNL343 at a late stage of disease in the LOF model reversed elevation in plasma biomarkers of neuroinflammation and neurodegeneration and prevented premature mortality. Several proteins and metabolites that are dysregulated in the LOF mouse brains were normalized by DNL343 treatment, and this response is detectable in human biofluids. Several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD), a neurodegenerative disease that is driven by eIF2B LOF and chronic ISR activation, supporting their potential translational relevance. This study demonstrates that DNL343 is a brain-penetrant ISR inhibitor capable of attenuating neurodegeneration in mouse models and identifies several biomarker candidates that may be used to assess treatment responses in the clinic.

Targeting the transferrin receptor to transport antisense oligonucleotides across the mammalian blood-brain barrier.

Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.

Urinary and Oral Microbiota Among Men Undergoing Buccal Urethroplasty.

OBJECTIVE: To assess changes in the urinary microbiota after buccal urethroplasty. METHODS: At the University of California San Francisco, we enrolled 9 adult males with urethral strictures undergoing buccal urethroplasty where we collected urine and oral swabs intraoperatively and 3 months postoperatively. 16S rRNA sequencing was used to profile the microbiota. RESULTS: At baseline, the mouth contains twice the number of unique bacteria (alpha diversity) and the microbial community is significantly distinct compared to the urinary tract. Despite having a buccal mucosa in the urinary tract after urethroplasty, the number of unique bacteria in the urine remained stable. However, the bacterial community composition and structure significantly changed in the urinary tract with the enrichment of Corynebacterium genus at 3 months post-urethroplasty procedure. CONCLUSION: In this pilot study, we showed that the alpha diversity in the urinary microbiota did not significantly change despite having a buccal tissue with the capacity to support high bacterial diversity in the urinary tract. To our surprise, the post-urethroplasty urinary microbiota was not a hybrid of baseline oral and urine microbiotas; the changes detected, such as an enrichment of the Corynebacterium genus, were more nuanced yet could profoundly impact surgical outcomes like graft changes and stricture recurrence. Our study not only established the feasibility but also outlined a blueprint for conducting a large-scale study to assess alterations in the urinary microbiome in relation to surgical outcomes.

Microbiome preterm birth DREAM challenge: Crowdsourcing machine learning approaches to advance preterm birth research.

Every year, 11% of infants are born preterm with significant health consequences, with the vaginal microbiome a risk factor for preterm birth. We crowdsource models to predict (1) preterm birth (PTB; <37 weeks) or (2) early preterm birth (ePTB; <32 weeks) from 9 vaginal microbiome studies representing 3,578 samples from 1,268 pregnant individuals, aggregated from public raw data via phylogenetic harmonization. The predictive models are validated on two independent unpublished datasets representing 331 samples from 148 pregnant individuals. The top-performing models (among 148 and 121 submissions from 318 teams) achieve area under the receiver operator characteristic (AUROC) curve scores of 0.69 and 0.87 predicting PTB and ePTB, respectively. Alpha diversity, VALENCIA community state types, and composition are important features in the top-performing models, most of which are tree-based methods. This work is a model for translation of microbiome data into clinically relevant predictive models and to better understand preterm birth.

Mild SARS-CoV-2 infection results in long-lasting microbiota instability.

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2-positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls. These results were confirmed and extended in the K18-humanized angiotensin-converting enzyme 2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the USA), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. IMPORTANCE Taken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2, it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

DR3 Regulates Intestinal Epithelial Homeostasis and Regeneration After Intestinal Barrier Injury.

BACKGROUND & AIMS: Tumor necrosis factor (TNF) superfamily member tumor necrosis factor-like protein 1A (TL1A) has been associated with the susceptibility and severity of inflammatory bowel diseases. However, the function of the tumor necrosis factor-like protein 1A and its receptor death receptor 3 (DR3) in the development of intestinal inflammation is incompletely understood. We investigated the role of DR3 expressed by intestinal epithelial cells (IECs) during intestinal homeostasis, tissue injury, and regeneration. METHODS: Clinical phenotype and histologic inflammation were assessed in C57BL/6 (wild-type), Tl1a-/- and Dr3-/- mice in dextran sulfate sodium (DSS)-induced colitis. We generated mice with an IEC-specific deletion of DR3 (Dr3ΔIEC) and assessed intestinal inflammation and epithelial barrier repair. In vivo intestinal permeability was assessed by fluorescein isothiocyanate dextran uptake. Proliferation of IECs was analyzed by bromodeoxyuridine incorporation. Expression of DR3 messenger RNA was assessed by fluorescent in situ hybridization. Small intestinal organoids were used to determine ex vivo regenerative potential. RESULTS: Dr3-/- mice developed more severe colonic inflammation than wild-type mice in DSS-induced colitis with significantly impaired IEC regeneration. Homeostatic proliferation of IECs was increased in Dr3-/- mice, but blunted during regeneration. Cellular localization and expression of the tight junction proteins Claudin-1 and zonula occludens-1 were altered, leading to increased homeostatic intestinal permeability. Dr3ΔIEC mice recapitulated the phenotype observed in Dr3-/- mice with increased intestinal permeability and IEC proliferation under homeostatic conditions and impaired tissue repair and increased bacterial translocation during DSS-induced colitis. Impaired regenerative potential and altered zonula occludens-1 localization also were observed in Dr3ΔIEC enteroids. CONCLUSIONS: Our findings establish a novel function of DR3 in IEC homeostasis and postinjury regeneration independent of its established role in innate lymphoid cells and T-helper cells.

VMAP: Vaginal Microbiome Atlas During Pregnancy.

The vaginal microbiome has been shown to be associated with pregnancy outcomes including preterm birth (PTB) risk. Here we present VMAP: Vaginal Microbiome Atlas during Pregnancy (http://vmapapp.org), an application to visualize features of 3,909 vaginal microbiome samples of 1,416 pregnant individuals from 11 studies, aggregated from raw public and newly generated sequences via an open-source tool, MaLiAmPi. Our visualization tool (http://vmapapp.org) includes microbial features such as various measures of diversity, VALENCIA community state types (CST), and composition (via phylotypes and taxonomy). This work serves as a resource for the research community to further analyze and visualize vaginal microbiome data in order to better understand both healthy term pregnancies and those associated with adverse outcomes.

Microbiome Preterm Birth DREAM Challenge: Crowdsourcing Machine Learning Approaches to Advance Preterm Birth Research.

Globally, every year about 11% of infants are born preterm, defined as a birth prior to 37 weeks of gestation, with significant and lingering health consequences. Multiple studies have related the vaginal microbiome to preterm birth. We present a crowdsourcing approach to predict: (a) preterm or (b) early preterm birth from 9 publicly available vaginal microbiome studies representing 3,578 samples from 1,268 pregnant individuals, aggregated from raw sequences via an open-source tool, MaLiAmPi. We validated the crowdsourced models on novel datasets representing 331 samples from 148 pregnant individuals. From 318 DREAM challenge participants we received 148 and 121 submissions for our two separate prediction sub-challenges with top-ranking submissions achieving bootstrapped AUROC scores of 0.69 and 0.87, respectively. Alpha diversity, VALENCIA community state types, and composition (via phylotype relative abundance) were important features in the top performing models, most of which were tree based methods. This work serves as the foundation for subsequent efforts to translate predictive tests into clinical practice, and to better understand and prevent preterm birth.

A TREM2-activating antibody with a blood-brain barrier transport vehicle enhances microglial metabolism in Alzheimer's disease models.

Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.

Mild SARS-CoV-2 infection results in long-lasting microbiota instability.

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of SARS-CoV-2 infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2 positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the United States), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila . Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. IMPORTANCE: Taken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2 it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

The systemic anti-microbiota IgG repertoire can identify gut bacteria that translocate across gut barrier surfaces.

Unique gut microbiota compositions have been associated with inflammatory diseases, but identifying gut bacterial functions linked to immune activation in humans remains challenging. Translocation of pathogens from mucosal surfaces into peripheral tissues can elicit immune activation, although whether and which gut commensal bacteria translocate in inflammatory diseases is difficult to assess. We report that a subset of commensal gut microbiota constituents that translocate across the gut barrier in mice and humans are associated with heightened systemic immunoglobulin G (IgG) responses. We present a modified high-throughput, culture-independent approach to quantify systemic IgG against gut commensal bacteria in human serum samples without the need for paired stool samples. Using this approach, we highlight several commensal bacterial species that elicit elevated IgG responses in patients with inflammatory bowel disease (IBD) including taxa within the clades Collinsella, Bifidobacterium, Lachnospiraceae, and Ruminococcaceae. These and other taxa identified as translocating bacteria or targets of systemic immunity in IBD concomitantly exhibited heightened transcriptional activity and growth rates in IBD patient gut microbiomes. Our approach represents a complementary tool to illuminate interactions between the host and its gut microbiota and may provide an additional method to identify microbes linked to inflammatory disease.

Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia.

BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Gestational diabetes: Optimizing Dx and management in primary care.

Understanding risk factors and glucose targets for gestational diabetes can help you identify at-risk patients and improve outcomes for mother and infant.

Gut microbiota mediate the FGF21 adaptive stress response to chronic dietary protein-restriction in mice.

Chronic dietary protein-restriction can create essential amino acid deficiencies and induce metabolic adaptation through the hepatic FGF21 pathway which serves to maintain host fitness during prolonged states of nutritional imbalance. Similarly, the gut microbiome undergoes metabolic adaptations when dietary nutrients are added or withdrawn. Here we confirm previous reports that dietary protein-restriction triggers the hepatic FGF21 adaptive metabolic pathway and further demonstrate that this response is mediated by the gut microbiome and can be tuned through dietary supplementation of fibers that alter the gut microbiome. In the absence of a gut microbiome, we discover that FGF21 is de-sensitized to the effect of protein-restriction. These data suggest that host-intrinsic adaptive pathways to chronic dietary protein-restriction, such as the hepatic FGF21 pathway, may in-fact be responding first to adaptive metabolic changes in the gut microbiome.

The new microbiology: cultivating the future of microbiome-directed medicine.

The discovery of human-associated microscopic life forms has captivated the scientific community since their first documentation in the 17th century. Subsequent isolation and cultivation of microorganisms have spurred great leaps in medicine, including the discovery of antibiotics, identifying pathogens that cause infectious diseases, and vaccine development. The realization that there is a vast discrepancy between the number of microscopic cell counts and how many could thrive in the laboratory motivated the advent of sequencing-based approaches to characterize the uncultured fraction of the microbiota, leading to an unprecedented view into their composition and putative function on all bodily surfaces. It soon became apparent that specific members of the microbiota can be our commensal partners with new implications on various aspects of health, as well as a rich source of therapeutic compounds and tools for biotechnology. Harnessing the immense repertoire of microbial properties, however, inadvertently requires pure cultures for validation and manipulation of candidate genes, proteins, or metabolic pathways, just as mammalian cell culture has become an indispensable tool for mechanistic understanding of host biology. Yet, this renewed interest in growing microorganisms, individually or as a consortium, is stalled by the laborious nature of conventional cultivation methods. Addressing this unmet need through implementation of improved media design and new cultivation techniques is arguably instrumental to future milestones in translational microbiome research.

Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans.

A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.

Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.