Applying Artificial Intelligence to Identify Common Targets for Treatment of Asthma, Eczema, and Food Allergy.

INTRODUCTION: Allergic disorders are common diseases marked by the abnormal immune response toward foreign antigens that are not pathogens. Often patients with food allergy also suffer from asthma and eczema. Given the similarities of these diseases and a shortage of effective treatments, developing novel therapeutics against common targets of multiple allergies would offer an efficient and cost-effective treatment for patients. METHODS: We employed the artificial intelligence-driven target discovery platform, PandaOmics, to identify common targets for treating asthma, eczema, and food allergy. Thirty-two case-control comparisons were generated from 15, 11, and 6 transcriptomics datasets related to asthma (558 cases, 315 controls), eczema (441 cases, 371 controls), and food allergy (208 cases, 106 controls), respectively, and allocated into three meta-analyses for target identification. Top-100 high-confidence targets and Top-100 novel targets were prioritized by PandaOmics for each allergic disease. RESULTS: Six common high-confidence targets (i.e., IL4R, IL5, JAK1, JAK2, JAK3, and NR3C1) across all three allergic diseases have approved drugs for treating asthma and eczema. Based on the targets' dysregulated expression profiles and their mechanism of action in allergic diseases, three potential therapeutic targets were proposed. IL5 was selected as a high-confidence target due to its strong involvement in allergies. PTAFR was identified for drug repurposing, while RNF19B was selected as a novel target for therapeutic innovation. Analysis of the dysregulated pathways commonly identified across asthma, eczema, and food allergy revealed the well-characterized disease signature and novel biological processes that may underlie the pathophysiology of allergies. CONCLUSION: Altogether, our study dissects the shared pathophysiology of allergic disorders and reveals the power of artificial intelligence in the exploration of novel therapeutic targets.

Protective Efficacy of Novel Engineered Human ACE2-Fc Fusion Protein Against Pan-SARS-CoV-2 Infection In Vitro and in Vivo.

Enduring occurrence of severe COVID-19 for unvaccinated, aged, or immunocompromised individuals remains an urgent need. Soluble human angiotensin-converting enzyme 2 (ACE2) has been used as a decoy receptor to inhibit SARS-CoV-2 infection, which is limited by moderate affinity. We describe an engineered, high-affinity ACE2 that is consistently effective in tissue cultures in neutralizing all strains tested, including Delta and Omicron. We also found that treatment of AC70 hACE2 transgenic mice with hACE2-Fc receptor decoys effectively reduced viral infection, attenuated tissue histopathology, and delayed the onset of morbidity and mortality caused by SARS-CoV-2 infection. We believe that using this ACE2-Fc protein would be less likely to promote the escape mutants of SARS-CoV-2 as frequently as did those neutralizing antibody therapies. Together, our results emphasize the suitability of our newly engineered hACE2-Fc fusion protein for further development as a potent antiviral agent against Pan-SARS-CoV-2 infection.

Antiaging Vaccines Targeting Senescent Cells.

The development of senomorphic drugs to attenuate the senescent phenotype and senolytics to clear pro-inflammatory senescent cells (SCs) to treat aging-associated disorders is being hotly pursued. The effort is complicated by the fact that SCs play a constructive role in some cellular processes such as tissue repair and wound healing. However, concerns about efficacy, which SCs to target, and unwanted side effects have created potential roadblocks. Chimeric antigen receptor T cells directed against urokinase-type plasminogen activator receptor, which is expressed on at least a subset of SCs in atherosclerotic plaques and fibrotic livers, removed SC and improved glucose metabolism. A vaccine targeting CD153-expressing senescent T cells also improved glucose metabolism in obese mice. Recent work to selectively target SCs associated with several pathologies has resulted in the creation of a peptide vaccine that primarily targets endothelial cells expressing high levels of GPNMB, recently identified as a biomarker of senescence. The vaccine reduces atherosclerotic plaque burden and metabolic dysfunction such as glucose intolerance in mouse models of obesity and atherosclerosis. For translation to humans the activity of the vaccine will need to be tightly controlled, as the target GPNMB has multiple roles in normal physiology, including acting to inhibit and possibly resolve inflammation. A promising alternative approach would be to use passive immunization with a monoclonal antibody directed against GPNMB.

Broad reactivity and enhanced potency of recombinant anti-EGFR × anti-CD3 bispecific antibody-armed activated T cells against solid tumours.

Introduction: Bispecific antibody (BiAb)-armed activated T cells (BATs) comprise an adoptive T cell therapy platform for treating cancer. Arming activated T cells (ATC) with anti-CD3 x anti-tumour associated antigen (TAA) BiAbs converts ATC into non-major histocompatibility complex (MHC)-restricted anti-tumour cytotoxic T lymphocytes (CTLs). Binding of target antigens via the BiAb bridge enables specific anti-tumour cytotoxicity, Th1 cytokines release, and T cell proliferation. Clinical trials in breast, prostate, and pancreatic cancer using BATs armed with chemically heteroconjugated BiAbs demonstrated safety, feasibility, induction of anti-tumour immune responses and potential increases in overall survival (OS).Objectives: The primary objective of this study was to develop a recombinant BiAb that confers enhanced anti-tumour activity of BATs against a broad range of solid tumours.Methods: A recombinant anti-epidermal growth factor receptor (EGFR) x anti-CD3 (OKT3) BiAb (rEGFRBi) was designed and expressed in CHO cells, used to arm ATC (rEGFR-BATs), and tested for specific cytotoxicity against breast, pancreatic and prostate cancers and glioblastoma.Results: rEGFR-BATs exhibit remarkably enhanced specific cytotoxicity and T1 cytokine secretion against a wide range of solid tumour cell lines vs. their respective chemically-heteroconjugated BATs.Conclusion: rEGFR-BATs may provide a "universal" T cell therapy for treating a wide range of solid tumours. KEY MESSAGEA (Gly4Ser)6 linker between the variable light and heavy chains of an scFv fused to the N-terminus of a heavy chain antibody confers unexpected stability to the heavy chain fusion protein and supports the efficient expression of the bispecific antibody.Arming of activated T cells with the rEGFRBi greatly enhances the relative cytotoxicity and Th1 cytokine secretion of theT cells relative to a chemically heteroconjugated BiAbs.rEGFR-BATs are promising candidates for the treatment of a broad range of solid tumours.

Stem Cell Rejuvenation by Restoration of Youthful Metabolic Compartmentalization.

Stem cell dysfunction is a hallmark of aging. Much recent study suggests that epigenetic changes play a critical role in the loss of stem cell function with age. However, the underlying mechanisms require elucidation. A recent report describes a process by which mild mitochondrial stress associated with aging causes lysosomal-mediated decreases in CiC, the mitochondrial citrate transporter, in bone marrow-derived mesenchymal stem cells (MSCs). This, in turn, results in a deficit of acetyl-CoA in the nucleus and hypoacetylation of histones. The altered epigenome results in skewered stem cell differentiation favoring adipogenesis and disfavoring osteogenesis, which is problematic given the role the MSCs play in maintaining the integrity of bone tissue. Restoration of nuclear acetyl-CoA by either ectopic expression of CiC or acetate supplementation of MSCs in culture rejuvenates the MSC, restoring the potential to efficiently differentiate along the osteogenic lineage. Citrate, which has recently been reported to extend lifespan in Drosophila, chemically incorporates acetyl-CoA and may prove useful to restore cytoplasmic and nuclear acetyl-CoA levels. The general applicability of the CiC defect in old cells, particularly stem cells, should be established.

Modulation of cGAS-STING Pathway by Nicotinamide Riboside in Alzheimer's Disease.

Numerous studies demonstrate a global decrease in nicotinamide adenine dinucleotide (NAD+) with aging. This decline is associated with the development of several of the hallmarks of aging such as reduced mitophagy and neuroinflammation, processes thought to play a significant role in the progression of Alzheimer's disease (AD). Augmentation of NAD+ by oral administration of a precursor, nicotinamide riboside (NR), reduces senescence of affected cells, attenuates DNA damage and neuroinflammation in the transgenic APP/PS1 murine model of AD. Inflammation mediated by microglial cells plays an important role in progression of AD and other neurodegenerative diseases. The cytoplasmic DNA sensor, cyclic GMP-AMP synthase (cGAS) and downstream stimulator of interferon genes (STING), generates an interferon signature characteristic of senescence and inflammaging in the brain of AD mice. Elevated cGAS-STING observed in the AD mouse brains and human AD fibroblasts was normalized by NR. This intervention also increased mitophagy with improved cognition and behavior in the APP/PS1 mice. These studies suggest that modulation of the cGAS-STING pathway may benefit AD patients and possibly other disorders characterized by compromised mitophagy and excessive neuroinflammation.

Response to Hypoxia in Cognitive Decline.

Inflammaging, the increase of proinflammatory processes with increasing age, has multiple mechanisms from increasing numbers of senescent cells secreting cytokines to changes in metabolic processes. Alterations of oxygen metabolism with aging, especially decreased levels of O2 with age resulting from endocrine and cardiovascular dysfunction as well as desensitization of cellular response to hypoxia, may exacerbate inflammaging, which in turn creates further oxygen metabolic dysfunction. During aging, decline in levels of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), BPG mutase, and adenosine A2B receptor, a key adenosine signaling receptor that can augment 2,3-BPG expression, may fail to protect sensitive brain tissue from subtly reduced O2 levels, in turn resulting in increased numbers of activated microglia and secretion of proinflammatory cytokines, ultimately promoting inflammaging and senescence of endothelial cells. Interventions to restore O2 levels directly or via increasing 2,3-BPG may help promote cognitive health in old age, but significant work to quantify the degree of reduced O2 during aging in mammals, and especially humans, needs to be pursued.

Beneficial Gut Microbiome Remodeled During Intermittent Fasting in Humans.

Intermittent fasting (IF) is the practice of restricting food intake for 12-48 hours per fasting cycle over a prolonged period of time. Previous study shows beneficial health effects such as weight loss and lower risk for cardiometabolic diseases. Although reduced calorie intake may account for some of the observed benefits of IF, exact mechanisms are still unclear. Recent evidence indicates that IF may lead to remodeling and increased taxonomic diversity in the human gut microbiome. In particular, the Lachnospiraceae family of anaerobic bacteria increased during fasting. This family, in the order Clostridiales, promotes butryogenesis in the gut, a process that is associated with healthful metabolic and prolongevity effects. IF-associated alterations to the microbiome may play a key role in the metabolic and potential healthspan-enhancing benefits of IF and dietary restriction.

Rejuvenation Through Oxygen, More or Less.

Modest modulation of oxygen intake, either by inducing mild intermittent hypoxia or hyperoxia appears to induce modest rejuvenative changes in mammals, in part, by activating key regulator hypoxia-induced factor 1a (HIF-1a). Interestingly both lower oxygen and transient higher oxygen levels induce this hypoxia regulator. Hyperbaric oxygen induces HIF-1a by the hyperoxic-hypoxic paradox that results from an overinduction of protective factors under intermittent hyperoxic conditions, leading to a state somewhat similar to that induced by hypoxia. A key difference being that SIRT1 is induced by hyperoxia, whereas it is reduced during hypoxia by the activity of HIF-1a. In a recent report, a small clinical trial employing 60 sessions of intermittent hyperbaric oxygen therapy (HBOT) studying old humans resulted in increased mean telomere length of immune cells including B cells, natural killer cells, T helper, and cytotoxic T lymphocytes. Moreover, there was a reduction in CD28null senescent T helper and cytotoxic T cells. In a parallel report, HBOT has been reported to enhance cognition in older adults, especially attention and information processing speed through increased cerebral blood flow (CBF) in brain regions where CBF tends to decline with age. The durability of these beneficial changes is yet to be determined. These preliminary results require follow-up, including more extensive characterization of changes in aging-associated biomarkers. An interesting avenue of potential work is to elucidate potential connections between hypoxia and epigenetics, especially the induction of the master pluripotent regulatory factors, which when expressed transiently have been reported to ameliorate some aging biomarkers and pathologies.

Metabolic Reprogramming Rejuvenates Aged Myeloid Cells Restoring Cognition.

Inflammaging is associated with aging-associated cognitive loss and neurodegeneration. Chronic nonsteroidal anti-inflammatory drug (NSAID) use has been reported to reduce the incidence of Alzheimer's disease (AD), presumably by inhibiting inflammation, although NSAIDs appear to not be good candidates for anti-AD therapeutics given disappointing clinical trial results. Prostaglandin E2 (PGE2) acts downstream of NSAID target COX-2, a cyclooxygenase, to activate several G-protein coupled receptors (GPCRs) including EP2, which is now reported to reduce glycolysis and oxidative phosphorylation during aging by increasing glycogen synthesis and polarizing myeloid cells toward the M1 proinflammatory phenotype. Inhibiting EP2 using small molecule drugs polarizes macrophages toward the anti-inflammatory phenotype, restores youthful metabolism and mitochondrial morphology as well as youthful hippocampus-based memory capability. EP2 may be a better target than COXs for the development of drugs that improve age-associated mild cognitive impairment and possibly even for the development of drugs to treat dementias.

SUMO Wrestles with Mitophagy to Extend Lifespan.

SUMOylation, a conserved protein post-translational modification that performs multiple functions including regulation of nuclear transport and transcription, is implicated in numerous biological processes including aging. RNAi knockdown of the sole Small Ubiquitin-like MOdifier (SUMO) gene, smo-1, in Caenorhabditis elegans shortened lifespan, whereas overexpression in the intestine modestly increased lifespan. Smo-1 is required for mitochondrial fission in a tissue-specific manner. Fission, in turn, is needed for mitophagy to maintain mitochondrial homeostasis during aging. SUMOlyation affects DAuer Formation (DAF)-16, which can be directly SUMOylated, and SKN-1, the homolog of mammalian Nrf2. These regulators play key roles in maintaining mitochondrial homeostasis. However, given the modest effect of overexpressing smo-1 on lifespan enhancement and potential interference with other genes that can promote increased lifespan, caution is advised in the translation of this study based on C. elegans. Although inhibitors of SUMOlyation have been developed for cancer and activators also have been identified, broad-acting biochemical pathway modifiers such as SUMO are often suboptimal drug targets and may not be as promising for antiaging applications as they first appear.

Contribution of Ferroptosis to Aging and Frailty.

Ferroptosis is a recently characterized cell death phenotype resulting from iron-catalyzed peroxidation of polyunsaturated fatty acid phospholipids. Increased dysfunctional iron metabolism is thought to lead to increased levels of iron and ferroptosis, which in turn leads to cell and organismal death at least in the nematode Caenorhabditis elegans. Drugs that block lipid peroxidation or scavenge intracellular iron extend healthspan and lifespan in C. elegans independently of other mechanisms such as the daf-1/daf-16 (insulin/insulin-like growth factor 1 [IGF-1]) pathway, but unlike many aging mechanisms do not alter temporal scaling across the life cycle of C. elegans, but rather act at specific late points in the organism's life history, temporarily blocking execution of critical dysfunction that results in listless worms. As such, inhibition of ferroptosis may be a means to extend healthspan and treat frailty and possibly neurodegenerative diseases that have a reported role for iron dyshomeostasis. However, a significant effort to understand ferroptosis in the context of mammalian and human biology is necessary. For example, some tumors block ferroptosis to survive. The constraints of balancing iron metabolism are significant and will require careful consideration in any drug development program.

Eosinophils and White Fat: Protection from Worms and Inflammaging.

Proinflammatory alterations of white adipose tissue (WAT) with increasing age play an important role in mammalian aging. WAT produced eotaxin-1 (CCL11-C-C motif chemokine ligand 11) and monocyte chemoattractant protein 1 (MCP-1) (CCL2) are elevated in old mammals. Obese and old adipose tissues produce excessive proinflammatory cytokines such as interleukin (IL)-6, CCL2, and IL-1-beta that contribute to inflammaging. WAT-based inflammaging involves an altered homeostatic equilibrium between proinflammatory cells such as activated type 1 macrophages, B cells (high IgJ) and T cells, and anti-inflammatory eosinophils and Tregs. Specifically, young and lean individuals exhibit a high eosinophil-to-macrophage ratio with an enrichment of alternative activated tissue macrophages that is reduced in the WAT of aging mice. Eosinophils from young animals adoptively transferred to old mice, home to WAT and reverse many of the immunoinflammatory signatures associated with aging. Whether eosinophil-based therapies for inflammaging could be created remains an open question.

Exercise Partially Rejuvenates Muscle Stem Cells.

Exercise has long been known to extend health and lifespan in humans and other mammals. However, typically exercise is thought to slow the loss of function that accompanies aging. Brett et al. have now shown that exercise restores functional competency to regenerate muscle stem cells (MuSCs) in mice as well as restore a significant portion of the transcriptional signature associated with young MuSCs. The mechanism involves the likely induction of plasma-borne factors that upregulate cell cycle regulator cyclin D1, which otherwise decreases with increasing age. Cyclin D1, in turn, through its noncanonical attenuation of TGF-beta/Smad3 signaling, helps maintain the regenerative capacity of MuSCs, which is lost as TGF-beta signaling increases with age. Interestingly, elevated levels of some proinflammatory regulators including NF-κB, TNF-alpha, and interleukin 6 (IL-6) are also reduced by exercise or ectopic expression of cyclin D1. Importantly, the rejuvenation is not complete, as Notch signaling, which also decreases with age, remains at old levels and the rejuvenative effect is not permanent: wearing off in ∼2 weeks after cessation of exercise. Understanding the limitations of the rejuvenative effect of exercise on MuSCs at the molecular level, including changes in the epigenome such as altered DNA methylation age, will be critical in developing more significant rejuvenative therapies including some for aged people wherein morbidities limit exercise.

Supplementation with Brush Border Enzyme Alkaline Phosphatase Slows Aging.

Diminished integrity of the intestinal epithelial barrier with advanced age is believed to contribute to aging-associated dysfunction and pathologies in animals. In mammals, diminished gut integrity contributes to inflammaging, the increase in inflammatory processes observed in old age. Recent work suggests that expression of intestinal alkaline phosphatase (IAP) plays a key role in maintaining gut integrity. IAP expression decreases with increasing age in mice and humans. Absence of IAP leads to liver inflammation and shortened life-spans in mice lacking the IAP gene. In normal mice, exogenous supplemental IAP reverses age-induced barrier dysfunction, improves aging-associated metabolic dysfunction, prevents microbiome dysbiosis (imbalance), and extends life-span. Consistent with IAP playing a conserved role in maintaining gut integrity, increased dietary IAP increases aging-diminished physical performance in flies. IAP helps maintain gut integrity in part by supporting the expression of tight junction proteins that maintain the intestinal epithelial barrier and by inactivating bacterial pro-inflammatory factors such as lipopolysaccharides (LPS) by dephosphorylation. Recombinant IAP is in late clinical trials for sepsis-associated acute kidney injury, suggesting it may soon become available as a therapeutic. Taken together, these reports support the idea that directly increasing IAP levels by supplemental recombinant IAP or by indirectly increasing IAP levels using dietary means to induce endogenous IAP may slow the development of aging-associated pathologies.

The Danger of Being Too Sympathetic: Norepinephrine in Alzheimer's Disease and Graying of Hair.

Although alterations in the sympathetic nervous system (SNS) with age have been reported, and serious degenerative diseases of the autonomic nervous system such as multiple system atrophy are more likely to strike older people, connections between dysregulated adrenergic receptors and age-associated diseases and phenotypes have not been well studied. Two recent reports suggest that SNS may be more closely connected than previously appreciated. First, low nanomolar concentrations of Alzheimer's disease (AD)-associated Aß42-amyloid oligomers alter signaling by SNS neurotransmitter norepinephrine (NE) to sufficiently activate kinase GSK3ß to hyperphosphorylate tau, a key mediator of neurotoxicity in AD. Connecting beta-amyloid to tau in AD has been a key quest in understanding AD and developing therapeutics. The α2 adrenergic receptor inhibitory drug idazoxan reduces GSK3ß activity and tau phosphorylation in AD mice with improved cognitive function, even in the presence of beta-amyloid deposits. In this study, SNS activation in the brain coupled with problematic Aß42-amyloid oligomers result in serious consequences that can be ameliorated by reducing SNS signaling. A second example of the detrimental effects of increased SNS signaling is the premature graying of hair in response to stress. Secretion of NE resulting from stress causes differentiation of most hair pigment melanocyte stem cells (MeSCs) into melanocytes, rapidly depleting the hair follicle of pigment-producing cells as mature melanocytes undergo apoptosis and MeSCs are eventually eliminated. Blockade of NE SNS signaling preserves hair coloration in stressed animals. Increased SNS activation has serious apparently irreversible effects on homeostasis in both situations. Although neither report directly addresses aging, given that AD and the loss of hair pigmentation have strong age associations, it is of interest to better understand the role that SNS has in promoting age-associated phenotypes generally and determine if tuning the SNS through drug-mediated attenuation of SNS signaling may be of medical benefit.

Increased REST to Optimize Life Span?

Reduced levels of neural activity are associated with a longer life span in the nematode Caenorhabditis elegans and in mice. Augmented neural activity is associated with a shorter life span. Recent studies show that levels of repressor element 1-silencing transcription factor (REST) increase with normal aging in mice and humans, and reduce neuronal excitation. In C. elegans, increased expression of spr-4, a functional REST homologue, increased the worm life span and is required for classical life span increase mediated by reduced DAF-2/insulin-IGF-1 and increased DAF-16. Preliminary evidence shows that REST and FOXO1, a DAF-16, homologue increase during mammalian aging, and that REST activity is needed for the age-related FOXO1 increase. On the contrary, REST is activated in epilepsy and plays a role in the pathogenesis of Huntington's disease. A simple unifying hypothesis suggests that REST is a "goldilocks-effect factor": too little REST promotes excitotoxic activity, which in turn leads to neurodegenerative diseases such as Alzheimer's. Appropriate increased levels of REST maintain the excitation/inhibition (E-I) balance by reducing potential excitotoxic activity. Increased levels of REST beyond this are toxic as neurons become dysfunctional due to loss of a neuronal phenotype.

Epigenetic Age Reversal by Cell-Extrinsic and Cell-Intrinsic Means.

Reversal of aging by factors or drugs that reprogram adult cells to induced pluripotent stem cells suggests that at least at the cellular level aging may be reversible by resetting somatic cell state to a "ground state." An open question has been whether such rejuvenation is possible in whole organisms, especially in mammals. A related key question is whether rejuvenation can be dissociated from dedifferentiation. Several recent reports suggest that one prominent biomarker of mammalian aging, age-associated DNA methylation (DNAm) state that has been used to create DNAm age (DNAma) clocks, can be partially reversed by intrinsic treatment of cells with sets of reprogramming factors without affecting cell fate. Partial reprogramming using a superset of reprogramming factors applied transiently or subset of Yamanaka factors applied continually can increase regenerative potential, and reverse DNAma, while maintaining cell identity. Alternatively, a cell-extrinsic manipulation can accomplish something similar. A small preliminary clinical trial in humans suggests that systemic treatment with a cocktail of growth hormone, dehydroepiandrosterone, and metformin could also partially reverse DNAma and at the same time regenerate the thymus, which shrinks with age. Important questions are raised: How completely does reversing DNAma clocks embody a reversal of other age-related phenotypes, such as functional decline in strength, cognition, or immunity? How universal are these epigenetic changes at the tissue and cell levels? For example, do populations of younger stem cells exist that respond to these manipulations and then only confer the appearance of decreasing DNAma as they proliferate and differentiate? Together, these studies have profound implications for the development of antiaging and healthspan-enhancing therapies. A combination of both intrinsic and extrinsic modalities will most likely provide an optimal benefit.

Roads to the Fountain of Youth? Rejuvenating Intestinal Stem Cells.

The intestinal stem cells (ISCs) of old mice and humans exhibit a reduced capacity for regeneration and repair. Compromised intestinal function may play a key role in systemic aging-related changes: not only in the affected gut, but also in the nervous and cardiovascular systems. For example, progression of age-related neurodegenerative diseases such as Alzheimer's and Parkinson's has been linked to increased inflammation from gut microbiota in old mammals, which, in turn, may be linked bidirectionally with reduced ISC function. Intestinal organoid formation has been used to dissect the mechanisms of decline of ISC function. Alterations of the Wnt pathway, including downregulation of Wnt ligands in ISCs and upregulation of Wnt ligand inhibitor Notum in Paneth cells, and dysregulation of mTORC1 contribute to the observed age-related decline. Short-term fasting, caloric restriction, and peroxisome proliferator-activated receptor delta agonists have been reported to increase ISC function in adult mice. Moreover, the mTOR inhibitor rapamycin, NAD+ precursor nicotinamide riboside, and ABC99, a small molecule Notum inhibitor, have all been reported to rejuvenate ISC function in old mice and thus may have promise in humans. However, there is some controversy over the key mechanisms involved in loss of function of ISCs, which likely results, in part, from differences in how the in vitro organoid assays are performed. Moreover, how the microbiome modulates the function of ISCs and vice versa remains to be elucidated.