Human immunodeficiency virus (HIV) can invade the central nervous system during the initial stages of infection and contribute to HIV-associated neurocognitive disorder, affecting up to 50% of people living with HIV (PLWH). To investigate HIV-1-induced immunometabolic changes in the brain, we used a three-dimensional microglia-embedded human neural organoid model. Transcriptomic analysis and genome-scale metabolic modeling revealed that HIV-1 infection led to more pronounced transcriptional changes in the presence of microglia, including upregulation of pro-inflammatory pathways. We identified CCR6, important for HIV-1 permissiveness, to be significantly upregulated upon infection. Metabolic analysis showed increased expression in metabolite transport-related genes, including solute carrier (SLC) genes and altered amino acid metabolism, particularly involving arginine, proline, and tyrosine. These microglia-driven immunometabolic changes may contribute to neuronal dysregulation and, subsequently, neurological complications, which are often observed in PLWH. Early detection of these alterations could support timely therapeutic intervention to improve HIV-related neurologic insult.

Human picornavirus infections remain without approved antiviral therapies. Antiviral testing performed in immortalised cell lines often fails to predict antiviral efficacy in vivo and subsequently clinical outcomes. Here, we assessed how antiviral responses to clinically approved compounds differ between immortalised intestinal cell lines and a physiologically relevant human intestinal organoid-derived epithelium (HIE) model. Posaconazole, itraconazole, ribavirin, remdesivir, and pocapavir, compounds with reported anti-picornaviral activity in cell lines, were evaluated against enterovirus A71 (EV-A71) and human parechovirus 1 (HPeV-1) in Caco-2 and HT-29 cell lines, and human foetal HIE monolayers from multiple donors. Remdesivir consistently inhibited EV-A71 replication in Caco-2 and HT-29 cell lines, and HPeV-1 replication in Caco-2 cells, whereas the other compounds showed limited or variable effects. In contrast, in HIE, remdesivir and ribavirin reduced EV-A71 replication in only one donor, while itraconazole and posaconazole exhibited donor-specific cytotoxicity. No compound consistently inhibited HPeV-1 replication across HIE donors despite detectable antiviral effect in cell lines. Comparative analysis of drug-metabolising enzyme expression did not fully account for donor-specific response variability. Collectively, these findings reveal substantial heterogeneity in antiviral responses that is not captured by standard cell lines and underscore the value of incorporating genetically and physiologically diverse human organoid-based models into preclinical antiviral screening pipelines to improve translational fidelity.

Human coronaviruses have been primarily associated with upper respiratory tract infections, yet cases of gastrointestinal symptoms in COVID-19 patients have highlighted their potential to cause systemic disease. Here, we detail the infection of intestinal epithelia by an endemic, low-pathogenic human coronavirus, human alphacoronavirus 229E, using patient-derived human intestinal enteroids (HIEs) from donors of various ages. Using fetal, pediatric, and adult HIEs, we investigated how physiologically relevant temperatures: 37 °C and 32 °C, reflecting gastrointestinal and upper-airway conditions, respectively, modulate epithelial responses and viral infection dynamics. We show that there is temperature-dependent transcriptional reprogramming, indicating strong temperature-dependent regulation of virus replication and epithelial responses. Among the seasonal coronaviruses tested, only HCoV-229E productively infects HIEs. At 32 °C, HCoV-229E replicates efficiently in enteroids from all donor ages and releases high titers of infectious progeny. In contrast, at 37 °C, productive replication is largely confined to fetal and a subset of pediatric tissues, revealing a developmental and temperature-sensitive restriction on infection. Confocal and flow cytometry analyses identify enterocytes as the primary target cells for HCoV-229E. Furthermore, we show that camostat, a serine protease inhibitor, significantly reduces HCoV-229E replication in HIEs, confirming a critical role for host serine protease activity. Collectively, these findings establish HIEs as a relevant model for HCoV-229E-host interactions and reveal temperature- and age-dependent determinants governing intestinal permissiveness to this seasonal coronavirus.

Enterovirus A71 (EV-A71) and enterovirus D68 (EV-D68) are recognised as causative agents of severe neurological complications, including acute flaccid myelitis (AFM). However, the molecular mechanisms underlying the neurovirulence and effects on neuromuscular integrity remain poorly understood. Here, we employed human induced pluripotent stem cell-derived neuromuscular organoids (NMOs) to investigate the cellular tropism and pathogenic effects of EV-A71 and EV-D68 in a human-relevant context. Both viruses infected neuronal populations within NMOs, with EV-A71 exhibiting higher levels of viral replication than EV-D68. Transcriptomic analysis revealed downregulation of neuronal and muscular gene networks following infection. EV-A71 preferentially suppressed neuronal pathways, while both viruses exerted comparable effects on muscle-associated gene expression. These transcriptional changes highlighted alterations in pathways governing neuronal and muscle function and communication, prompting examination of synaptic vesicle machinery components. At the protein level, both viruses were associated with sporadic cleavage of the neuronal SNARE protein synaptosomal-associated protein 25 (SNAP25). In addition, infection with either virus increased cleaved caspase-3 levels, consistent with activation of apoptotic signalling. Together, these findings indicate virus-specific downstream effects and establish NMOs as a robust platform for dissecting enterovirus-host interactions relevant to AFM.

Enteroviruses (EV) are worldwide circulating viruses causing a broad range of disease manifestations. Sero-surveillance, by measuring neutralising antibodies against specific EV types, can provide valuable information on viral spread and community circulation. EV neutralisation is generally regarded as type-specific, providing the foundation for the reliability of EV sero-surveillance. Recent data has challenged the assumption of type-specific neutralisation. To study the extent of cross-neutralisation, we measured neutralising antibody titres in EV type-specific polyclonal animal sera against twelve recently circulating homotypic and heterotypic clinical and prototype strains belonging to all four EV species, making use of virus neutralisation assays. We found no cross-neutralising Abs against EV-A71, CVA4, CVB2, CVB5, CVA24, and EV-D68. No cross-neutralising antibodies were found in poliovirus 1, 2 and 3 anti-sera. However, cross-neutralising antibodies were found in anti-CVA20 and anti-CVA24 animal sera against the included echovirus types, E11 and E30. Our study demonstrates that cross-neutralisation among EV is generally uncommon, though not absent. To enhance the reliability and comparability of sero-surveillance studies, cross-neutralisation between geno- and subgenotypes should be further evaluated in humans and virus neutralisation assays (VNA) should be further standardised.

IntroductionThe intestinal mucosa plays a vital role in nutrient absorption, drug metabolism, and pathogen defence. Advances in single-cell technologies have highlighted the specialised roles of various cell types that execute these diverse functions. Beyond intestinal epithelial cells, fibroblasts regulate the extracellular matrix (ECM) and modulate pro-inflammatory signalling, while antigen-presenting cells (macrophages and dendritic cells) maintain intestinal homeostasis and immune responses. However, the incorporation of such cellular complexity within existing in vitro models of the human intestine is currently lacking.MethodsWe developed a human intestinal co-culture model that incrementally mimics the mucosal cellular environment, comprising intestinal epithelial cells, intestinal fibroblasts, and antigen-presenting cells. The model incorporated co-cultures of both adult and foetal cells to facilitate studies of intestinal development, barrier function, inflammation, and viral infections.ResultsWe successfully established an advanced multi-cellular intestinal co-culture model that recapitulates key features of the native mucosal environment. This model demonstrated the capacity for ECM deposition, Paneth cell differentiation, and functional immune interactions. Notably, the co-culture system effectively modelled immune responses during viral infections, demonstrating the utility of incorporating multiple cell types and developmental stages.DiscussionOur co-culture model improves the physiological relevance of in vitro studies by incorporating multi-cellular complexity previously lacking in existing systems. This advancement enables the exploration of epithelial-mesenchymal-immune crosstalk in intestinal health and disease, providing a powerful platform for studying intestinal development, barrier dysfunction, and infection-related pathology.

Enteroviruses (EV) usually cause acute, mild, self-limiting disease. Chronic infections with EVs are rare, and typically occur in patients with immunodeficiency, posing a high risk of severe outcomes. We report a rare case of chronic diarrhea caused by coxsackievirus A1 (CVA1) (from EV-C species) infection in a patient with a common variable immunodeficiency, who was on treatment with pooled intravenous immunoglobulin (IVIG) from the Netherlands. To explore treatment options, we assessed the presence of neutralizing antibodies (nAbs) against CVA1 in pooled IVIG from South Africa, where EV-Cs are prevalent, and tested the antiviral efficacy of US Food and Drug Administration-approved drugs like fluoxetine, itraconazole, ribavirin, and remdesivir (RDV) against CVA1 in vitro. Both Dutch and South African IVIG showed low nAb titers against CVA1. The patient, treated with Dutch IVIG, also received a combination of amantadine and fluoxetine, which were discontinued due to side effects. Among the drugs tested, only RDV significantly inhibited CVA1 replication in rhabdomyosarcoma (RD) cells. This in vitro efficacy was not reflected by a favorable clinical response after treatment of the patient with RDV. In concordance with unfavorable antiviral response in the patient, preliminary tests on a co-culture model containing isogenic human intestinal cells and intestinal fibroblasts showed no significant reduction in CVA1 RNA copies after RDV administration. In conclusion, our results showed that repurposing of drugs that have shown in vitro efficacy does not translate well to the patients, and this is also reflected in a more physiologically relevant model of the human intestine.

Parechovirus ahumpari 3 (HPeV-3) is among the main agents causing severe neonatal neurological infections such as encephalitis and meningitis. However, the underlying molecular mechanisms and changes to the host cellular landscape leading to neurological disease has been understudied. Through quantitative proteomic analysis of HPeV-3 infected neural organoids, we identified unique metabolic changes following HPeV-3 infection that indicate immunometabolic dysregulation. Protein and pathway analyses showed significant alterations in neurotransmission and potentially, neuronal excitotoxicity. Elevated levels of extracellular glutamate, lactate dehydrogenase (LDH), and neurofilament light (NfL) confirmed glutamate excitotoxicity to be a key mechanism contributing to neuronal toxicity in HPeV-3 infection and can lead to apoptosis induced by caspase signaling. These insights are pivotal in delineating the metabolic landscape following severe HPeV-3 CNS infection and may identify potential host targets for therapeutic interventions.

Introduction:The process of transition from paediatric to adult care is a crucial step towards self-management of healthcare for young adults with chronic health conditions. In the Netherlands, the On Your Own Feet program offers an extensive framework for supporting optimal transition. In this national study, we aimed to evaluate the transition experiences of young adults with perinatal HIV who transferred from a paediatric to an adult HIV treatment centre. Methods:Participants who transferred to adult care less than six years ago received questionnaires on transition experiences, the validated On Your Own Feet - Transition Experiences Scale (OYOF-TES), and demographics. Demographic and healthcare-related variables were collected from patients' medical files. We explored correlations between OYOF-TES scores and other variables. Results:Of 44 participants, 29 responded (65.1 %). Their median age was 20 (IQR 19-23). Overall, high scores were found on the subscales 'Reception in adult care' (median 4.8, IQR 4.0-5.0), 'Alliance paediatric and adult care' (median 4.0, IQR 3.4-4.4), and 'Transfer readiness' (median 4.2, IQR 3.8-4.8). The lowest scores were found in the subscales 'Preparation for transfer' (median 3.3, IQR 2.7-4.0) and 'Youth involvement' (median 3.5, IQR 2.5-4.5). Higher scores on transition experiences were correlated with younger age and female sex, while lower scores were correlated with those of whom one or both biological parents died during childhood. Conclusion:Transition experiences in our population were positive. Younger participants felt better prepared for the transfer, which indicates that transition is increasingly becoming a joint effort between young adults and healthcare professionals and that pathways accustomed to individual needs improve the transition experience for young adults with perinatal HIV.

Youth with perinatally acquired HIV (PHIV) are at risk for cardiovascular disease (CVD) despite combination anti-retroviral therapy (cART). Longitudinal data on the impact of HIV and cART on lipid metabolism and CVD risk in PHIV youth is limited. We investigated lipid and lipoprotein levels in PHIV youth and matched controls over time and examined associations with cART and metabolic syndrome (MetS) markers. We included 32 PHIV and 36 controls at three time points: 2013, 2018 and 2023. In 2023, we assessed lipid profiles cross-sectionally in a larger cohort of 53 PHIV participants and 45 controls. Measurements included lipoprotein (a) (Lp(a)), apolipoprotein B (ApoB), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), reduced high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TC) and markers related to MetS risk. The median age was 21.7 years (IQR 16.7–25.2) for PHIV participants and 21.2 years (16.8—22.3) for controls in 2023 for longitudinal assessment. No significant differences in lipid or lipoprotein levels were observed over time (p values > 0.05). TG levels were significantly higher in PHIV participants at second assessment (p = 0.043), but other levels were comparable (p values > 0.05). Higher Lp(a) levels were associated with higher LDL-C and ApoB levels, however associations were significantly weakened among PHIV participants. Furthermore, protease inhibitor (PI) use was associated with elevated TC, TG and LDL-C. During cross-sectional assessment median age was 17.4 years (IQR 12.7–22.4) and 19.1 years (IQR 15.0—21.8) for PHIV youth and controls. Lipid and MetS markers were comparable between groups (p values > 0.05). PHIV youth on cART showed similar lipid and lipoprotein levels over time compared to matched controls. Lp(a) associations with lipid markers were weakened for PHIV youth and PI use was associated with lipid alterations. Our results imply that while lipid profiles, including Lp(a), are important components of cardiovascular health monitoring, the increased CVD risk observed in PHIV youth may be more substantially influenced by disease-specific or broader pathophysiological mechanisms related to HIV-infection and treatment. Dutch clinical trial registration: Overview of Medical Research in the Netherlands (OMON) (ID: NL-OMON53727).

Background:With effective antiretroviral treatment, more children with perinatally acquired human immunodeficiency virus (HIV) reach adulthood. We assessed their long-term socioeconomic outcomes-educational level, reliance on social welfare or absence of income, and living in poverty-using a sibling comparison design to disentangle biological from familial and environmental influences. Methods:We conducted a retrospective cohort study from the Netherlands using data from the ATHENA cohort and nonpublic microdata from Statistics Netherlands (CBS). We included individuals aged ≥18 years with perinatally acquired HIV and siblings without HIV (identified through maternal CBS data). Logistic regression evaluated associations between sociodemographic and HIV-related factors with outcomes. Generalized estimating equations assessed differences between groups. Results:Among 145 individuals with HIV, 12% had low educational level, 17% relied on social welfare or had no income, and 15% lived in poverty. Receiving HIV care before 1996 was associated with low educational level (odds ratio [OR], 4.58 [95% confidence interval {CI}, 1.46-14.43]; P = .01), while older age increased odds of having no income or reliance on social welfare (OR, 1.24/year [95% CI, 1.10-1.39]; P = .0001). Older age at HIV diagnosis was linked to living in poverty (OR, 1.20/year [95% CI, 1.06-1.34]; P = .003). Compared to 94 siblings, individuals with HIV had higher odds of low education (adjusted OR [aOR], 6.59 [95% CI, 1.91-22.73]; P < .01) and having no income or social welfare reliance (aOR, 2.54 [95% CI, 1.05-6.12]; P = .04). Poverty rates did not differ significantly between groups. Conclusions:Adults with perinatally acquired HIV face educational and economic disadvantages compared to their siblings without HIV, highlighting the lasting impact of perinatal HIV beyond familial or environmental background.

Human cytomegalovirus (CMV) infections can cause severe neurological complications, particularly in newborns and immunocompromised patients. Children affected with congenital CMV infection may develop long-term neurological damage and intellectual disabilities. Currently, postnatal antiviral therapies are limited and there are no prenatal options available. Research on antivirals against congenital CMV infections is, at least partly, restricted due to the lack of physiologically relevant models and the use of lab-adapted CMV strains with limited clinical relevance. In this study, we evaluated the toxicity and antiviral efficacy of three FDA-approved anti-CMV drugs against two CMV strains, a clinical and a lab-adapted strain, using two human induced pluripotent stem cell (iPSC-)derived central nervous system models, viz. neural progenitor cells (NPCs) and dorsal forebrain regionalized neural organoids (RNOs). We found iPSC line-dependent differences in antiviral toxicity. We observed that antiviral treatment restored NPCs and RNOs gene expression after CMV infection and reduced CMV copy numbers. Infection of NPCs and RNOs with the clinical CMV strain, but not with the lab-adapted strain, led to an impaired expression of cortical development markers. Our findings highlight the value of using physiologically relevant human models and clinical CMV strains to understand the neuropathogenesis of congenital CMV and to test therapeutic strategies.

Halofuginone hydrobromide has shown potent antiviral efficacy against a variety of viruses such as SARS-CoV-2, dengue, or chikungunya virus, and has, therefore, been hypothesized to have broad-spectrum antiviral activity. In this paper, we tested this broad-spectrum antiviral activity of Halofuginone hydrobomide against viruses from different families (Picornaviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae, and Flaviviridae). To this end, we used relevant human models of the airway and intestinal epithelium and regionalized neural organoids. Halofuginone hydrobomide showed antiviral activity against SARS-CoV-2 in the airway epithelium with no toxicity at equivalent concentrations used in human clinical trials but not against any of the other tested viruses.

The gastrointestinal tract is a prominent portal of entry for HIV-1 during sexual or perinatal transmission, as well as a major site of HIV-1 persistence and replication. Elucidation of underlying mechanisms of intestinal HIV-1 infection are thus needed for the advancement of HIV-1 curative therapies. Here, we present a human 2D intestinal immuno-organoid system to model HIV-1 disease that recapitulates tissue compartmentalization and epithelial-immune cellular interactions. Our data demonstrate that apical exposure of intestinal epithelium to HIV-1 results in viral internalization, with subsequent basolateral shedding of replication-competent viruses, in a manner that is impervious to antiretroviral treatment. Incorporation of subepithelial dendritic cells resulted in HIV-1 luminal sampling and amplification of residual viral replication of lab-adapted and transmitted-founder (T/F) HIV-1 variants. Markedly, intraepithelial viral capture ensued an altered distribution of specialized endosomal pathways alongside durable sequestration of infectious HIV-1 within lysobisphosphatidic acid (LPBA)-rich vesicles. Therapeutic neutralization of LBPA-dependent trafficking limited productive HIV-1 infection, and thereby demonstrated the pivotal role of intraepithelial multivesicular endosomes as niches for virulent HIV-1 within the intestinal mucosa. Our study showcases the application of primary human 2D immune-competent organoid cultures in uncovering mechanisms of intestinal HIV-1 disease as well as a platform for preclinical antiviral drug discovery.

Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease, the highly prevalent PeV-A1 is not associated with CNS disease. Here, we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively, our findings align with clinical observations and suggest a role for neuroinflammation, rather than viral replication, in PeV-A3 (and E11) infection.

Children with perinatally acquired human immunodeficiency virus (PHIV) are growing into adulthood with HIV and treatment-associated comorbidities, such as dyslipidemia and insulin resistance. HIV is identified as independent risk factor for cardiovascular disease (CVD). The hypothesis behind increased CVD risk associated with HIV includes vascular inflammation, dyslipidemia and combination antiretroviral therapy (cART) metabolomic toxicity. To investigate differences in lipid profiles and pathophysiological mechanisms of CVD risk in adolescents with PHIV, we compared the plasma lipidome of PHIV adolescents and HIV-negative controls. We additionally investigated the influence of current cART regimens and increased lipoprotein(a) (Lp(a)) levels on the plasma lipidome. We included 20 PHIV-infected adolescents and 20 HIV-negative controls matched for age, sex, ethnic origin and socio-economic status. Plasma lipidome was measured using Thermo Scientific Ultimate 3000 binary high-performance liquid chromatography (HPLC)–mass spectrometry. We evaluated the plasma lipidome in PHIV adolescents using different cART regimens (including those known to be associated with lipid alterations). The median age was 17.5 years (15.5–20.7) and 16.5 years (15.7–19.8) for PHIV adolescents and controls, respectively. Of PHIV adolescents, 45% used a non-nucleotide reverse transcriptase inhibitor (NNRTI)-based (25%) or protease inhibitor (PI)-based (20%) cART regimen. In this pilot study, we observed no significant differences between lipidomic profiles between PHIV adolescents and controls. We observed no differences in the plasma lipidome in participants with increased versus normal Lp(a) levels. Different cART regimens appear to influence chain length differences in the plasma lipidome of PHIV adolescents; however, the significance and causality of this observation remains undetermined. Further research on the influence of cART on lipid composition could further identify these alterations.

Non-polio enteroviruses (NPEV) cause significant disease worldwide. Population-based sero-surveillance, by measuring antibodies against specific NPEV types, provides additional information on past circulation and the prediction for future upsurges. Virus neutralisation assays (VNA), the current method of choice for measuring NPEV type specific antibodies, are not entirely standardised. Via the European Non-Polio Enterovirus Network, we organised a VNA quality assessment in which twelve laboratories participated. We provided five echovirus (E) types (E1, E18, E30 G2, E30 G6 and E6) and intravenous immunoglobulins (IVIG) as a sample for the NPEV VNA quality assessment. Differences in VNA protocols and neutralising Ab (nAb) titres were found between the participating laboratories with geometric coefficients of variation ranging from 10.3-62.9 %. Mixed-effects regression analysis indicated a small but significant effect of type of cell line used. Harmonisation of cell line passage number, however, did not improve variation between laboratories. Calibration by making use of a reference sample, reduced variation between laboratories but differences in nAb titres remained higher than two log2 dilution steps. In conclusion, sero-surveillance data from different laboratories should be compared with caution and standardised protocols are needed.

Introduction:Health-related quality of life (HRQoL) in adult people with HIV is lower than that of the general population. Previously, no differences were detected in HRQoL of Dutch children with perinatal HIV (PHIV) compared with norm groups. In this study, we compared HRQoL of PHIV young adults (PHIV-YA, aged 18-30 years) with 2 norm groups, the healthy Dutch YA population and YA with various chronic conditions.Methods:Participants received questionnaires on HRQoL, adherence, and demographics. Additional social and health care-related variables were collected from patients' medical files. We explored correlations between HRQoL and demographic characteristics. Effect sizes (ES, Hedges 'g with confidence intervals) were calculated to quantify the difference between PHIV-YA and norm groups.Results:Of 81 participants, 53 filled out the questionnaires. Compared with the healthy Dutch YA population, PHIV-YA aged 18-30 years had significantly lower HRQoL scores in the school/work subscale. PHIV-YA aged 26-30 years had significantly lower total, physical and psychosocial HRQoL scores as well. Participants in the older age category had lower HRQoL scores throughout all subcategories as compared with the younger age group. For PHIV-YA aged 18-25 years, lower scores on the school/work subscale were correlated with substance use and being born outside the Netherlands.Conclusions:PHIV-YA had low HRQoL scores in school/work functioning compared with the healthy Dutch YA population. The circumstances driving these outcomes are likely to be multidimensional, including HIV infection, social background, and challenges in growing up with a chronic condition.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive neuromuscular degeneration resulting from mutations in the survival motor neuron (SMN1) gene. The availability of disease-modifying therapies for SMA therapies highlights the pressing need for easily accessible and cost-effective blood biomarkers to monitor treatment response and for better disease management. Additionally, the wide implementation of newborn genetic screening programs in Western countries enables presymptomatic diagnosis of SMA and immediate treatment administration. However, the absence of monitoring and prognostic blood biomarkers for neurodegeneration in SMA hinders effective disease management. Neurofilament light protein (NfL) is a promising biomarker of neuroaxonal damage in SMA and reflects disease progression in children with SMA undergoing treatment. Recently, the European Medicines Agency issued a letter of support endorsing the potential utilization of NfL as a biomarker of pediatric neurological diseases, including SMA. Within this review, we comprehensively assess the potential applications of NfL as a monitoring biomarker for disease severity and treatment response in pediatric-onset SMA. We provide reference ranges for normal levels of serum based NfL in neurologically healthy children aged 0-18 years. These reference ranges enable accurate interpretation of NfL levels in children and can accelerate the implementation of NfL into clinical practice.

The intestinal mucosa plays a vital role in nutrient absorption, drug metabolism, and pathogen defence. Advances in single-cell technologies have highlighted the specialised roles of various cell types that execute these diverse functions. Aside from intestinal epithelial cells, fibroblasts play an essential role in regulating the extracellular matrix and controlling pro-inflammatory signalling, and antigen-presenting cells (macrophages and dendritic cells) maintain intestinal homeostasis and immune responses. The incorporation of such cellular complexity within the existing in vitro models of the human intestine is currently challenging. To address this, we developed a human intestinal model that accurately mimics the mucosal cellular environment comprising intestinal epithelial cells, intestinal fibroblasts, and antigen presenting cells. This model includes co-cultures of adult and foetal cells, facilitating studies on barrier function, inflammation, and viral infections. It replicates extracellular matrix deposition, Paneth cell differentiation, immune interactions, and can be used to model host-pathogen interactions. Our advanced co-culture model improves the physiological relevance of in vitro studies, enabling the exploration of epithelial-mesenchymal-immune crosstalk and its role in intestinal health and disease. ### Competing Interest Statement A.D., A.E., and S.S. are employees of STEMCELL Technologies Ltd., Cambridge, UK. A.E., S.L., R.K.C, W.C. are employees of STEMCELL Technologies Inc., Vancouver, Canada. A.E. is the founder and CEO of STEMCELL Technologies Inc., Vancouver, Canada. A.D. and S.S. have provisional patent applications related to this research. C.C., J.K., N.J., E.F., D.P., K.C.W. and A.S. declare no competing interests