Loss of ARID3A perturbs intestinal epithelial proliferation-differentiation ratio and regeneration.

Intestinal stem cells at the crypt divide and give rise to progenitor cells that proliferate and differentiate into various mature cell types in the transit-amplifying (TA) zone. Here, we showed that the transcription factor ARID3A regulates intestinal epithelial cell proliferation and differentiation at the TA progenitors. ARID3A forms an expression gradient from the villus tip to the upper crypt mediated by TGF-ß and WNT. Intestinal-specific deletion of Arid3a reduces crypt proliferation, predominantly in TA cells. Bulk and single-cell transcriptomic analysis shows increased enterocyte and reduced secretory differentiation in the Arid3a cKO intestine, accompanied by enriched upper-villus gene signatures of both cell lineages. We find that the enhanced epithelial differentiation in the Arid3a-deficient intestine is caused by increased binding and transcription of HNF1 and HNF4. Finally, we show that loss of Arid3a impairs irradiation-induced regeneration with sustained cell death and reprogramming. Our findings imply that Arid3a functions to fine-tune the proliferation-differentiation dynamics at the TA progenitors, which are essential for injury-induced regeneration.

Retrospective analysis of hospital electronic health records reveals unseen cases of acute hepatitis with unknown aetiology in adults in Oxfordshire.

BACKGROUND: An outbreak of acute severe hepatitis of unknown aetiology (AS-Hep-UA) in children during 2022 was subsequently linked to infections with adenovirus-associated virus 2 and other 'helper viruses', including human adenovirus. It is possible that evidence of such an outbreak could be identified at a population level based on routine data captured by electronic health records (EHR). METHODS: We used anonymised EHR to collate retrospective data for all emergency presentations to Oxford University Hospitals NHS Foundation Trust in the UK, between 2016-2022, for all ages from 18 months and older. We investigated clinical characteristics and temporal distribution of presentations of acute hepatitis and of adenovirus infections based on laboratory data and clinical coding. We relaxed the stringent case definition adopted during the AS-Hep-UA to identify all cases of acute hepatitis with unknown aetiology (termed AHUA). We compared events within the outbreak period (defined as 1st Oct 2021-31 Aug 2022) to the rest of our study period. RESULTS: Over the study period, there were 903,433 acute presentations overall, of which 391 (0.04%) were classified as AHUA. AHUA episodes had significantly higher critical care admission rates (p < 0.0001, OR = 41.7, 95% CI:26.3-65.0) and longer inpatient admissions (p < 0.0001) compared with the rest of the patient population. During the outbreak period, significantly more adults (≥ 16 years) were diagnosed with AHUA (p < 0.0001, OR = 3.01, 95% CI: 2.20-4.12), and there were significantly more human adenovirus (HadV) infections in children (p < 0.001, OR = 1.78, 95% CI:1.27-2.47). There were also more HAdV tests performed during the outbreak (p < 0.0001, OR = 1.27, 95% CI:1.17-1.37). Among 3,707 individuals who were tested for HAdV, 179 (4.8%) were positive. However, there was no evidence of more acute hepatitis or increased severity of illness in HadV-positive compared to negative cases. CONCLUSIONS: Our results highlight an increase in AHUA in adults coinciding with the period of the outbreak in children, but not linked to documented HAdV infection. Tracking changes in routinely collected clinical data through EHR could be used to support outbreak surveillance.

Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

COVID-19 in non-hospitalised adults caused by either SARS-CoV-2 sub-variants Omicron BA.1, BA.2, BA.4/5 or Delta associates with similar illness duration, symptom severity and viral kinetics, irrespective of vaccination history.

BACKGROUND: SARS-CoV-2 variant Omicron rapidly evolved over 2022, causing three waves of infection due to sub-variants BA.1, BA.2 and BA.4/5. We sought to characterise symptoms and viral loads over the course of COVID-19 infection with these sub-variants in otherwise-healthy, vaccinated, non-hospitalised adults, and compared data to infections with the preceding Delta variant of concern (VOC). METHODS: In a prospective, observational cohort study, healthy vaccinated UK adults who reported a positive polymerase chain reaction (PCR) or lateral flow test, self-swabbed on alternate weekdays until day 10. We compared participant-reported symptoms and viral load trajectories between infections caused by VOCs Delta and Omicron (sub-variants BA.1, BA.2 or BA.4/5), and tested for relationships between vaccine dose, symptoms and PCR cycle threshold (Ct) as a proxy for viral load using Chi-squared (χ2) and Wilcoxon tests. RESULTS: 563 infection episodes were reported among 491 participants. Across infection episodes, there was little variation in symptom burden (4 [IQR 3-5] symptoms) and duration (8 [IQR 6-11] days). Whilst symptom profiles differed among infections caused by Delta compared to Omicron sub-variants, symptom profiles were similar between Omicron sub-variants. Anosmia was reported more frequently in Delta infections after 2 doses compared with Omicron sub-variant infections after 3 doses, for example: 42% (25/60) of participants with Delta infection compared to 9% (6/67) with Omicron BA.4/5 (χ2 P < 0.001; OR 7.3 [95% CI 2.7-19.4]). Fever was less common with Delta (20/60 participants; 33%) than Omicron BA.4/5 (39/67; 58%; χ2 P = 0.008; OR 0.4 [CI 0.2-0.7]). Amongst infections with an Omicron sub-variants, symptoms of coryza, fatigue, cough and myalgia predominated. Viral load trajectories and peaks did not differ between Delta, and Omicron, irrespective of symptom severity (including asymptomatic participants), VOC or vaccination status. PCR Ct values were negatively associated with time since vaccination in participants infected with BA.1 (ß = -0.05 (CI -0.10-0.01); P = 0.031); however, this trend was not observed in BA.2 or BA.4/5 infections. CONCLUSION: Our study emphasises both the changing symptom profile of COVID-19 infections in the Omicron era, and ongoing transmission risk of Omicron sub-variants in vaccinated adults. TRIAL REGISTRATION: NCT04750356.

Mosaic BRAF Fusions Are a Recurrent Cause of Congenital Melanocytic Nevi Targetable by MAPK Pathway Inhibition.

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.

Healthcare Worker Characteristics Associated with SARS-CoV-2 Vaccine Uptake in Ireland; a Multicentre Cross-Sectional Study.

The prevention of SARS-CoV-2 acquisition and transmission among healthcare workers is an ongoing challenge. Vaccination has been introduced to mitigate these risks. Vaccine uptake varies among healthcare workers in the absence of vaccine mandates. We investigated engagement with SARS-CoV-2 vaccination among healthcare workers and identified characteristics associated with lower vaccine uptake. This multi-site cross-sectional study recruited n = 1260 healthcare workers in both clinical and non-clinical roles over a three-month period from November 2022. Participants reported their engagement with the primary SARS-CoV-2 vaccination programme and subsequent booster programmes, as well as providing demographic, occupational and personal medical history information. Multivariable linear regression identified characteristics associated with vaccine uptake. Engagement with vaccination programmes was high, with 88% of participants receiving at least one booster dose after primary vaccination course. Younger age and female sex were associated with reduced vaccine uptake. Healthcare workers in non-clinical roles also had reduced vaccine uptake. These findings should inform vaccination strategies across healthcare settings and target populations with reduced vaccine uptake directly, in particular young, female, and non-clinical healthcare workers, both for SARS-CoV-2 and other healthcare-associated vaccine-preventable infections.

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalization in ALS motor neurons and is restored by VCP ATPase inhibition.

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition.

Corrigendum: Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature.

[This corrects the article DOI: 10.3389/fimmu.2023.1043631.].

Integrated transcriptome landscape of ALS identifies genome instability linked to TDP-43 pathology.

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response.

Sotrovimab restores neutralization against current Omicron subvariants in patients with blood cancer.

Wu et al. report that patients with hematologic malignancies have reduced immunity against SARS-CoV-2 Omicron subvariants and Sotrovimab retains neutralizing capacity against all tested Omicron subvariants.

Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature.

Effective secondary response to antigen is a hallmark of immunological memory. However, the extent of memory CD8 T cell response to secondary boost varies at different times after a primary response. Considering the central role of memory CD8 T cells in long-lived protection against viral infections and tumors, a better understanding of the molecular mechanisms underlying the changing responsiveness of these cells to antigenic challenge would be beneficial. We examined here primed CD8 T cell response to boost in a BALB/c mouse model of intramuscular vaccination by priming with HIV-1 gag-encoding Chimpanzee adenovector, and boosting with HIV-1 gag-encoding Modified Vaccinia virus Ankara. We found that boost was more effective at day(d)100 than at d30 post-prime, as evaluated at d45 post-boost by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-expression (as a guide to memory status) and in vivo killing. RNA-sequencing of splenic gag-primed CD8 T cells at d100 revealed a quiescent, but highly responsive signature, that trended toward a central memory (CD62L+) phenotype. Interestingly, gag-specific CD8 T cell frequency selectively diminished in the blood at d100, relative to the spleen, lymph nodes and bone marrow. These results open the possibility to modify prime/boost intervals to achieve an improved memory CD8 T cell secondary response.

The dietary sweetener sucralose is a negative modulator of T cell-mediated responses.

Artificial sweeteners are used as calorie-free sugar substitutes in many food products and their consumption has increased substantially over the past years1. Although generally regarded as safe, some concerns have been raised about the long-term safety of the consumption of certain sweeteners2-5. In this study, we show that the intake of high doses of sucralose in mice results in immunomodulatory effects by limiting T cell proliferation and T cell differentiation. Mechanistically, sucralose affects the membrane order of T cells, accompanied by a reduced efficiency of T cell receptor signalling and intracellular calcium mobilization. Mice given sucralose show decreased CD8+ T cell antigen-specific responses in subcutaneous cancer models and bacterial infection models, and reduced T cell function in models of T cell-mediated autoimmunity. Overall, these findings suggest that a high intake of sucralose can dampen T cell-mediated responses, an effect that could be used in therapy to mitigate T cell-dependent autoimmune disorders.

CD1d-dependent rewiring of lipid metabolism in macrophages regulates innate immune responses.

Alterations in cellular metabolism underpin macrophage activation, yet little is known regarding how key immunological molecules regulate metabolic programs in macrophages. Here we uncover a function for the antigen presenting molecule CD1d in the control of lipid metabolism. We show that CD1d-deficient macrophages exhibit a metabolic reprogramming, with a downregulation of lipid metabolic pathways and an increase in exogenous lipid import. This metabolic rewiring primes macrophages for enhanced responses to innate signals, as CD1d-KO cells show higher signalling and cytokine secretion upon Toll-like receptor stimulation. Mechanistically, CD1d modulates lipid import by controlling the internalization of the lipid transporter CD36, while blocking lipid uptake through CD36 restores metabolic and immune responses in macrophages. Thus, our data reveal CD1d as a key regulator of an inflammatory-metabolic circuit in macrophages, independent of its function in the control of T cell responses.

Published Support for Wellness, Diversity, Equity, and Inclusion Among Internal Medicine Residency Program Websites.

INTRODUCTION: The objective of this study was to review internal medicine residency program websites in the United States based on their published support for wellness, diversity, equity, and inclusion concepts. Inclusion of wellness, diversity, equity, and inclusion on program websites can serve as critical student benchmarks, and it may be paramount to optimize residency program websites accordingly. METHODS: This is a cross-sectional study of the websites of 597 internal medicine residency programs accredited by the Accreditation Council for Graduate Medical Education between March 25 and April 25, 2022. The websites were assessed based on 22 characteristics consisting of wellness verbiage, gender and underrepresented in medicine evaluation of faculty and residents, and diversity, equity, and inclusion-related semantics. Website photos were used to assess ethnic/sex representation. These attributes were devised by two sequentially set up focus groups consisting of 49 racially, ethnically, and gender-diverse medical students. RESULTS: A total of 579 internal medicine programs were reviewed. Only 239 (41%) had a dedicated page for resident wellness activities and efforts, while 134 (19%) had no mention of the concept throughout their web pages. Similarly, only 136 (23%) had a dedicated wellness officer, whether faculty or resident, who was focused on departmental interests. Gender diversity could be determined in 445 (77%) and 459 (79%) websites for faculty and residents, respectively. Underrepresented in medicine faculty and residents was noted in 293 (51%) and 393 (68%) of websites, respectively. A diversity, equity, and inclusion section was present in 172 (30%) of programs, with 93 (16%) having an assigned faculty or resident. Chairpersons or program directors stressed diversity, equity, and inclusion in up to 456 (79%) of the websites, with 181 (31%) having program mission statements or goals that include diversity, equity, and inclusion verbiage. CONCLUSION: A deficit of various essential wellness, diversity, equity, and inclusion attributes persists across internal medicine residency websites. Residency programs would benefit from optimizing their websites to attract more diverse applicants.

Functional immune responses against SARS-CoV-2 variants of concern after fourth COVID-19 vaccine dose or infection in patients with blood cancer.

Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.

RAS oncogenic activity predicts response to chemotherapy and outcome in lung adenocarcinoma.

Activating mutations in KRAS occur in 32% of lung adenocarcinomas (LUAD). Despite leading to aggressive disease and resistance to therapy in preclinical studies, the KRAS mutation does not predict patient outcome or response to treatment, presumably due to additional events modulating RAS pathways. To obtain a broader measure of RAS pathway activation, we developed RAS84, a transcriptional signature optimised to capture RAS oncogenic activity in LUAD. We report evidence of RAS pathway oncogenic activation in 84% of LUAD, including 65% KRAS wild-type tumours, falling into four groups characterised by coincident alteration of STK11/LKB1, TP53 or CDKN2A, suggesting that the classifications developed when considering only KRAS mutant tumours have significance in a broader cohort of patients. Critically, high RAS activity patient groups show adverse clinical outcome and reduced response to chemotherapy. Patient stratification using oncogenic RAS transcriptional activity instead of genetic alterations could ultimately assist in clinical decision-making.