Developmental Programming Mediated by Complementary Roles of Imprinted Grb10 in Mother and Pup

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

T cell epitope peptide therapy for allergic diseases

Careful selection of dominant T cell epitope peptides of major allergens that display degeneracy for binding to a wide array of MHC class II molecules allows induction of clinical and immunological tolerance to allergen in a refined treatment strategy. From the original concept of peptide-induced T cell anergy arising from in vitro studies, proof-of-concept murine models and flourishing human trials followed. Current randomized, double-blind, placebo-controlled clinical trials of mixtures of T cell-reactive short allergen peptides or long contiguous overlapping peptides are encouraging with intradermal administration into non-inflamed skin a preferred delivery. Definitive immunological mechanisms are yet to be resolved but specific anergy, Th2 cell deletion, immune deviation, and Treg induction seem implicated. Significant efficacy, particularly with short treatment courses, in a range of aeroallergen therapies (cat, house dust mite, grass pollen) with inconsequential non-systemic adverse events likely heralds a new class of therapeutic for allergy, Synthetic Peptide Immuno-Regulatory Epitopes (SPIRE)

Costimulatory molecule expression on stimulated basophils.

<p>CD40 (A), CD80 (B), CD86 (C) and MHC Class II (D) expression on IL-3, IFN-γ and GM-CSF stimulated basophils of 2 atopic HDM-allergic (i,ii) and 1 non-atopic (iii) donor. CpG-stimulated B cells served as a positive control for costimulatory molecule detection (iv). Gates were determined using corresponding isotype controls.</p

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

T cell proliferation assay.

<p>(i) Percentage of MHC Class II positive basophils after 72 hours culture with IL-3, IFN-γ and GM-CSF prior to co-culture with T cells. HDM extract (10 µg/ml) was also added to the basophil stimulation culture for A. (ii) Percentage of CD25⁺ CFSE^low proliferating CD3⁺CD4⁺ T cells after culture of T cells alone or with autologous basophils or monocytes in the presence of 10 µg/ml HDM (A, non-atopic donor; similar results for 2 HDM-allergic donors) for 72 hours or 10 µg/ml peptide (B) for 120 hours. T cell: APC ratio indicated below. (C) Representative dot plot showing gating strategy for identification of proliferating CD3⁺CD4⁺ T cells.</p

Isolated basophil purity and viability.

<p>Representative dot plots showing proportion of basophils in PBMC and after isolation assessed by antibodies specific for (A) IgE and CD123, (B) FcεRI and CD203c, (C) FcεRI and Lineage-1. Example of basophil viability after culture for 72 hours with (D) IL-3 (10 ng/ml) or (E) medium alone. (F) Percentage of viable basophils (7AAD⁻, Annexin V⁻) after 72 hours of culture with a panel of cytokines and TLR ligands. Representative of 2 separate experiments.</p

Gene expression of MHC Class II components and costimulatory molecules.

<p>RT-PCR of RNA from freshly isolated basophils (1), MHC Class II negative (2) and MHC Class II positive basophils (3) after 72 hours of culture with IL-3, IFN-γ, GM-CSF and HDM extract, compared with CD123⁺ IgE^low pDC (4), CD19⁺ B cells from freshly isolated PBMC (5) and CD19⁺ B cells from CpG stimulated PBMC (6) as positive controls, and a RT negative control (7). Data for a non-atopic donor are shown, representative of 5 separate experiments.</p

MHC Class II expression on stimulated basophils.

<p>Representative dot plots showing MHC Class II expression on basophils freshly isolated (A), and after 72 hours culture with medium alone (B) or IL-3 (10 ng/ml), IFN-γ and GM-CSF (100 ng/ml) (C). Corresponding isotype control (D). Percentage of MHC Class II positive basophils for individual atopic HDM-allergic (open shapes) and non-atopic (closed shapes) donors after 72 hours culture with various stimuli (E). Differences between groups were calculated with One-way ANOVA and Dunett's Multiple comparison post-test with IL-3 as the control group for complete data sets of the 6 donors, * p&lt;0.05, ** p&lt;0.01.</p

Analysis of activation of MLN DC and lung Treg after intranasal OVA.

<p>(A) Experimental design for diet regimen and induction of respiratory tolerance via intranasal administration of saline or ovalbumin (OVA). (B) Gating strategy for migratory DC in MLN within live singlet cells. (C-F) Expression of CCR7, CD86, PDL2 and ICOSL on migratory DC in MLN measured as geometric mean fluorescence intensity. (G) Gating strategy for Treg within live singlet cells. (H-I) Percentage of Treg within live singlet lymphocytes in MLN and lung. (J-M) Expression of GITR, ICOS, IL-10 on lung Treg measured as geometric mean fluorescence intensity and % CTLA-4 ⁺ Treg. * P<0.05 and ** P<0.01 using Kruskal-Wallis with Dunn’s post-test. Each symbol represents one mouse and mean values are indicated. This pilot experiment was performed once. Insufficient cells were obtained from one mouse in the ND/OVA group to perform the staining for Fig 2C, E.</p