The Pediatric Cell Atlas: defining the growth phase of human development at single-cell resolution

Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan

Humanized mouse models of genetic immune disorders and hematological malignancies

The immune system is quite remarkable having both the ability to tolerate innocuous and self-antigens while possessing a robust capacity to recognize and eradicate infectious pathogens and foreign entities. The genetics that encode this delicate balancing act include multiple genes and specialized cell types. Over the past several years, whole exome and whole genome sequencing has uncovered the genetics driving many human immune-mediated diseases including monogenic disorders and hematological malignancies. With the advent of genome editing technologies, the ability to correct genetic immune defects in autologous cells holds great promise for a number of conditions. Since assessment of novel therapeutic strategies have been difficult in mice, in recent years, immunodeficient mice capable of engrafting human cells and tissue have been developed and utilized for a variety of research applications. In this review, we discuss immune-humanized mice as a research tool to study human immunobiology and genetic immune disorders in vivo and the promise of future applications.Biological, physical and clinical aspects of cancer treatment with ionising radiatio

Citrobacter rodentium Requires Intestinal Neural Wiskott-Aldrich Syndrome Protein (N-WASp) for Actin Pedestal Formation, Colonization and Epithelial Barrier Disruption In Vivo

Background: Citrobacter rodentium is a natural mouse pathogen that is genetically closelyrelated to the human enteric pathogens enteropathogenic and enterohemorrhagic E. coli.Among the repertoire of conserved virulence factors that these pathogens deliver via typeIII secretion, Tir and EspF are responsible for the formation of characteristic actin-richpedestals and disruption of tight junction integrity, respectively. There is evidence In Vitrothese effectors accomplish this, at least in part, by subverting the normal host cellularfunctions of N-WASP, a critical regulator of branched chain actin assembly. Although NWASPhas been shown to be involved in pedestal formation In Vitro, the requirements ofN-WASP-mediated actin pedestals for intestinal colonization by attaching/effacing (A/E)pathogens In Vivo is not known. Furthermore, it is not known whether N-WASP is requiredfor EspF-mediated tight junction disruption. Methods: To investigate the role of N-WASPin the gut epithelium, we generated mice with intestine-specific deletion of N-WASP(iNWKO), by mating mice homozygous for a floxed N-WASP allele (N-WASPL2L/L2L) tomice expressing Cre recombinase under the villin promoter. Separately housed groups ofWT and iNWKO mice were inoculated with 5x108 GFP-expressing C. rodentium by intragastriclavage. Stool was collected 2, 4, 7, and 12 days after infection, and recoverablecolony forming units (CFUs) of C. rodentium were quantified by plating serial dilutions ofhomogenized stool on MacConkey's agar. GFP+ colonies were counted after 24 hoursincubation at 37°C. The presence of actin pedestals was investigated by electron microscopy(EM), and tight junction morphology was assessed by immunofluorescence staining ofoccludin, ZO-1 and claudin-2. Results: C. rodentium infection did not result in mortalityin WT or iNWKO mice. Compared to controls, iNWKO mice exhibited higher levels ofbacterial shedding during the first 4 days of infection (day 4 average: WT 5.2x104 CFU/gvs. iNWKO 4.7x105 CFU/g, p=0.08), followed by a more rapid clearance of C. rodentium, (day7-12 average: WT 2x106 CFU/g vs. iNWKO 2.7x105, p=0.01). EM and immunofluorescencerevealed the complete lack of actin pedestals in iNWKO mice and no mucosa-associatedGFP+ C. rodentium by day 7. WT controls exhibited tight junction disruption, reflected byaltered distribution of ZO-1, whereas iNWKO mice had no change in the pattern of ZO-1.Conclusion: Intestinal N-WASP is required for actin pedestal formation by C. rodentium InVivo, and ablation of N-WASP is associated with more rapid bacterial clearance and decreasedability of C. rodentium to disrupt intercellular junctions

Spontaneous food allergy in Was(-/-) mice occurs independent of Fc epsilon RI-mediated mast cell activation

Developmen

Human MD2 deficiency-an inborn error of immunity with pleiotropic features.

BACKGROUND: Toll-like receptors (TLRs) are important pattern recognition receptors that sense microbes and control host defense. Myeloid differentiation protein 2 (MD2) is the indispensable coreceptor for TLR4, facilitating the binding to the gram-negative bacterial cell wall component LPS and activation of downstream signaling. OBJECTIVE: We sought to provide phenotypic and mechanistic insights into human MD2 deficiency. METHODS: To elucidate the genetic cause in a patient with very early onset inflammatory bowel disease, we performed whole-exome sequencing and studied the functional consequences of the identified mutation in LY96 (encoding for MD2) in genetically engineered induced pluripotent stem cell-derived macrophages with knockout of MD2 or knockin of the patient-specific mutation, including TLR4-mediated signaling, cytokine production, and bacterial handling. RESULTS: Whole-exome sequencing identified a homozygous in-frame deletion in the LY96 gene (c.347_349delCAA; p.Thr116del) in a patient with very early onset inflammatory bowel disease and a sibling presenting with pneumonia and otitis media. Induced pluripotent stem cell-derived macrophages with knockout of MD2 or expression of the Thr116del mutation showed impaired activation of nuclear factor kappa B and mitogen-activated protein kinase signaling as well as TLR4 endocytosis on challenge with LPS or bacteria. In addition, MD2-deficient macrophages showed decreased cytokine expression (eg, IL-6, TNF, and IL-10) in response to LPS or gram-negative but not gram-positive bacteria. CONCLUSIONS: Human MD2 deficiency causes defective TLR4 signaling in response to LPS or gram-negative bacteria. The clinical manifestations and expressivity might be variable due to unknown secondary risk factors. Because TLR4 represents a therapeutic target for multiple inflammatory conditions, our study may provide insights into potential side effects of pharmacological TLR4 targeting

Pathogenic interleukin-10 receptor alpha variants in humans - balancing natural selection and clinical implications.

Balancing natural selection is a process by which genetic variants arise in populations that are beneficial to heterozygous carriers, but pathogenic when homozygous. We systematically investigated the prevalence, structural, and functional consequences of pathogenic IL10RA variants that are associated with monogenic inflammatory bowel disease. We identify 36 non-synonymous and non-sense variants in the IL10RA gene. Since the majority of these IL10RA variants have not been functionally characterized, we performed a systematic screening of their impact on STAT3 phosphorylation upon IL-10 stimulation. Based on the geographic accumulation of confirmed pathogenic IL10RA variants in East Asia and in Northeast China, the distribution of infectious disorders worldwide, and the functional evidence of IL-10 signaling in the pathogenesis, we identify Schistosoma japonicum infection as plausible selection pressure driving variation in IL10RA. Consistent with this is a partially augmented IL-10 response in peripheral blood mononuclear cells from heterozygous variant carriers. A parasite-driven heterozygote advantage through reduced IL-10 signaling has implications for health care utilization in regions with high allele frequencies and potentially indicates pathogen eradication strategies that target IL-10 signaling