Complete analysis of the B-cell response to a protein antigen, from in vivo germinal centre formation to 3-D modelling of affinity maturation

Somatic hypermutation of immunoglobulin variable region genes occurs within germinal centres (GCs) and is the process responsible for affinity maturation of antibodies during an immune response. Previous studies have focused almost exclusively on the immune response to haptens, which may be unrepresentative of epitopes on protein antigens. In this study, we have exploited a model system that uses transgenic B and CD4<sup>+</sup> T cells specific for hen egg lysozyme (HEL) and a chicken ovalbumin peptide, respectively, to investigate a tightly synchronized immune response to protein antigens of widely differing affinities, thus allowing us to track many facets of the development of an antibody response at the antigen-specific B cell level in an integrated system <i>in</i> <i>vivo</i>. Somatic hypermutation of immunoglobulin variable genes was analysed in clones of transgenic B cells proliferating in individual GCs in response to HEL or the cross-reactive low-affinity antigen, duck egg lysozyme (DEL). Molecular modelling of the antibody–antigen interface demonstrates that recurring mutations in the antigen-binding site, selected in GCs, enhance interactions of the antibody with DEL. The effects of these mutations on affinity maturation are demonstrated by a shift of transgenic serum antibodies towards higher affinity for DEL in DEL-cOVA immunized mice. The results show that B cells with high affinity antigen receptors can revise their specificity by somatic hypermutation and antigen selection in response to a low-affinity, cross-reactive antigen. These observations shed further light on the nature of the immune response to pathogens and autoimmunity and demonstrate the utility of this novel model for studies of the mechanisms of somatic hypermutation

Zinc oxide enriched peat influence Escherichia coli infection related diarrhea, growth rates, serum and tissue zinc levels in Norwegian piglets around weaning: five case herd trials

Abstract Background Zinc oxide (ZnO), commonly used to control post-weaning diarrhea in piglets, has been highlighted as of potential concern from an environmental perspective. The aim of this field trial was to examine effects of different sources and levels of ZnO added to peat on average daily weight gain (ADG), fecal score in pens and serum and tissue zinc (Zn) levels around time of weaning in order to reduce the environmental impact without loss of the beneficial effect of ZnO on intestinal health and growth. Five case herds with enterotoxic colibacillosis challenges were included. The piglets entered the study aged three or five weeks. All piglets received a commercial diet containing <150 mg Zn/ per kg of complete feed. Four treatment groups received commercial peat added A: uncoated ZnO, B: lipid microencapsulated ZnO, C: solely commercial peat or D: no peat (Farms 2 and 3). Results At Farms 1, 2 and 3, a significant effect of treatment was identified for fecal score (P < 0.05). Treatment A led to lower fecal scores compared to treatments C (P < 0.05) and D (P < 0.01). At Farms 2 and 3, there was a significant difference in individual average daily weight gain (iADG) between treatment A and D (P < 0.05). The iADG of piglets receiving treatment B did not differ significantly from treatment A. Conclusions In 2016, The European Medicines Agency’s Committee on Veterinary Medicinal Products concluded that the benefits of ZnO for the prevention of diarrhea in pigs do not outweigh the risks to the environment. Effective alternative measures to reduce the accumulation of Zn in the environment have not been identified. Our results imply that peat added low concentration of both coated and uncoated ZnO influences the gut health of weaned piglets reflected by enhanced weight gain and reduced occurrence of diarrhea. This preventive approach certainly represents a favourable alternative in the “One Health” perspective. It will also contribute to reduced antibiotic use in pig farming while diminishing the environmental consequences caused by ZnO

A three-dimensional model of human lung development and disease from pluripotent stem cells

Author ManuscriptRecapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.NIH HL120046-01 (H.-W.S.), 1U01HL134760-01 (H.-W.S.) RO1 AI031971 (A.M.), and RO1 AI114736 (A.M.), as well as a sponsored research and agreement from Northern Biologics Inc. (H.-W.S.), and funding from the Thomas R Kully IPF Research Fund (H.-W.S.). RUES2-HPS1 cells were generated by the Columbia Stem Cell Core Facility. We thank NYULMC OCS Microscopy core C. Petzold and K. Dancel for their assistance with transmission electron microscopy. We thank M. Peeples (Ohio State University) for providing the original recombinant RSV. Flow cytometry was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD02005