Villification in the mouse: Bmp signals control intestinal villus patterning.

In the intestine, finger-like villi provide abundant surface area for nutrient absorption. During murine villus development, epithelial Hedgehog (Hh) signals promote aggregation of subepithelial mesenchymal clusters that drive villus emergence. Clusters arise first dorsally and proximally and spread over the entire intestine within 24 h, but the mechanism driving this pattern in the murine intestine is unknown. In chick, the driver of cluster pattern is tensile force from developing smooth muscle, which generates deep longitudinal epithelial folds that locally concentrate the Hh signal, promoting localized expression of cluster genes. By contrast, we show that in mouse, muscle-induced epithelial folding does not occur and artificial deformation of the epithelium does not determine the pattern of clusters or villi. In intestinal explants, modulation of Bmp signaling alters the spatial distribution of clusters and changes the pattern of emerging villi. Increasing Bmp signaling abolishes cluster formation, whereas inhibiting Bmp signaling leads to merged clusters. These dynamic changes in cluster pattern are faithfully simulated by a mathematical model of a Turing field in which an inhibitor of Bmp signaling acts as the Turing activator. In vivo, genetic interruption of Bmp signal reception in either epithelium or mesenchyme reveals that Bmp signaling in Hh-responsive mesenchymal cells controls cluster pattern. Thus, unlike in chick, the murine villus patterning system is independent of muscle-induced epithelial deformation. Rather, a complex cocktail of Bmps and Bmp signal modulators secreted from mesenchymal clusters determines the pattern of villi in a manner that mimics the spread of a self-organizing Turing field.

Estimation of the lag time in a subsequent monomer addition model for fibril elongation.

Fibrillogenesis, the production or development of protein fibers, has been linked to protein folding diseases. The progress curve of fibrils or aggregates typically takes on a sigmoidal shape with a lag phase, a rapid growth phase, and a final plateau regime. The study of the lag phase and the estimation of its critical timescale provide insight into the factors regulating the fibrillation process. However, methods to estimate a quantitative expression for the lag time rely on empirical expressions, which cannot connect the lag time to kinetic parameters associated with the reaction mechanisms of protein fibrillation. Here we introduce an approach for the estimation of the lag time using the governing rate equations of the elementary reactions of a subsequent monomer addition model for protein fibrillation as a case study. We show that the lag time is given by the sum of the critical timescales for each fibril intermediate in the subsequent monomer addition mechanism and therefore reveals causal connectivity between intermediate species. Furthermore, we find that single-molecule assays of protein fibrillation can exhibit a lag phase without a nucleation process, while dyes and extrinsic fluorescent probe bulk assays of protein fibrillation do not exhibit an observable lag phase during template-dependent elongation. Our approach could be valuable for investigating the effects of intrinsic and extrinsic factors to the protein fibrillation reaction mechanism and provides physicochemical insights into parameters regulating the lag phase.

Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity.

Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleural fluid) from hospitalized COVID-19 patients and non-COVID-19 controls. Cellular, humoral and cytokine responses were analysed and correlated with clinical data. SARS-CoV-2-specific IgM, IgG and IgA antibodies were detected using ELISA and multiplex assay in both the respiratory tract and blood of COVID-19 patients, although a higher receptor binding domain (RBD)-specific IgM and IgG seroconversion level was found in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples was detected only when high levels of RBD-specific antibodies were present. Strikingly, cytokine/chemokine levels and profiles greatly differed between respiratory samples and plasma, indicating that inflammation needs to be assessed in respiratory specimens for the accurate assessment of SARS-CoV-2 immunopathology. Diverse immune cell subsets were detected in respiratory samples, albeit dominated by neutrophils. Importantly, we also showed that dexamethasone and/or remdesivir treatment did not affect humoral responses in blood of COVID-19 patients. Overall, our study unveils stark differences in innate and adaptive immune responses between respiratory samples and blood and provides important insights into effect of drug therapy on immune responses in COVID-19 patients.

Systems serology detects functionally distinct coronavirus antibody features in children and elderly.

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.

Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19.

Compared to adults, children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly mild or asymptomatic infections, but the underlying immunological differences remain unclear. Here, we describe clinical features, virology, longitudinal cellular, and cytokine immune profile, SARS-CoV-2-specific serology and salivary antibody responses in a family of two parents with PCR-confirmed symptomatic SARS-CoV-2 infection and their three children, who tested repeatedly SARS-CoV-2 PCR negative. Cellular immune profiles and cytokine responses of all children are similar to their parents at all timepoints. All family members have salivary anti-SARS-CoV-2 antibodies detected, predominantly IgA, that coincide with symptom resolution in 3 of 4 symptomatic members. Plasma from both parents and one child have IgG antibody against the S1 protein and virus-neutralizing activity detected. Using a systems serology approach, we demonstrate higher levels of SARS-CoV-2-specific antibody features of these family members compared to healthy controls. These data indicate that children can mount an immune response to SARS-CoV-2 without virological confirmation of infection, raising the possibility that immunity in children can prevent the establishment of SARS-CoV-2 infection. Relying on routine virological and serological testing may not identify exposed children, with implications for epidemiological and clinical studies across the life-span.

Coordination of signaling and tissue mechanics during morphogenesis of murine intestinal villi: a role for mitotic cell rounding.

Efficient digestion and absorption of nutrients by the intestine requires a very large apical surface area, a feature that is enhanced by the presence of villi, fingerlike epithelial projections that extend into the lumen. Prior to villus formation, the epithelium is a thick pseudostratified layer. In mice, villus formation begins at embryonic day (E)14.5, when clusters of mesenchymal cells form just beneath the thick epithelium. At this time, analysis of the flat lumenal surface reveals a regular pattern of short apical membrane invaginations that form in regions of the epithelium that lie in between the mesenchymal clusters. Apical invaginations begin in the proximal intestine and spread distally, deepening with time. Interestingly, mitotically rounded cells are frequently associated with these invaginations. These mitotic cells are located at the tips of the invaginating membrane (internalized within the epithelium), rather than adjacent to the apical surface. Further investigation of epithelial changes during membrane invagination reveals that epithelial cells located between mesenchymal clusters experience a circumferential compression, as epithelial cells above each cluster shorten and widen. Using a computational model, we examined whether such forces are sufficient to cause apical invaginations. Simulations and in vivo data reveal that proper apical membrane invagination involves intraepithelial compressive forces, mitotic cell rounding in the compressed regions and apico-basal contraction of the dividing cell. Together, these data establish a new model that explains how signaling events intersect with tissue forces to pattern apical membrane invaginations that define the villus boundaries.