Anna Elizabeth Stella Brooks

Rotaviruses (RVs) are unique among non-enveloped animal viruses encoding a structural and a nonstructural glycoprotein in their genome. The outer capsid of the virion is formed by 260 trimers of VP7, which is glycosylated at an asparagine residue(s) in the endoplasmic reticulum of infected cells, where it is retained until a late stage of virion morphogenesis. We investigated the fate of VP7 produced during synchronous infection of polarized Caco-2 cells and observed that a previously undetected form with a molecular weight greater than virion-associated VP7 was secreted into the medium. This secreted form of VP7 (sVP7) was not associated with virions and was insensitive to endoglycosidase H treatment consistent with the dominance of complex type N-glycans revealed in structural details using LC-MS/MS. We also detected core 1 and 2-type O-glycans on sVP7 contributing to the higher-than-predicted molecular weight. Purification of sVP7 from the medium of virus-infected cells revealed that it was trimeric and retained conformationally sensitive epitopes recognized by neutralizing monoclonal antibodies. Like the secreted nonstructural protein 4(NSP4), sVP7 exhibited agonism of TLR2 and TLR4 and caused activation of innate immune cells in human blood. Our results identify a novel form of the rotavirus capsid glycoprotein previously undetected and suggest that VP7 may possess extracellular functions beyond its role in the virion as a modulator of innate and adaptive antiviral immunity. IMPORTANCE: Rotavirus (RV) exhibits a mode of assembly unique among non-enveloped animal viruses whereby virion morphogenesis occurs within an endomembrane compartment. The outer shell of rotavirus is assembled within the lumen of the ER where a glycoprotein VP7 is transferred to immature particles that bud from cytoplasmic inclusions. Lytic release of virions from non-polarized cells has obscured the fate of rotavirus glycoproteins from cells with a differentiated phenotype. This study reveals that in polarized cells, a significant proportion of VP7 is secreted, independently of virions, and acquires novel glycan signatures through extensive modification of N- and O-linked glycosylation within the medial Golgi complex. Secreted VP7 exhibited agonism of Toll-like receptors and activated several distinct subsets of innate immune cells while retaining conformationally dependent neutralizing epitopes associated with a trimeric structure indicating a potential role for the secreted capsid glycoprotein in modulating both innate and adaptive immune responses to rotavirus infection.

Immune dysfunction is reported to play a significant role in the etiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). To gain an understanding of the underlying immune abnormalities associated with this complex condition, a comprehensive approach for characterizing immune cell subsets and their inferred functional states is essential. We developed a high-dimensional flow cytometry method that enables detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) from ME/CFS patients. By simultaneously measuring over 40 markers on individual cells within one sample, this approach provides a comprehensive assessment of immune cell subsets, incorporating effector or functional states, to enable assessment of their potential roles in disease pathogenesis.

Problem: Considerable evidence suggests that placental extracellular vesicles (EVs) interact with most types of leukocytes in vitro but in vivo biodistribution studies question whether these interactions are reflective of the situation in vivo.Method of Study: CellTracker Red CMTPX stained human placental micro‐EVs were isolated from first trimester placental explant cultures. Equivalent amounts of micro‐EVs were cultured with murine leukocytes in vitro or injected into pregnant or non‐pregnant mice. After intravenous injection, on day 12.5 of gestation, major organs and blood samples were harvested 30 min or 24 h post injection.Results: We screened cryosections of the organs and confirmed that human placental EVs were specifically localised to the spleen, liver and the lungs 30 min or 24 h after injection. Immunohistochemistry showed that most of the EVs interacted with macrophages in those three organs and some of them also associated with T and B lymphocytes in the spleen or endothelial cells in the lungs and liver. Flow cytometry demonstrated that there was very little interaction between circulating leukocytes and EVs in vivo. While minimal, significantly more EVs interacted with leukocytes in pregnant than nonpregnant mice.Conclusion: The major interaction between human placental micro‐EVs and maternal leukocytes appear to be with macrophages predominantly in the splenic marginal zone, liver and lungs with little interaction between EVs and circulating leukocytes. Since marginal zone macrophages induce tolerance after phagocytosing apoptotic bodies it is likely that phagocytosis of placental EVs by marginal zone macrophages may also contribute to maternal immune tolerance.

Skeletal stem and progenitor cells are critical for bone homeostasis and healing, but their identity and diversity in humans are not well understood. In this study, we compared stromal populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-level analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow stromal populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+ population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. In conclusion, our study indicates considerable diversity in the stromal cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal stem cells reside within the CD90+CD34+ population.

Single-cell technologies (RNA-sequencing, flow cytometry) are critical tools to reveal how cell heterogeneity impacts developmental pathways. The placenta is a fetal exchange organ, containing a heterogeneous mix of mesenchymal cells (fibroblasts, myofibroblasts, perivascular, and progenitor cells). Placental mesenchymal stromal cells (pMSC) are also routinely isolated, for therapeutic and research purposes. However, our understanding of the diverse phenotypes of placental mesenchymal lineages, and their relationships remain unclear. We designed a 23-colour flow cytometry panel to assess mesenchymal heterogeneity in first-trimester human placentae. Four distinct mesenchymal subsets were identified; CD73⁺CD90⁺ mesenchymal cells, CD146⁺CD271⁺ perivascular cells, podoplanin⁺CD36⁺ stromal cells, and CD26⁺CD90⁺ myofibroblasts. CD73⁺CD90⁺ and podoplanin + CD36+ cells expressed markers consistent with cultured pMSCs, and were explored further. Despite their distinct ex-vivo phenotype, in culture CD73⁺CD90⁺ cells and podoplanin⁺CD36⁺ cells underwent phenotypic convergence, losing CD271 or CD36 expression respectively, and homogenously exhibiting a basic MSC phenotype (CD73⁺CD90⁺CD31^-CD144^-CD45^-). However, some markers (CD26, CD146) were not impacted, or differentially impacted by culture in different populations. Comparisons of cultured phenotypes to pMSCs further suggested cultured pMSCs originate from podoplanin⁺CD36⁺ cells. This highlights the importance of detailed cell phenotyping to optimise therapeutic capacity, and ensure use of relevant cells in functional assays.

Background. The profile of cellular immunological responses of children across the spectrum of COVID-19, ranging from acute SARS-CoV-2 infection to full recovery or Long COVID, has not yet been fully investigated. Methods. We examined and compared cytokines in sera and cell subsets in peripheral blood mononuclear cells (B and regulatory T lymphocytes) collected from four distinct groups of children, distributed as follows: younger than 18 years of age with either acute SARS-CoV-2 infection (n = 49); fully recovered from COVID-19 (n = 32); with persistent symptoms (Long COVID, n = 51); and healthy controls (n = 9). Results. In the later stages after SARS-CoV-2 infection, the cohorts of children, both with recovered and persistent symptoms, showed skewed T and B subsets, with remarkable differences when compared with children at the onset of the infection and with controls. The frequencies of IgD⁺CD27⁻ naïve B cells, IgD⁺IgM⁺ and CD27⁻IgM⁺CD38^dim B cells were higher in children with recent infection than in those with an older history of disease (p < 0.0001 for all); similarly, the total and natural Tregs compartments were more represented in children at onset when compared with Long COVID (p < 0.0001 and p = 0.0005, respectively). Despite the heterogeneity, partially due to age, sex and infection incidence, the susceptibility of certain children to develop persistent symptoms after infection appeared to be associated with the imbalance of the adaptive immune response. Following up and comparing recovered versus Long COVID patients, we analyzed the role of circulating naïve and switched B and regulatory T lymphocytes in counteracting the evolution of the symptomatology emerged, finding an interesting correlation between the amount and ability to reconstitute the natural Tregs component with the persistence of symptoms (linear regression, p = 0.0026). Conclusions. In this study, we suggest that children affected by Long COVID may have a compromised ability to switch from the innate to the adaptive immune response, as supported by our data showing a contraction of naïve and switched B cell compartment and an unstable balance of regulatory T lymphocytes occurring in these children. However, further prospective immunological studies are needed to better clarify which factors (epigenetic, diet, environment, etc.) are involved in the impairment of the immunological mechanisms in the Long COVID patients.

B‐cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B‐cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13‐low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68⁺CD11c⁺PU.1⁺ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine‐1‐phosphate (S1P) gradient that mediates B‐cell egress from LNs, through their expression of the S1P‐degrading enzyme, S1P lyase. Based on our findings, we present a model of B‐cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Background: Trichomonas vaginalis, the causative agent of a prevalent urogenital infection in humans, is an evolutionarily divergent protozoan. Protein-coding genes in T. vaginalis are largely controlled by two core promoter elements, producing mRNAs with short 5′ UTRs. The specific mechanisms adopted by T. vaginalis to fine-tune the translation efficiency (TE) of mRNAs remain largely unknown. Results: Using both computational and experimental approaches, this study investigated two key factors influencing TE in T. vaginalis: codon usage and mRNA secondary structure. Statistical dependence between TE and codon adaptation index (CAI) highlighted the impact of codon usage on mRNA translation in T. vaginalis. A genome-wide interrogation revealed that low structural complexity at the 5′ end of mRNA followed closely by a highly structured downstream region correlates with TE variation in this organism. To validate these findings, a synthetic library of 15 synonymous iLOV genes was created, representing five mRNA folding profiles and three codon usage profiles. Fluorescence signals produced by the expression of these synonymous iLOV genes in T. vaginalis were consistent with and validated our in silico predictions. Conclusions: This study demonstrates the role of codon usage bias and mRNA secondary structure in TE of T. vaginalis mRNAs, contributing to a better understanding of the factors that influence, and possibly regulate, gene expression in this human pathogen.

The extensively branched vascular network within the placenta is vital for materno-fetal exchange, and inadequate development of this network is implicated in the pregnancy disorder fetal growth restriction (FGR), where babies are born pathologically small. Placental mesenchymal stem/stromal cells (pMSCs) and placental macrophages both reside in close proximity to blood vessels within the placenta, where they are thought to promote angiogenesis via paracrine mechanisms. However, the relationship between pMSCs, macrophages and placental vascular development has not yet been examined. We aimed to determine if inadequate paracrine stimulation of placental vascular development by pMSCs and macrophages during pregnancy may contribute to the inadequate vascularisation seen in FGR. Media conditioned by MSCs from FGR placentae significantly inhibited endothelial tube formation, compared to conditioned media derived from normal pMSCs. Similarly, macrophages exposed to media conditioned by FGR pMSCs were less able to stimulate endothelial tube formation in comparison to macrophages exposed to media conditioned by normal pMSCs. While MSCs from normal placentae produce a combination of angiogenic and anti-angiogenic cytokines, there were no significant differences in the secretion of the anti-angiogenic cytokines thrombospondin-1, insulin growth factor binding protein-4, or decorin between normal and FGR pMSCs that could explain how FGR pMSCs inhibited endothelial tube formation. Together, these data suggest a dysregulation in the secretion of paracrine factors by pMSCs in FGR placentae. These findings illustrate how cross talk between pro-angiogenic cell types in the placenta may be crucial for adequate angiogenesis.