Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19.

Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy.

Modelling Toxoplasma gondii infection in a 3D cell culture system In Vitro: Comparison with infection in 2D cell monolayers.

Three-dimensional (3D) cell culture models bridge the gap between two-dimensional (2D) monolayer cultures and animal models. Physiologically relevant, 3D culture models have significantly advanced basic cell science and provide unique insights into host-pathogen interactions intrinsically linked to cell morphology. Toxoplasma gondii is an obligate intravacuolar parasite that chronically infects a large portion of the global human population. Our current understanding of Toxoplasma infection is largely based on 2D cell cultures, in which mammalian cells are grown on flat surfaces. However, 2D cell cultures may not recapitulate key conditions of in vivo infections as they introduce artificial pressures and tensions, which may subsequently alter infectious processes that are dependent on spatiality, e.g., invasion, replication and egress. In this study, we adapted a collagen-based 3D cell culture system to reproduce the 3D environment of T. gondii natural infections for investigation of the replication and egress of the parasite from the parasitophorous vacuole. Suspended in the 3D matrix, Toxoplasma-infected VERO cells have round morphology, as opposed to infected VERO cells in 2D monolayers. The doubling time of Toxoplasma in VERO cells within the matrix is comparable to that of parasites cultivated in VERO cell monolayers. In the absence of the pressure of flattened host cells grown in 2D cultures, the parasitophorous vacuole of T. gondii has a globular shape, with intravacuolar parasites distributed radially, forming 3D spherical 'rosette' structures. Parasites egress radially away from the ruptured host cell in 3D matrices, in contrast to Toxoplasma cultivated in 2D monolayer cultures, where the parasites escape perpendicularly from the flat surface below the host cells. These observations demonstrate the utility of collagen matrices for studying parasite modes of infection as these 3D assays more closely mimic in vivo conditions.

Host Mitochondrial Association Evolved in the Human Parasite Toxoplasma gondii via Neofunctionalization of a Gene Duplicate.

In Toxoplasma gondii, an intracellular parasite of humans and other animals, host mitochondrial association (HMA) is driven by a gene family that encodes multiple mitochondrial association factor 1 (MAF1) proteins. However, the importance of MAF1 gene duplication in the evolution of HMA is not understood, nor is the impact of HMA on parasite biology. Here we used within- and between-species comparative analysis to determine that the MAF1 locus is duplicated in T. gondii and its nearest extant relative Hammondia hammondi, but not another close relative, Neospora caninum Using cross-species complementation, we determined that the MAF1 locus harbors multiple distinct paralogs that differ in their ability to mediate HMA, and that only T. gondii and H. hammondi harbor HMA(+) paralogs. Additionally, we found that exogenous expression of an HMA(+) paralog in T. gondii strains that do not normally exhibit HMA provides a competitive advantage over their wild-type counterparts during a mouse infection. These data indicate that HMA likely evolved by neofunctionalization of a duplicate MAF1 copy in the common ancestor of T. gondii and H. hammondi, and that the neofunctionalized gene duplicate is selectively advantageous.

Circannual rhythm in body temperature, torpor, and sensitivity to A1 adenosine receptor agonist in arctic ground squirrels.

A1 adenosine receptor (A1AR) activation within the central nervous system induces torpor, but in obligate hibernators such as the arctic ground squirrel (AGS; Urocitellus parryii), A1AR stimulation induces torpor only during the hibernation season, suggesting a seasonal increase in sensitivity to A1AR signaling. The purpose of this research was to investigate the relationship between body temperature (Tb) and sensitivity to an adenosine A1 receptor agonist in AGS. We tested the hypothesis that increased sensitivity in A1AR signaling would lead to lower Tb in euthermic animals during the hibernation season when compared with the summer season. We further predicted that if a decrease in euthermic Tb reflects increased sensitivity to A1AR activation, then it should likewise predict spontaneous torpor. We used subcutaneous IPTT-300 transponders to monitor Tb in AGS housed under constant ambient conditions (12:12 L:D, 18 °C) for up to 16 months. These animals displayed an obvious rhythm in euthermic Tb that cycled with a period of approximately 8 months. Synchrony in the Tb rhythm within the group was lost after several months of constant L:D conditions; however, individual rhythms in Tb continued to show clear sine wave-like waxing and waning. AGS displayed spontaneous torpor only during troughs in euthermic Tb. To assess sensitivity to A1AR activation, AGS were administered the A1AR agonist N(6)-cyclohexyladenosine (CHA, 0.1 mg/kg, ip), and subcutaneous Tb was monitored. AGS administered CHA during a seasonal minimum in euthermic Tb showed a greater drug-induced decrease in Tb (1.6 ± 0.3 °C) than did AGS administered CHA during a peak in euthermic Tb (0.4 ± 0.3 °C). These results provide evidence for a circannual rhythm in Tb that is associated with increased sensitivity to A1AR signaling and correlates with the onset of torpor.