The Spectrum of Postacute Sequelae of COVID-19 in Children: From MIS-C to Long COVID.

The effects of SARS-CoV-2 infection on children continue to evolve following the onset of the COVID-19 pandemic. Although life-threatening multisystem inflammatory syndrome in children (MIS-C) has become rare, long-standing symptoms stemming from persistent immune activation beyond the resolution of acute SARS-CoV-2 infection contribute to major health sequelae and continue to pose an economic burden. Shared pathophysiologic mechanisms place MIS-C and long COVID within a vast spectrum of postinfectious conditions characterized by intestinal dysbiosis, increased gut permeability, and varying degrees of immune dysregulation. Insights obtained from MIS-C will help shape our understanding of the more indolent and prevalent postacute sequelae of COVID and ultimately guide efforts to improve diagnosis and management of postinfectious complications of SARS-CoV-2 infection in children.

Early host immune responses in a human organoid-derived gallbladder monolayer to Salmonella Typhi strains from patients with acute and chronic infections: a comparative analysis.

Salmonella enterica serovar Typhi (S. Typhi), a human-restricted pathogen, invades the host through the gut to cause typhoid fever. Recent calculations of the typhoid fever burden estimated that more than 10 million new typhoid fever cases occur in low and middle-income countries, resulting in 65,400-187,700 deaths yearly. Interestingly, if not antibiotic-treated, upon the resolution of acute disease, 1%-5% of patients become asymptomatic chronic carriers. Chronically infected hosts are not only critical reservoirs of infection that transmit the disease to naive individuals but are also predisposed to developing gallbladder carcinoma. Nevertheless, the molecular mechanisms involved in the early interactions between gallbladder epithelial cells and S. Typhi remain largely unknown. Based on our previous studies showing that closely related S. Typhi strains elicit distinct innate immune responses, we hypothesized that host molecular pathways activated by S. Typhi strains derived from acutely and chronically infected patients would differ. To test this hypothesis, we used a novel human organoid-derived polarized gallbladder monolayer model, and S. Typhi strains derived from acutely and chronically infected patients. We found that S. Typhi strains derived from acutely and chronically infected patients differentially regulate host mitogen-activated protein kinase (MAPK) and S6 transcription factors. These variations might be attributed to differential cytokine signaling, predominantly via TNF-α and IL-6 production and appear to be influenced by the duration the isolate was subjected to selective pressures in the gallbladder. These findings represent a significant leap in understanding the complexities behind chronic S. Typhi infections in the gallbladder and may uncover potential intervention targets.

Implementation of a robot-mediated upper limb rehabilitation protocol for a customized treatment after stroke: A retrospective analysis.

BACKGROUND: Many authors have emphasized the need for individualized treatments in rehabilitation, but no tailored robotic rehabilitation protocol for stroke patients has been established yet. OBJECTIVE: To evaluate the effectiveness of a robot-mediated upper limb rehabilitation protocol based on clinical assessment for customized treatment of stroke patients. METHODS: Clinical data from 81 patients with subacute stroke, undergoing an upper limb robot-mediated rehabilitation, were analyzed retrospectively. 49 patients were treated using a customized robotic protocol (experimental group, EG) based on a clinically guided flowchart, while 32 were treated without it (control group, CG). Fugl-Meyer Assessment for Upper Extremity (FMA-UE), Motricity Index (MI), modified Barthel Index (mBI) and Numerical Rating Scale (NRS) measured before (T0) and after (T1) rehabilitation intervention were used as clinical outcomes. RESULTS: There was statistically significant improvement in both groups in terms of FMA-UE, MI, and mBI, while no change in NRS. Intergroup analysis showed significantly greater improvement of the FMA-UE (P = 0.002) and MI (P <  0.001) in the EG, compared with the CG. CONCLUSION: The implementation of our robotic protocol for customized treatment of stroke patients yielded greater recovery in upper limb motor function and strength over robotic treatment without a defined protocol.

Assessment of Postural Control in Children with Movement Disorders by Means of a New Technological Tool: A Pilot Study.

Considering the variability and heterogeneity of motor impairment in children with Movement Disorders (MDs), the assessment of postural control becomes essential. For its assessment, only a few tools objectively quantify and recognize the difference among children with MDs. In this study, we use the Virtual Reality Rehabilitation System (VRRS) for assessing the postural control in children with MD. Furthermore, 16 children (mean age 10.68 ± 3.62 years, range 4.29-18.22 years) were tested with VRRS by using a stabilometric balance platform. Postural parameters, related to the movements of the Centre of Pressure (COP), were collected and analyzed. Three different MD groups were identified according to the prevalent MD: dystonia, chorea and chorea-dystonia. Statistical analyses tested the differences among MD groups in the VRRS-derived COP variables. The mean distance, root mean square, excursion, velocity and frequency values of the dystonia group showed significant differences (p < 0.05) between the chorea group and the chorea-dystonia group. Technology provides quantitative data to support clinical assessment: in this case, the VRRS detected differences among the MD patterns, identifying specific group features. This tool could be useful also for monitoring the longitudinal trajectories and detecting post-treatment changes.

SARS-CoV-2 infection elucidates unique features of pregnancy-specific immunity.

Pregnancy is a risk factor for increased severity of SARS-CoV-2 and other respiratory infections. The mechanisms underlying this risk have not been well-established, partly due to a limited understanding of how pregnancy shapes immune responses. To gain insight into the role of pregnancy in modulating immune responses at steady state and upon perturbation, we collected peripheral blood mononuclear cells (PBMC), plasma, and stool from 226 women, including 152 pregnant individuals (n = 96 with SARS-CoV-2 infection and n = 56 healthy controls) and 74 non-pregnant women (n = 55 with SARS-CoV-2 and n = 19 healthy controls). We found that SARS-CoV-2 infection was associated with altered T cell responses in pregnant compared to non-pregnant women. Differences included a lower percentage of memory T cells, a distinct clonal expansion of CD4-expressing CD8 + T cells, and the enhanced expression of T cell exhaustion markers, such as programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain-3 (Tim-3), in pregnant women. We identified additional evidence of immune dysfunction in severely and critically ill pregnant women, including a lack of expected elevation in regulatory T cell (Treg) levels, diminished interferon responses, and profound suppression of monocyte function. Consistent with earlier data, we found maternal obesity was also associated with altered immune responses to SARS-CoV-2 infection, including enhanced production of inflammatory cytokines by T cells. Certain gut bacterial species were altered in pregnancy and upon SARS-CoV-2 infection in pregnant individuals compared to non-pregnant women. Shifts in cytokine and chemokine levels were also identified in the sera of pregnant individuals, most notably a robust increase of interleukin-27 (IL-27), a cytokine known to drive T cell exhaustion, in the pregnant uninfected control group compared to all non-pregnant groups. IL-27 levels were also significantly higher in uninfected pregnant controls compared to pregnant SARS-CoV-2-infected individuals. Using two different preclinical mouse models of inflammation-induced fetal demise and respiratory influenza viral infection, we found that enhanced IL-27 protects developing fetuses from maternal inflammation but renders adult female mice vulnerable to viral infection. These combined findings from human and murine studies reveal nuanced pregnancy-associated immune responses, suggesting mechanisms underlying the increased susceptibility of pregnant individuals to viral respiratory infections.

Humoral profiles of toddlers and young children following SARS-CoV-2 mRNA vaccination.

Although young children generally experience mild symptoms following infection with SARS-CoV-2, severe acute and long-term complications can occur. SARS-CoV-2 mRNA vaccines elicit robust immunoglobulin profiles in children ages 5 years and older, and in adults, corresponding with substantial protection against hospitalizations and severe disease. Whether similar immune responses and humoral protection can be observed in vaccinated infants and young children, who have a developing and vulnerable immune system, remains poorly understood. To study the impact of mRNA vaccination on the humoral immunity of infant, we use a system serology approach to comprehensively profile antibody responses in a cohort of children ages 6 months to 5 years who were vaccinated with the mRNA-1273 COVID-19 vaccine (25 µg). Responses are compared with vaccinated adults (100 µg), in addition to naturally infected toddlers and young children. Despite their lower vaccine dose, vaccinated toddlers elicit a functional antibody response as strong as adults, with higher antibody-dependent phagocytosis compared to adults, without report of side effects. Moreover, mRNA vaccination is associated with a higher IgG3-dependent humoral profile against SARS-CoV-2 compared to natural infection, supporting that mRNA vaccination is effective at eliciting a robust antibody response in toddlers and young children.

Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study.

BACKGROUND AND AIMS: We have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions. METHODS: We identified, isolated, cultivated, and sequenced a unique novel strain (20220303-A2) of B. vulgatus found only in control subjects. Using a human gut organoid system developed from pre-celiac patients, we monitored epithelial phenotype and innate immune cytokines at baseline, after exposure to gliadin, or gliadin plus B. vulgatus cell free supernatant (CFS). RESULTS: Following gliadin exposure, we observed increases in epithelial cell death, epithelial monolayer permeability, and secretion of pro-inflammatory cytokines. These effects were mitigated upon exposure to B. vulgatus 20220303-A2 CFS, which had matched phenotype gene product mutations. These protective effects were mediated by epigenetic reprogramming of the organoids treated with B. vulgatus CFS. CONCLUSIONS: We identified a unique strain of B. vulgatus that may exert a beneficial role by protecting CeD epithelium against a gluten-induced break of epithelial tolerance through miRNA reprogramming. IMPACT: Gut dysbiosis precedes the onset of celiac disease in genetically at-risk infants. This dysbiosis is characterized by the loss of protective bacterial strains in those children who will go on to develop celiac disease. The paper reports the mechanism by which one of these protective strains, B. vulgatus, ameliorates the gluten-induced break of gut epithelial homeostasis by epigenetically re-programming the target intestinal epithelium involving pathways controlling permeability, immune response, and cell turnover.

Zonulin as a Biomarker for the Development of Celiac Disease.

OBJECTIVES: Increased intestinal permeability seems to be a key factor in the pathogenesis of autoimmune diseases, including celiac disease (CeD). However, it is unknown whether increased permeability precedes CeD onset. This study's objective was to determine whether intestinal permeability is altered before celiac disease autoimmunity (CDA) in at-risk children. We also examined whether environmental factors impacted zonulin, a widely used marker of gut permeability. METHODS: We evaluated 102 children in the CDGEMM study from 2014-2022. We included 51 CDA cases and matched controls, who were enrolled for 12 months or more and consumed gluten. We measured serum zonulin from age 12 months to time of CDA onset, and the corresponding time point in controls, and examined clinical factors of interest. We ran a mixed-effects longitudinal model with dependent variable zonulin. RESULTS: Children who developed CDA had a significant increase in zonulin in the 18.3 months (range 6-78) preceding CDA compared to those without CDA (slope differential = ß = 0.1277, 95% CI: 0.001, 0.255). Among metadata considered, zonulin trajectory was only influenced by increasing number of antibiotic courses, which increased the slope of trajectory of zonulin over time in CDA subjects (P = .04). CONCLUSIONS: Zonulin levels significantly rise in the months that precede CDA diagnosis. Exposure to a greater number of antibiotic courses was associated with an increase in zonulin levels in CDA subjects. This suggests zonulin may be used as a biomarker for preclinical CeD screening in at-risk children, and multiple antibiotic courses may increase their risk of CDA by increasing zonulin levels.

Antigen-specific T cell responses in SARS-CoV-2 mRNA-vaccinated children.

SARS-CoV-2 mRNA vaccines elicit humoral responses in children that are comparable to those in adults. However, early-life T cell responses are distinct from adult ones, and questions remain about the nature and kinetics of mRNA vaccine-induced T cell responses in children. We report that Pfizer BNT162b2 mRNA vaccination elicits a significant antigen-specific CD4+ T cell response in the ≥12-year-old cohort. This response is weaker in magnitude in the 5- to 11-year-old cohort and is not improved by a higher vaccine dose (Moderna mRNA1273, 100 µg), suggesting distinct developmental programming that may underscore early-life T cell immunity. Increased effector phenotypes of antigen-specific T cells in younger children correspond with elevated anti-receptor binding domain antibody levels, albeit at the cost of memory generation. These studies highlight aspects of age-specific adaptive immune responses and the need for careful consideration of priming conditions including vaccine dose and adjuvant in the pediatric population.

Evaluating Prophylactic Effect of Bovine Colostrum on Intestinal Barrier Function in Zonulin Transgenic Mice: A Transcriptomic Study.

The intestinal barrier comprises a single layer of epithelial cells tightly joined to form a physical barrier. Disruption or compromise of the intestinal barrier can lead to the inadvertent activation of immune cells, potentially causing an increased risk of chronic inflammation in various tissues. Recent research has suggested that specific dietary components may influence the function of the intestinal barrier, potentially offering a means to prevent or mitigate inflammatory disorders. However, the precise mechanism underlying these effects remains unclear. Bovine colostrum (BC), the first milk from cows after calving, is a natural source of nutrients with immunomodulatory, anti-inflammatory, and gut-barrier fortifying properties. This novel study sought to investigate the transcriptome in BC-treated Zonulin transgenic mice (Ztm), characterized by dysbiotic microbiota, intestinal hyperpermeability, and mild hyperactivity, applying RNA sequencing. Seventy-five tissue samples from the duodenum, colon, and brain of Ztm and wild-type (WT) mice were dissected, processed, and RNA sequenced. The expression profiles were analyzed and integrated to identify differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). These were then further examined using bioinformatics tools. RNA-seq analysis identified 1298 DEGs and 20,952 DETs in the paired (Ztm treatment vs. Ztm control) and reference (WT controls) groups. Of these, 733 DEGs and 10,476 DETs were upregulated, while 565 DEGs and 6097 DETs were downregulated. BC-treated Ztm female mice showed significant upregulation of cingulin (Cgn) and claudin 12 (Cldn12) duodenum and protein interactions, as well as molecular pathways and interactions pertaining to tight junctions, while BC-treated Ztm males displayed an upregulation of transcripts like occludin (Ocln) and Rho/Rac guanine nucleotide exchange factor 2 (Arhgf2) and cellular structures and interfaces, protein-protein interactions, and organization and response mechanisms. This comprehensive analysis reveals the influence of BC treatment on tight junctions (TJs) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway gene expressions. The present study is the first to analyze intestinal and brain samples from BC-treated Ztm mice applying high-throughput RNA sequencing. This study revealed molecular interaction in intestinal barrier function and identified hub genes and their functional pathways and biological processes in response to BC treatment in Ztm mice. Further research is needed to validate these findings and explore their implications for dietary interventions aimed at improving intestinal barrier integrity and function. The MGH Institutional Animal Care and Use Committee authorized the animal study (2013N000013).

SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a 'reservoir'. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated.

In vitro differentiation of macrophages from peripheral blood cells of celiac patients.

Macrophages have both a protective and pathological role in many autoimmune and inflammatory diseases. Macrophages phenotype is regulated by the environment that affects their polarization toward a pro- or anti-inflammatory phenotype. We describe a protocol for in vitro differentiation of macrophages from blood peripheral monocytes, that may be adopted to study different pathologies. Here, we are interested to study the phenotype of macrophages differentiated from patients affected by acute celiac disease (CD) or subjects following a gluten free diet (GFD), after in vitro gliadin challenge. We assess the pro-inflammatory phenotype of these macrophages by cytokines quantization on the cell supernatant. Moreover, our proposed protocol allows the preparation of total RNA to analyze the expression profile of many genes.

Gastrointestinal distress as a potential mediator between stress and periodontal inflammation.

OBJECTIVES: Periodontal disease is multifactorial in its aetiology, which encompasses biopsychosocial contributors, including psychological stress. Gastrointestinal distress and dysbiosis have been associated with several chronic inflammatory diseases yet have rarely been investigated with respect to oral inflammation. Given the implications of gastrointestinal distress on extraintestinal inflammation, this study aimed to evaluate the potential role of such distress as a mediator between psychological stress and periodontal disease. METHODS: Utilizing a cross-sectional, nationwide sample of 828 adults in the USA generated via Amazon Mechanical Turk, we evaluated data collected from a series of validated self-report psychosocial questionnaires on stress, gut-specific anxiety around current gastrointestinal distress and periodontal disease, including periodontal disease subscales targeted at physiological and functional factors. Structural equation modelling was used to determine total, direct and indirect effects, while controlling for covariates. RESULTS: Psychological stress was associated with gastrointestinal distress (ß = .34) and self-reported periodontal disease (ß = .43). Gastrointestinal distress also was associated with self-reported periodontal disease (ß = .10). Gastrointestinal distress likewise mediated the relation between psychological stress and periodontal disease (ß = .03, p = .015). Given the multifactorial nature of periodontal disease(s), similar results were demonstrated using the subscales of the periodontal self-report measure. CONCLUSIONS: Associations exist between psychological stress and overall reports of periodontal disease as well as more specific physiological and functional components. Additionally, this study provided preliminary data supporting the potential mechanistic role that gastrointestinal distress plays in connecting the gut-brain and the gut-gum pathways.

Long-term humoral signatures following acute pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children.

BACKGROUND: Although most children experience mild symptoms during acute SARS-CoV-2 infection, some develop the severe post-COVID-19 complication, Multisystem Inflammatory Syndrome in Children (MIS-C). While acute presentations of COVID-19 and MIS-C have been well immunophenotyped, little is known about the lasting immune profile in children after acute illness. METHODS: Children 2 months-20 years of age presenting with either acute COVID-19 (n = 9) or MIS-C (n = 12) were enrolled in a Pediatric COVID-19 Biorepository at a single medical center. We deeply profiled humoral immune responses and circulating cytokines following pediatric COVID-19 and MIS-C. RESULTS: Twenty-one children and young adults provided blood samples at both acute presentation and 6-month follow-up (mean: 6.5 months; standard deviation: 1.77 months). Pro-inflammatory cytokine elevations resolved after both acute COVID-19 and MIS-C. Humoral profiles continue to mature after acute COVID-19, displaying decreasing IgM and increasing IgG over time, as well as stronger effector functions, including antibody-dependent monocyte activation. In contrast, MIS-C immune signatures, especially anti-Spike IgG1, diminished over time. CONCLUSIONS: Here, we show the mature immune signature after pediatric COVID-19 and MIS-C, displaying resolving inflammation with recalibration of the humoral responses. These humoral profiles highlight immune activation and vulnerabilities over time in these pediatric post-infectious cohorts. IMPACT: The pediatric immune profile matures after both COVID-19 and MIS-C, suggesting a diversified anti-SARS-CoV-2 antibody response after resolution of acute illness. While pro-inflammatory cytokine responses resolve in the months following acute infection in both conditions, antibody-activated responses remain relatively heightened in convalescent COVID-19. These data may inform long-term immunoprotection from reinfection in children with past SARS-CoV-2 infections or MIS-C.

Secretory-IgA binding to intestinal microbiota attenuates inflammatory reactions as the intestinal barrier of preterm infants matures.

Previous work has shown that Secretory-IgA (SIgA) binding to the intestinal microbiota is variable and may regulate host inflammatory bowel responses. Nevertheless, the impact of the SIgA functional binding to the microbiota remains largely unknown in preterm infants whose immature epithelial barriers make them particularly susceptible to inflammation. Here, we investigated SIgA binding to intestinal microbiota isolated from stools of preterm infants <33 weeks gestation with various levels of intestinal permeability. We found that SIgA binding to intestinal microbiota attenuates inflammatory reactions in preterm infants. We also observed a significant correlation between SIgA affinity to the microbiota and the infant's intestinal barrier maturation. Still, SIgA affinity was not associated with developing host defenses, such as the production of mucus and inflammatory calprotectin protein, but it depended on the microbiota shifts as the intestinal barrier matures. In conclusion, we reported an association between the SIgA functional binding to the microbiota and the maturity of the preterm infant's intestinal barrier, indicating that the pattern of SIgA coating is altered as the intestinal barrier matures.

Humoral profiles of toddlers and young children following SARS-CoV-2 mRNA vaccination.

Although young children generally experience mild symptoms following infection with SARS-CoV-2, severe acute and long-term complications can occur. SARS-CoV-2 mRNA vaccines elicit robust immunoglobulin profiles in children ages 5 years and older, and in adults, corresponding with substantial protection against hospitalizations and severe disease. Whether similar immune responses and humoral protection can be observed in vaccinated infants and young children, who have a developing and vulnerable immune system, remains poorly understood. To study the impact of mRNA vaccination on the humoral immunity of infant, we used a system serology approach to comprehensively profile antibody responses in a cohort of children ages 6 months to 5 years who were vaccinated with the mRNA-1273 COVID-19 vaccine (25 µg). Responses were compared with vaccinated adults (100 µg), in addition to naturally infected toddlers and young children. Despite their lower vaccine dose, vaccinated toddlers elicited a stronger functional antibody response than adults, including against variant of concerns (VOCs), without report of side effects. Moreover, mRNA vaccination was associated with a higher IgG3-dependent humoral profile against SARS-CoV-2 compared to natural infection, supporting that mRNA vaccination is effective at eliciting a robust antibody response in toddlers and young children.

Alterations of the intestinal mucus layer correlate with dysbiosis and immune dysregulation in human Type 1 Diabetes.

BACKGROUND: In preclinical models of Type 1 Diabetes (T1D) the integrity of the gut barrier (GB) is instrumental to avoid dysregulated crosstalk between the commensal microbiota and immune cells and to prevent autoimmunity. The GB is composed of the intestinal epithelial barrier (IEB) and of the mucus layer containing mucins and antimicrobial peptides (AMPs) that are crucial to maintain immune tolerance. In preclinical models of T1D the alterations of the GB primarily affect the mucus layer. In human T1D increased gut permeability and IEB damage have been demonstrated but the integrity of the mucus layer was never assessed. METHODS: We evaluated GB integrity by measuring serological markers of IEB damage (serological levels of zonulin) and bacterial translocation such as lipopolysaccharide binding protein (LBP) and myeloid differentiation protein 2 (MD2), and mRNA expression of tight junction proteins, mucins and AMPs in intestinal tissue of T1D patients and healthy controls (HC). Simultaneously, we performed immunological profiling on intestinal tissue and 16S rRNA analysis on the mucus-associated gut microbiota (MAGM). FINDINGS: Our data show a GB damage with mucus layer alterations and reduced mRNA expression of several mucins (MUC2, MUC12, MUC13, MUC15, MUC20, MUC21) and AMPs (HD4 and HD5) in T1D patients. Mucus layer alterations correlated with reduced relative abundance of short chain fatty acids (SCFA)-producing bacteria such as Bifidobacterium dentium, Clostridium butyricum and Roseburia intestinalis that regulate mucin expression and intestinal immune homeostasis. In T1D patients we also found intestinal immune dysregulation with higher percentages of effector T cells such as T helper (Th) 1, Th17 and TNF-α+ T cells. INTERPRETATION: Our data show that mucus layer alterations are present in T1D subjects and associated with dysbiosis and immune dysregulation. FUNDING: Research Grants from the Juvenile Diabetes Foundation (Grant 1-INO-2018-640-A-N to MF and 2-SRA-2019-680-S-B to JD) and from the Italian Ministry of Health (Grant RF19-12370721 to MF).