An Organ Culture Technique for Maintaining the Pulp Tissue of Intact Human Teeth

The size of explants in organ culture is limited by their ability to feed, respire, and excrete by diffusion. The perfusion procedure described in this report is not limited to a particular explant size. The test tissue was the adult human tooth. In order to maintain the pulp tissue of the intact human tooth in organ culture, special precautions were taken to prevent internal necrosis and to allow diffusion of nutrients and waste products. Using aseptic technique, a standard tissue culture medium plus appropriate antibiotics were delivered to a glass manifold housed in an incubator with an atmosphere at 100% humidity containing about 5% CO2. Tubing from the manifold led to small needles which were threaded from the apex of each tooth to the pulp horns. The teeth covered with a layer of saturated gauze rested in modified petri dishes. Adenosine triphosphate concentrations in perfused, nonperfused and immediately extracted teeth were compared to determine the success of the procedure. The mean ATP level in 25 immediately extracted teeth was 0.15 micrograms ATP/mg wet weight. The mean ATP level in 24 teeth successfully perfused for up to 11 days was 0.14 micrograms ATP/mg wet weight, while the ATP level in all non-perfused teeth was 0. 01 micrograms ATP/mg. Eighty-six percent of all teeth were considered successfully perfused. A histologic study on perfused, non-perfused, and immediately fixed teeth revealed good cellular detail, collagen fiber integrity, and normal appearing odontoblasts in many areas of the pulp in teeth perfused for periods up to 14 days. The non-perfused counterparts demonstrated little cellular detail, no odontoblastic layer, and frank necrosis in many areas. In some areas of the pulp of perfused teeth numerous tissue histiocytes could be seen. Using standard tissue culture techniques, explants of pulp perfused for 72 hours showed extensive monolayer outgrowth of fibroblast-like cells. Non-perfused pulp tissue after 72 hours in saline showed no such outgrowth. It is concluded that the perfusion procedure maintained viability of the pulp tissue of intact teeth for extended periods of time

Time and Out-of-Pocket Costs Associated with Respiratory Syncytial Virus Hospitalization of Infants

AbstractObjectiveThe objective of this study was to quantify time spent plus out-of-pocket costs associated with confirmed respiratory syncytial virus (RSV) hospitalization of infants not prophylaxed against RSV.MethodsA prospective survey was carried out at multiple tertiary care hospitals in the United States.PatientsThe patients consisted of a consecutive sample of infants <12 months, born between 33 and 35 weeks of gestation. One site also enrolled full-term infants hospitalized with confirmed RSV. Daily patient census identified eligible patients. Consenting caregivers of eligible subjects (n=84, 1 refusal) were interviewed on discharge day and by telephone ∼30 days following discharge regarding time and out-of-pocket costs due to RSV.ResultsTotal average out of pocket expenses were $643.69 (range$21–$16,867; SD$2,403) for premature and $214.42 (range$6–$827; SD$218) (P=.0158) for full-term subjects. Total average economic burden per admission was $4517.07 for premature and$2135.30 for full-term infants, including the value of lost productivity but excluding inpatient hospital and physician bills and lost income. Premature infants (n=48) had longer hospital stays (mean 6.9 days; SD 7.5 vs. 3.4 days; SD 2.6 days) (P=.001) with an associated mean total time spent by up to 5 adults of 281.7 hours (range 25–2819.7 hours; SD 465.8 hours) versus a mean of 139.7 hours (range 31.8–561.3 hours; SD 118.1 hours) for term infants (P=.109). Time and out-of-pocket costs continued after discharge.ConclusionsRSV hospitalization of infants is associated with substantial, previously unmeasured time and monetary losses. These losses continued following discharge. The economic burden on families and society appears heavier for infants born at 33 to 35 weeks of gestation than for full-term infants

Elevated levels of type 2 respiratory innate lymphoid cells in human infants with severe respiratory syncytial virus bronchiolitis

© 2019 by the American Thoracic Society. Rationale: Studies of the immune responses at the site of respiratory syncytial virus (RSV) infection are sparse despite nearly five decades of research into understanding RSV disease. Objectives: To investigate the role of mucosal innate immune responses to RSV and respiratory viral load in infants hospitalized with the natural disease. Methods: Cytokines, viral load, and type 2 innate lymphoid cell (ILC2) levels in nasal aspirates, collected within 24 hours of enrollment, from infants hospitalized with RSV infection were quantified. Measurements and Main Results: RSV severity in infants was categorized based on admission to the general ward (moderate) or the pediatric ICU (severe). Evaluable subjects included 30 patients with severe and 63 patients with moderate disease (median age, 74 d; range, 9-297 d). ILC2s were found in the nasal aspirates of patients with severe disease (0.051% of total respiratory CD451 cells) to a significantly greater extent than in patients with moderate disease (0.018%, P = 0.004). Levels of IL-4, IL-13, IL-33, and IL-1b were significantly higher in nasal aspirates of patients with severe disease compared with those of patients with moderate disease. Factors associated with disease severity were gestational age (odds ratio, 0.49; 95% confidence interval, 0.29-0.82; P = 0.007) and IL-4 (odds ratio, 9.67; 95% confidence interval, 2.45-38.15; P = 0.001). Conclusions: This study shows, for the first time, that elevated levels of ILC2s is associated with infant RSV severity. The findings highlight the dominance of type-2 responses to RSV infection in infants and suggest an important role of ILC2 in shaping the immune response early during RSV infection

Altered gut microbiota in infants is associated with respiratory syncytial virus disease severity

© 2020 The Author(s). Background: Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infections in infants. There are still no vaccines or specific antiviral therapies against RSV, mainly due to the inadequate understanding of RSV pathogenesis. Recent data suggest a role for gut microbiota community structure in determining RSV disease severity. Our objective was to determine the gut microbial profile associated with severe RSV patients, which could be used to help identify at-risk patients and develop therapeutically protective microbial assemblages that may stimulate immuno-protection. Results: We enrolled 95 infants from Le Bonheur during the 2014 to 2016 RSV season. Of these, 37 were well-babies and 58 were hospitalized with RSV. Of the RSV infected babies, 53 remained in the pediatric ward (moderate) and 5 were moved to the pediatric intensive care unit at a later date (severe). Stool samples were collected within 72 h of admission; and the composition of gut microbiota was evaluated via 16S sequencing of fecal DNA. There was a significant enrichment in S24_7, Clostridiales, Odoribacteraceae, Lactobacillaceae, and Actinomyces in RSV (moderate and severe) vs. controls. Patients with severe RSV disease had slightly lower alpha diversity (richness and evenness of the bacterial community) of the gut microbiota compared to patients with moderate RSV and healthy controls. Beta diversity (overall microbial composition) was significantly different between all RSV patients (moderate and severe) compared to controls and had significant microbial composition separating all three groups (control, moderate RSV, and severe RSV). Conclusions: Collectively, these data demonstrate that a unique gut microbial profile is associated with RSV disease and with severe RSV disease with admission to the pediatric intensive care unit. More mechanistic experiments are needed to determine whether the differences observed in gut microbiota are the cause or consequences of severe RSV disease

Respiratory syncytial virus disease severity is associated with distinct CD8\u3csup\u3e+\u3c/sup\u3e T-cell profiles

Copyright © 2020 by the American Thoracic Society. Rationale: Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Although T-helper type 2 (Th2) cell pathology is implicated in severe disease, the mechanisms underlying the development of immunopathology are incompletely understood. Objectives: We aimed to identify local immune responses associated with severe RSV in infants. Our hypothesis was that disease severity would correlate with enhanced Th2 cellular responses. Methods: Nasal aspirates were collected from infants hospitalized with severe (admitted to the pediatric ICU) or moderate (maintained in the general ward) RSV disease at 5 to 9 days after enrollment. The immune response was investigated by evaluating T-lymphocyte cellularity, cytokine concentration, and viral load. Measurements and Main Results: Patients with severe disease had increased proportions of CD8 (cluster of differentiation 8)- positive T cells expressing IL-4 (Tc2) and reduced proportions of CD8+ T cells expressing IFNγ (Tc1). Nasal aspirates from patients with severe disease had reduced concentrations of IL-17. Patients with greater frequencies of Tc1, CD8+ T cells expressing IL-17 (Tc17), and CD4+ T cells expressing IL-17 (Th17) had shorter durations of hospitalization. Conclusions: Severe RSV disease was associated with distinct T-cell profiles. Tc1, Tc17, and Th17 were associated with shorter hospital stay and may play a protective role, whereas Tc2 cells may play a previously underappreciated role in pathology

Limited type I interferons and plasmacytoid dendritic cells during neonatal respiratory syncytial virus infection permit immunopathogenesis upon reinfection

Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN- α-treated neonatal mice, suggesting dual mechanisms of action. © 2014, American Society for Microbiology

Prolonged viral replication and longitudinal viral dynamic differences among respiratory syncytial virus infected infants

© 2017 2017 International Pediatric Research Foundation, Inc. BackgroundLongitudinal respiratory syncytial virus (RSV) dynamics have not been well studied despite the existence of factors favoring prolonged RSV replication including high mutation rates allowing rapid evolution and potential escape from immune control. We therefore measured viral load in previously RSV-naive infants over prolonged time spans.MethodsDuring 2014-2015, quantitative nasal aspirates were collected from 51 RSV-PCR+ infants. Multiple parallel assessments of viral loads were quantified at each collected time point using a well-validated real-time quantitative reverse transcriptase polymerase chain reaction assay. After observing viral load rebound phenomenon in some infants, the viral dynamics of 27 infants with sufficient longitudinal viral load data points were analyzed using the pre-defined criteria for viral rebound. Additional analyses were performed comparing age with viral rebound, viral clearance rates, and viral load area-under-the-curve (AUC VL).ResultsThe 51 infants (303 nasal aspirate samples; mean of 5.9 per patient) exhibited slower than expected viral clearance. Lower age trended toward slower viral clearance and greater AUC VL. Six infants had detectable viral loads ≥1 month after symptom onset. Ten of twenty-seven evaluable subjects exhibited viral rebound and this rebound was age-dependent (P=0.0259). All but one rebounder were rebound; likely representing viral mutational immune escape

RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease