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The tonsils have been identified as a site of replication for Epstein-Barr virus, adenovirus, human papillomavirus, and other respiratory viruses. Human tonsil epithelial cells (HTECs) are a heterogeneous group of actively differentiating cells. Here, we investigated the cellular features and susceptibility of differentiated HTECs to specific influenza viruses, including expression of avian-type and mammalian-type sialic acid (SA) receptors, viral replication dynamics, and the associated cytokine secretion profiles. We found that differentiated HTECs possess more abundant α2,3-linked SA (preferentially bound by avian influenza viruses) than α2,6-linked SA (preferentially bound by mammalian strains). This dual receptor expression suggests a role in influenza virus adaptation and tropism within the tonsils by facilitating the binding and entry of multiple influenza virus strains. Our results indicated the susceptibility of differentiated HTECs to a wide range of influenza viruses from human, swine, and avian hosts. Virus production for most strains was detected as early as 1 day post-infection (dpi), and typically peaked by 3 dpi. However, pandemic H1N1 virus showed remarkably delayed replication kinetics that did not peak until at least 7 dpi. Notably, influenza virus infection impacted the expression of cytokeratins in HTEC cultures, which correlated with altered cytokine secretion patterns. These patterns varied within the strains but were most distinct in swine H3N2 infection. In conclusion, differentiated HTECs exhibited a strain-specific pattern of influenza virus replication and innate immune responses that included changes in cytokeratin and cytokine expression. These studies shed light on the complex interplay between influenza viruses and host cells in the tonsils., Importance: To develop effective interventions against influenza, it is important to identify host factors affecting pathogenesis and immune responses. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and the immune response. Influenza virus tropism in the human upper respiratory tract is a key determinant of host-range, pathogenesis, and transmission. Accordingly, experimental models using primary cells from the human respiratory tract are relevant for assessing virus tropism and replication competence. Our study addresses the dynamics of influenza virus replication in HTECs, including cellular tropism, infectivity, and cytokeratin and cytokine expression. The results of this study highlight the complex interplay between structural proteins and immune signaling pathways, all of which provide valuable insights into host-virus interactions., Competing Interests: The authors declare no conflict of interest.

Although variation in effect sizes and predicted values among studies of similar phenomena is inevitable, such variation far exceeds what might be produced by sampling error alone. One possible explanation for variation among results is differences among researchers in the decisions they make regarding statistical analyses. A growing array of studies has explored this analytical variability in different fields and has found substantial variability among results despite analysts having the same data and research question. Many of these studies have been in the social sciences, but one small "many analyst" study found similar variability in ecology. We expanded the scope of this prior work by implementing a large-scale empirical exploration of the variation in effect sizes and model predictions generated by the analytical decisions of different researchers in ecology and evolutionary biology. We used two unpublished datasets, one from evolutionary ecology (blue tit, Cyanistes caeruleus, to compare sibling number and nestling growth) and one from conservation ecology (Eucalyptus, to compare grass cover and tree seedling recruitment). The project leaders recruited 174 analyst teams, comprising 246 analysts, to investigate the answers to prespecified research questions. Analyses conducted by these teams yielded 141 usable effects (compatible with our meta-analyses and with all necessary information provided) for the blue tit dataset, and 85 usable effects for the Eucalyptus dataset. We found substantial heterogeneity among results for both datasets, although the patterns of variation differed between them. For the blue tit analyses, the average effect was convincingly negative, with less growth for nestlings living with more siblings, but there was near continuous variation in effect size from large negative effects to effects near zero, and even effects crossing the traditional threshold of statistical significance in the opposite direction. In contrast, the average relationship between grass cover and Eucalyptus seedling number was only slightly negative and not convincingly different from zero, and most effects ranged from weakly negative to weakly positive, with about a third of effects crossing the traditional threshold of significance in one direction or the other. However, there were also several striking outliers in the Eucalyptus dataset, with effects far from zero. For both datasets, we found substantial variation in the variable selection and random effects structures among analyses, as well as in the ratings of the analytical methods by peer reviewers, but we found no strong relationship between any of these and deviation from the meta-analytic mean. In other words, analyses with results that were far from the mean were no more or less likely to have dissimilar variable sets, use random effects in their models, or receive poor peer reviews than those analyses that found results that were close to the mean. The existence of substantial variability among analysis outcomes raises important questions about how ecologists and evolutionary biologists should interpret published results, and how they should conduct analyses in the future., Competing Interests: Declarations. Ethics approval and consent to particpate: We obtained permission to conduct this research from the Whitman College Institutional Review Board (IRB). As part of this permission, the IRB approved the consent form ( https://osf.io/xyp68/ ) that all participants completed prior to joining the study. The authors declare that they have no competing interests. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

Background: Clindamycin resistance among community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) complicates the management of a challenging infection. Little data exist to guide clinicians in the management of invasive clindamycin-resistant CA-MRSA infections in children and studies using oral regimens such as trimethoprim-sulfamethoxazole (TMP-SMX) and linezolid for treatment of these infections are limited. We sought to reevaluate antibiotic management among invasive CA-MRSA at a tertiary children's hospital., Methods: Cases of invasive clindamycin-resistant MRSA infections in children were identified through an ongoing S. aureus surveillance study. Eligible cases were those occurring in otherwise healthy children from 2011-2021. Medical records were reviewed., Results: Thirty-four subjects met inclusion criteria. The most common diagnoses were osteomyelitis (n = 17) and deep abscess (n = 7). The median duration of IV therapy was 11.5 days (IQR 6-42 days) and total therapy (IV + oral) was 32 days (IQR 23-42). Overall, 50% of patients were transitioned to oral therapy. Definitive antibiotics used for treatment included vancomycin (n = 15), TMP/SMX (n = 9), linezolid (n = 7), ceftaroline (n = 2), and doxycycline (n = 1). Cure rates were similar across definitive antibiotic therapies (vancomycin-73.3%; TMP/SMX-88.9%; ceftaroline 50%; linezolid and doxycycline-100%). Three subjects died of MRSA disease; two definitively treated with vancomycin and one with ceftaroline., Conclusions: Vancomycin is the most commonly used agent in the treatment of invasive clindamycin-resistant CA-MRSA in children at our center. However, TMP/SMX and linezolid can be considered as oral options when completing treatment in select cases. Further work is needed to evaluate the optimal management of these infections.

Monitoring for antibiotic-related lab abnormalities (ARLA), including hematologic, renal, and/or hepatic toxicity, in pediatric osteomyelitis is common. In 240 cases of osteomyelitis with outpatient laboratory monitoring, ARLA occurred in 13.3% with the most common finding being neutropenia. ARLA impacted antibiotic therapy in <1% of subjects, however, raising questions about the value of such monitoring being performed routinely., Competing Interests: J.C.M. receives support through AHRQ R01HS026896 and Merck for unrelated work. He is also a site investigator on a clinical trial sponsored by Nabriva also unrelated to the work under consideration. S.L.K. receives grant funding from Pfizer for unrelated research. All other authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The need for echocardiography in pediatric Staphylococcus aureus bacteremia (SAB) remains uncertain. We reviewed 331 pediatric SAB cases. Nine subjects, all with comorbidities, met the echocardiogram criteria for infective endocarditis (IE). IE was associated with congenital heart disease and prolonged bacteremia, suggesting that echocardiography is unnecessary in most children with SAB., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Shortly after its introduction into clinical practice, Staphylococcus aureus isolates gained resistance to penicillin via the acquisition of β-lactamases. A number of centers have recently described an increase in the proportion of invasive methicillin-susceptible S. aureus (MSSA), which are also susceptible to penicillin (PSSA). Little data are available regarding the prevalence or impact of PSSA in skin and soft tissue infections (SSTI). Community-acquired MSSA SSTI isolates were obtained through a surveillance study at Texas Children's Hospital from January 2017 to December 2021. A total of 200 random isolates underwent PCR for blaZ β-lactamase; blaZ -negative isolates then underwent penicillin susceptibility testing using macrobroth dilution. Isolates which were blaZ negative and had a penicillin MIC ≤0.125 µg/mL were regarded as PSSA with the remainder regarded as penicillin-resistant MSSA (PR-MSSA). All PSSA underwent multilocus sequence typing. Medical records were reviewed. The median age of subjects was 4.2 years (IQR: 1.6-10.5). PSSA accounted for 9% of isolates during the study period. PSSA and PR-MSSA cases were similar with respect to age, demographics, and rates of prior antibiotic exposure. PSSA isolates less often had vancomycin MIC ≥1.5 µg/mL. Furthermore, 39% of PSSA were variants of sequence type 1. In multivariable analyses, penicillin susceptibility was independently associated with both hospital admission and surgical intervention. PSSA account for a small but significant proportion of MSSA SSTI in children. Clinically distinguishing patients with PSSA and PR-MSSA SSTI is challenging. However, PSSA SSTI were independently associated with higher rates of hospital admission as well as the need for surgical intervention suggesting a significant clinical impact.IMPORTANCEThe vast majority of Staphylococcus aureus in the US are penicillin resistant with most clinical labs no longer reporting penicillin susceptibility for this organism. A number of centers, however, have reported increasing penicillin susceptibility among invasive S. aureus infections. Skin and soft tissue infections (SSTI) are far more common than invasive infections, yet the frequency and impact of penicillin-susceptible S. aureus (PSSA) in this population are uncertain. Through active surveillance at a children's hospital, we found that 9% of methicillin-susceptible S. aureus SSTI isolates were PSSA. PSSA were independently associated with hospital admission for the management of SSTI as well as the need for debridement in the operating room. Given that most SSTI are managed in the outpatient setting, these findings suggest a clinical impact of this phenotype and the need for a reassessment of the value in susceptibility testing and potentially even treatment with penicillin., Competing Interests: J.C.M. receives grant support through Merck for unrelated research and is a site investigator on a clinical trial sponsored by Nabriva (also unrelated to the work under consideration). S.L.K. and K.G.H. receive support through Pfizer for unrelated work.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce robust CD4 + T cell responses. Using single-cell transcriptomics, here, we evaluated CD4 + T cells specific for the SARS-CoV-2 spike protein in the blood and draining lymph nodes (dLNs) of individuals 3 months and 6 months after vaccination with the BNT162b2 mRNA vaccine. We analyzed 1,277 spike-specific CD4 + T cells, including 238 defined using Trex, a deep learning-based reverse epitope mapping method to predict antigen specificity. Human dLN spike-specific CD4 + follicular helper T (T FH ) cells exhibited heterogeneous phenotypes, including germinal center CD4 + T FH cells and CD4 + IL-10 + T FH cells. Analysis of an independent cohort of SARS-CoV-2-infected individuals 3 months and 6 months after infection found spike-specific CD4 + T cell profiles in blood that were distinct from those detected in blood 3 months and 6 months after BNT162b2 vaccination. Our findings provide an atlas of human spike-specific CD4 + T cell transcriptional phenotypes in the dLNs and blood following SARS-CoV-2 vaccination or infection., (© 2024. Springer Nature America, Inc.)

The differentiation and specificity of human CD4 + T follicular helper cells (T FH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4 + T FH cell response. The first vaccination induced an increase in the frequency of circulating T FH (cT FH ) and LN T FH cells at week 1 postvaccination. This increase was transient for cT FH cells, whereas the LN T FH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of T FH cells in the LN, including pre-T FH cells, memory T FH cells, germinal center (GC) T FH cells and interleukin-10 + T FH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC T FH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific T FH cell clonal lineages, including multiple responses targeting internal influenza virus proteins, and found that each T FH cell state was attainable within a clonal lineage. Thus, human T FH cells form a durable and dynamic multitissue network., (© 2024. The Author(s).)

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah -/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease., Competing Interests: Declaration of interests H.A.M. and B.Y.C. consult for Ena Respiratory. A.G.R. received research support from Illumina. P.G.T. is on the SAB of Immunoscape and Cytoagents, consulted for JNJ, received travel support/honoraria from Illumina and 10× Genomics, and has patents related to TCR discovery. J.C.C. and P.G.T. have patents related to treating or reducing severity of viral infections, including SARS-CoV-2., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The Streptococcus anginosus group (SAG) pathogens have the potential to cause head and neck space infections, including intracranial abscesses. Several centers noted an increase in intracranial abscesses in children during the SARS-CoV-2 pandemic, prompting a Centers for Disease Control and Prevention health alert in May 2022. We examined the epidemiology of pediatric intracranial abscesses at a tertiary care center with a focus on SAG pre- and post-pandemic., Methods: Cases of intracranial abscesses of any microbiologic etiology admitted from January 2011 to December 2022 were identified using International Classification of Diseases 10 codes. Subjects were cross-referenced with culture results from the microbiology laboratory at Texas Children's Hospital. Cases included were those associated with either otitis media, mastoiditis or sinusitis and medical records were reviewed., Results: A total of 157 cases were identified and 59.9% (n = 94) were caused by SAG. The incidence of all sinogenic/otogenic intracranial infections ( P = 0.002), and SAG-specific infections ( P = 0.004), increased from 2011 to 2022. SAG infection was more often associated with multiple surgeries, and these subjects were more likely to require craniotomy or craniectomy. Among sinogenic abscesses, S. intermedius was the most common pathogen, while among otogenic cases, S. pyogenes predominated. From March 2020 to Dec 2022, 9/49 cases tested positive for SARS-CoV-2 (18.4%); characteristics of infection were not significantly different among cases with and without SARS-CoV-2., Conclusions: Over the last decade, intracranial complications of sinusitis/otitis have been increasing, specifically those caused by SAG; this trend, however, predated the SARS-CoV-2 pandemic. SAG was associated with a greater need for surgical intervention, specifically neurosurgery. Further work is necessary to determine the cause for these rising infections., Competing Interests: J.C.McN: receives funding from AHRQ R01HS026896 for work unrelated to this manuscript; J.C.McN. also serves as the site investigator for a clinical trial sponsored by Nabriva unrelated to this work. A.R.F. receives funding through NIAID R21AI153663 and R21AI159059 and B.M.H. receives funding through NIAID K01AI148593-01 which in part supported the genomic studies in this project. S.L.K. receives funding from Pfizer for unrelated work. All other authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Perturbation of the apoptosis and necroptosis pathways critically influences embryogenesis. Receptor-associated protein kinase-1 (RIPK1) interacts with Fas-associated via death domain (FADD)-caspase-8-cellular Flice-like inhibitory protein long (cFLIP L ) to regulate both extrinsic apoptosis and necroptosis. Here, we describe Ripk1-mutant animals (Ripk1 R588E [RE]) in which the interaction between FADD and RIPK1 is disrupted, leading to embryonic lethality. This lethality is not prevented by further removal of the kinase activity of Ripk1 (Ripk1 R588E K45A [REKA]). Both Ripk1 RE and Ripk1 REKA animals survive to adulthood upon ablation of Ripk3. While embryonic lethality of Ripk1 RE mice is prevented by ablation of the necroptosis effector mixed lineage kinase-like (MLKL), animals succumb to inflammation after birth. In contrast, Mlkl ablation does not prevent the death of Ripk1 REKA embryos, but animals reach adulthood when both MLKL and caspase-8 are removed. Ablation of the nucleic acid sensor Zbp1 largely prevents lethality in both Ripk1 RE and Ripk1 REKA embryos. Thus, the RIPK1-FADD interaction prevents Z-DNA binding protein-1 (ZBP1)-induced, RIPK3-caspase-8-mediated embryonic lethality, affected by the kinase activity of RIPK1., Competing Interests: Declaration of interests D.R.G. consulted for Sonata Pharmaceuticals, Ventus Pharmaceuticals, and ASHA Therapeutics and received grant support from Amgen during this research., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Pelvic involvement has been reported in 3%-14% of acute hematogenous osteomyelitis (AHO) cases in children. One guideline suggests need for a longer antibiotic course in pelvic AHO, however, recent data are lacking. We describe the clinical course of children with pelvic AHO and compare it to nonpelvic AHO., Methods: A retrospective review of patients with a diagnosis of AHO admitted to Texas Children's Hospital from January 2012 to December 2020 was conducted. Patients 6 months-<19 years old and with ≤14 days of symptoms at admission were eligible. Patients with sickle cell disease or immunocompromised were excluded. Wilcoxon rank-sum test assessed for differences between continuous variables and Fisher exact for categorical variables using STATA 17., Results: We compared 104 cases of pelvic AHO to 314 cases of nonpelvic AHO. Patients had similar microbiology, length of stay and length of antibiotic therapy. Patients with pelvic AHO had pyomyositis identified by magnetic resonance imaging more often (28.8 vs. 9.4%, P < 0.001) and bone abscess less often (22.1 vs. 46.5%, P < 0.001). Rates of chronic complications were comparable between patients with pelvic AHO and nonpelvic AHO (8.4% vs. 15.1%, P = 0.1). Nineteen patients (18.3%) with pelvic AHO received ≤30 antibiotic days without complications, but they had less need for intensive care or bone abscesses than patients treated longer., Conclusions: Pelvic AHO in children may be more frequent than previously reported but is not associated with more complications. Four weeks of therapy may be sufficient in selected patients. Prospective studies to compare outcomes with different lengths of therapy are needed., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome 1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection 6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies. We observe that fusion-specific CD8 T cells are rare and that FLC patient TCR repertoires lack large clusters of related TCR sequences characteristic of potent antigen-specific responses, potentially explaining why endogenous immune responses are insufficient to clear FLC tumors. Nevertheless, we define two functional fusion-specific TCRs, one of which has strong anti-tumor activity in vivo. Together, our results provide insights into the fragmented nature of neoantigen-specific repertoires in humans and indicate routes for clinical development of successful immunotherapies for FLC., Competing Interests: Declaration of interests J.C.C., A.M.K., A.E.Z., S.E.S., and P.G.T. have a patent application for TCRs for treating FLC. J.C.C. has additional patent applications in the field of immunotherapy. A.W.P. is a member of the scientific advisory board (SAB) of Bioinformatics Solutions Inc. (Canada), shareholder and SAB member of Evaxion Biotech (Denmark), consultant for Grey Wolf Therapeutics (UK), and cofounder of Resseptor Therapeutics (Melbourne). S.G. is a co-inventor on patent applications in the fields of cell/gene therapy for cancer, consultant of TESSA Therapeutics, member of the Data and Safety Monitoring Board of Immatics, and SAB member of Be Biopharma and has received honoraria from Tidal, Catamaran Bio, and Sanofi within the last 2 years. S.E.S. is a cofounder, stockholder, and paid consultant for Gliknik Inc., serves on the SAB and holds stock options for Virion Inc., and receives royalties from the Mayo Clinic for licensed IP surrounding manipulation of the PD-1:PD-L1 pathway for cancer treatment. P.G.T. is on the SAB of Immunoscape and Shennon Bio, received personal fees and research support from Elevate Bio, and consulted for 10×, Illumina, Pfizer, Cytoagents, and JNJ., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The incidence of invasive Group A Streptococcus (iGAS) has varied throughout the COVID-19 pandemic. We reviewed iGAS infections in infants ≤1 year from 2012 to 2022. Twenty-five percent of cases occurred in the last quarter of 2022. Pneumonia (21.8%) was the most common presentation. Twenty-one patients (65.6%) were successfully transitioned to oral antibiotics., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Acute hematogenous osteomyelitis (AHO) can be associated with severe complications which can be difficult to predict in the clinical setting. The previously published predictive acute complication score ("A-SCORE") and chronic complication score ("C-SCORE") show promise, however, further external validation is needed., Methods: We performed a retrospective study of 418 children with AHO and analyzed the performance of A-SCORE (variables included bone abscess, fever after 48 h of starting antibiotics, suppurative arthritis, disseminated disease, and delayed source control) to predict risk for acute complicated course (treatment failure, prolonged admission, and/or need for ≥2 bone debridements) and C-SCORE (includes disseminated disease, bone debridement, and CRP ≥10 mg/dL at 2-4 days after starting antibiotics) to predict chronic complications (growth restriction, pathologic fracture, chronic osteomyelitis, avascular necrosis, joint deformity, and/or frozen joint)., Results: An acute complicated course occurred in 106/418 (25.4%); 51/380 (13.5%) with complete follow-up data had a chronic complication. The A-SCORE performed with similar specificity (78%) and negative predictive value (NPV) (92%), and higher sensitivity (81%) and increased area under the receiver operating curve (AUC) (0.87) in our population. The C-SCORE performed with similar sensitivity (64%) and NPV (94%) but had lower specificity (86%) and AUC (0.71) than originally reported. Other variables associated with development of complications such as tibia involvement and bacteremia ≥2 days were identified but did not result in significantly improved predictive scores., Conclusions: Predictive A-SCORE and C-SCORE for AHO complications in children may help guide acute management and long-term follow-up decisions. Prospective studies are needed to determine their applicability., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T cell-based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, costimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of antitumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 41BB and CD28 costimulation through transgenic expression of 41BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful antitumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell antitumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma., Significance: CAR T-cell immunotherapies hold great potential for treating brain cancers; however, they are hindered by a challenging immune environment that dampens their effectiveness. In this study, we show that the CAR design influences the makeup of the immune environment in brain tumors, underscoring the need to target specific immune components to improve CAR T-cell performance, and highlighting the significance of using models with functional immune systems to optimize this therapy., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background: Invasive infections caused by Streptococcus pyogenes (invasive group A streptococcus [iGAS]) and Streptococcus pneumoniae (invasive pneumococcal disease [IPD]) decreased substantially at the beginning of the COVID-19 pandemic. Our study sought to evaluate the extent of this decrease and the trends of these infections since reversion of societal adjustments incident to the pandemic. We also wanted to compare the frequency of these infections with invasive community-onset Staphylococcus aureus (I-CO-SA) infections and common respiratory viral infections in this period., Methods: Cases of iGAS, IPD, and I-CO-SA infections were identified prospectively and retrospectively at 2 large US children's hospitals by positive cultures from July 2018 through December 2022. Admission data were used to estimate frequency. For comparison, rates of respiratory syncytial virus (RSV), influenza, and SARS-CoV-2 were estimated by the number of positive viral test results at each institution., Results: I-CO-SA infections showed little variation in the study period. Rates of iGAS infection and IPD decreased by 46% and 44%, respectively, from 2019 to 2020, coinciding with a substantial decrease in RSV and influenza. In 2022, RSV and influenza infection rates increased to prepandemic winter season rates, coinciding with a return to prepandemic rates of IPD (225% increase from 2021 to 2022) and a surge above prepandemic rates of iGAS infections (543% increase from 2021 to 2022)., Conclusions: The COVID-19 pandemic had an unexpected influence on IPD and iGAS infections that was temporally related to changes in rates of viral infections., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

CD8 + T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8 + T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M1 58-66 (A2/M1 58 ) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M1 58 + CD8 + T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8 + T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8 + T cell responses toward viral infections., (© 2023. The Author(s).)

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6) 1 . Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4 + T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration 2 or are in clinical studies 3-5 , we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials 1,6-8 and may influence the design and production of autologous and allogeneic cell therapies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections., Competing Interests: The authors declare no conflict of interest.

We profiled blood and draining lymph node (LN) samples from human volunteers after influenza vaccination over two years to define evolution in the T follicular helper cell (TFH) response. We show LN TFH cells expanded in a clonal-manner during the first two weeks after vaccination and persisted within the LN for up to six months. LN and circulating TFH (cTFH) clonotypes overlapped but had distinct kinetics. LN TFH cell phenotypes were heterogeneous and mutable, first differentiating into pre-TFH during the month after vaccination before maturing into GC and IL-10+ TFH cells. TFH expansion, upregulation of glucose metabolism, and redifferentiation into GC TFH cells occurred with faster kinetics after re-vaccination in the second year. We identified several influenza-specific TFH clonal lineages, including multiple responses targeting internal influenza proteins, and show each TFH state is attainable within a lineage. This study demonstrates that human TFH cells form a durable and dynamic multi-tissue network.

Integrins are essential surface receptors that sense extracellular changes to initiate various intracellular signaling cascades. The rapid activation of the epithelial-intrinsic β6 integrin during influenza A virus (IAV) infection has been linked to innate immune impairments. Yet, how β6 regulates epithelial immunity remains undefined. Here, we identify the role of β6 in mediating the Toll-like receptor 7 (TLR7) through the regulation of intracellular trafficking. We demonstrate that deletion of the β6 integrin in lung epithelial cells significantly enhances the TLR7-mediated activation of the type I interferon (IFN) response during homeostasis and respiratory infection. IAV-induced β6 facilitates TLR7 trafficking to lysosome-associated membrane protein (LAMP2a) components, leading to a reduction in endosomal compartments and associated TLR7 signaling. Our findings reveal an unappreciated role of β6-induced autophagy in influencing epithelial immune responses during influenza virus infection.

Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido -/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1 -/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Influenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection., Methods: We measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR q<0.05)., Results: Comparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation ( RETN, TCN1, OLFM4, MMP8, LCN2, BPI, LTF, S100A12, GUSB) compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways ( RPL3, MRPL3, HLA-DMB, EEF1G , CD8A ) were associated with MODS recovery within a week., Conclusion: Thus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome., Competing Interests: AGR, TN, and MMN: NIH/NIAID as declared in the funding statement - grant support paid to Boston Children’s Hospital. Also current CDC grant for COVID-19 work unrelated to current manuscript also paid to Boston Children’s Hospital under PI AGR. AGR also receives Royalties from UpToDate, Inc. as Section editor of Pediatric Critical Care Medicine. Honoraria: Grand Rounds presentations on MIS-C not related to current manuscript. Participation on a Data Safety Monitoring/Advisory Board: NIH Grace Study. Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: Medical Advisory Board for Families Fight Flu and Chair of International Sepsis Forum. JCC: additionally declares support from ALSAC for the current work. Other entities not related to the current work: U01AI150747, R01AI136514, U01AI144616; American Association of Immunologists Abstract award 2021, Patent: Methods for Treating or Reducing the Severity of a Viral Infection Publication no. WO 2021/226174 A1. MWH declared Kiadis, Licensing income, unrelated to the content of this manuscript, American Board of Pediatrics Payment, Abbive payment. Partner Therapeutics Provision of study drug, unrelated to the current manuscript, Sobi Provision of study drug, unrelated to the current manuscript. JC: NIH, paid to institution not related to this work; GLG Group payment to her, Elsevier payment made to her for work done as Associate Editor; Patent pending for Methods and compositions for treating and preventing T cell-driven diseases. BMC: Not related to current work: receives grants paid to institution from American Lung Association. Sobi Participation on a Data Safety Monitoring Board/Advisory Board. SLW: Receives royalties from UpToDate, Inc. Has grants/contracts with Pennsylvania Dept of Health not related to current manuscript. PGT: declares support from ALSAC and NIAID R01 AI154470 for the current work. Has several grants not associated with current work: NIAID 5R01AI128805-05; 1R01AI154470; NIAID 75N93019C00052; 5R01AI136514; 5R01AI35025; NIAID 5U01AI144616; NIAID U01AI150747. Royalties/licenses paid to both PGT and his institution: TCR cloning technology—Miltenyi Biotec & TCR amplification technology—Shennon Bio. Consulting fees paid to him: Johnson and Johnson, Cytoagents, Immunoscape, Shennon Bio. Payment or honoraria for lectures, presentations, or other events: Illumina—Future Genomic Advances, Yale University, CZ Biohub, PACT Pharma, UCSD, Tufts, University of Arizona, Mt. Sinai, Umass, OSU, Korean Association of Immunology, SISMID, University of Washington, MSKCC, Washington University, University of Missouri, Fred Hutch, UNM Illumina Single Cell, Cincinnati Childrens, University of Toronto, Purdue University, Iowa State University, University of Georgia. Patent: Methods for Treating or Reducing the Severity of a Viral Infection Publication no. WO 2021/226174 A1. (continued disclosures for PGT:) Support for attending meetings and/or travel: NIH Study Section, Keystone Viral Immunity, NIAID CEIRR, CEIRS and CIVIC meetings, GRC, 10X Users Symposium, Illumina Symposia, ImmunOktoberfest, Options for the Control of Influenza XI, Carghese Workshop, FOCIS, Max Planck for Complex Systems, Santa Fe Institute, AAI, Weizmann Institute, APS Society, Banff Institute, ISIRV meeting, University of Melbourne, British Society of Immunology. Stock or stock options: Shennon Bio Scientific advisory board payments. (Additional patents:) US20170304293A1 Coordinated metabolic reprogramming in response to productive viral infections; Cloning and expression system for t-cell receptors; License payments made to me and institution; Method for detecting SARS-CoV-2 infection. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Novak, Crawford, Hahn, Hall, Thair, Newhams, Chou, Mourani, Tarquinio, Markovitz, Loftis, Weiss, Higgerson, Schwarz, Pinto, Thomas, Gedeit, Sanders, Mahapatra, Coates, Cvijanovich, Ackerman, Tellez, McQuillen, Kurachek, Shein, Lange, Thomas and Randolph.)

Previous studies have identified cytokines associated with respiratory virus infection illness outcome. However, few studies have included comprehensive cytokine panels, longitudinal analyses, and/or simultaneous assessment across the severity spectrum. This, coupled with subjective definitions of cytokine storm syndrome (CSS), have contributed to inconsistent findings of cytokine signatures, particularly with COVID severity. Here, we measured 38 plasma cytokines and compared profiles in healthy, SARS-CoV-2 infected, and multisystem inflammatory syndrome in children (MIS-C) patients (n = 169). Infected patients spanned the severity spectrum and were classified as Asymptomatic, Mild, Moderate or Severe. Our results showed acute cytokine profiles and longitudinal dynamics of IL1Ra, IL10, MIP1b, and IP10 can differentiate COVID severity groups. Only 4% of acutely infected patients exhibited hypercytokinemia. Of these subjects, 3 were Mild, 3 Moderate, and 1 Severe, highlighting the lack of association between CSS and COVID severity. Additionally, we identified IL1Ra and TNFa as potential biomarkers for patients at high risk for long COVID. Lastly, we compare hypercytokinemia profiles across COVID and influenza patients and show distinct elevated cytokine signatures, wherein influenza induces the most elevated cytokine profile. Together, these results identify key analytes that, if obtained at early time points, can predict COVID illness outcome and/or risk of complications, and provide novel insight for improving the conceptual framework of hypercytokinemia, wherein CSS is a subgroup that requires concomitant severe clinical manifestations, and including a list of cytokines that can distinguish between subtypes of hypercytokinemia.

Acute respiratory distress syndrome (ARDS), termed pediatric ARDS (pARDS) in children, is a severe form of acute respiratory failure (ARF). Pathologic immune responses are implicated in pARDS pathogenesis. Here, we present a description of microbial sequencing and single cell gene expression in tracheal aspirates (TAs) obtained longitudinally from infants with ARF. We show reduced interferon stimulated gene (ISG) expression, altered mononuclear phagocyte (MNP) transcriptional programs, and progressive airway neutrophilia associated with unique transcriptional profiles in patients with moderate to severe pARDS compared to those with no or mild pARDS. We additionally show that an innate immune cell product, Folate Receptor 3 (FOLR3), is enriched in moderate or severe pARDS. Our findings demonstrate distinct inflammatory responses in pARDS that are dependent upon etiology and severity and specifically implicate reduced ISG expression, altered macrophage repair-associated transcriptional programs, and accumulation of aged neutrophils in the pathogenesis of moderate to severe pARDS caused by RSV., (© 2023. The Author(s).)

Microbial components have a range of direct effects on the fetal brain. However, little is known about the cellular targets and molecular mechanisms that mediate these effects. Neural progenitor cells (NPCs) control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. We identify ventricular radial glia (vRG), the primary NPC, as the target of bacterial cell wall (BCW) generated during the antibiotic treatment of maternal pneumonia. BCW enhanced proliferative potential of vRGs by shortening the cell cycle and increasing self-renewal. Expanded vRGs propagated to increase neuronal output in all cortical layers. Remarkably, Toll-like receptor 2 (TLR2), which recognizes BCW, localized at the base of primary cilia in vRGs and the BCW-TLR2 interaction suppressed ciliogenesis leading to derepression of Hedgehog (HH) signaling and expansion of vRGs. We also show that TLR6 is an essential partner of TLR2 in this process. Surprisingly, TLR6 alone was required to set the number of cortical neurons under healthy conditions. These findings suggest that an endogenous signal from TLRs suppresses cortical expansion during normal development of the neocortex and that BCW antagonizes that signal through the TLR2/cilia/HH signaling axis changing brain structure and function. IMPORTANCE Fetal brain development in early gestation can be impacted by transplacental infection, altered metabolites from the maternal microbiome, or maternal immune activation. It is less well understood how maternal microbial subcomponents that cross the placenta, such as bacterial cell wall (BCW), directly interact with fetal neural progenitors and neurons and affect development. This scenario plays out in the clinic when BCW debris released during antibiotic therapy of maternal infection traffics to the fetal brain. This study identifies the direct interaction of BCW with TLR2/6 present on the primary cilium, the signaling hub on fetal neural progenitor cells (NPCs). NPCs control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. Within a window of vulnerability before the appearance of fetal immune cells, the BCW-TLR2/6 interaction results in the inhibition of ciliogenesis, derepression of Sonic Hedgehog signaling, excess proliferation of neural progenitors, and abnormal cortical architecture. In the first example of TLR signaling linked to Sonic Hedgehog, BCW/TLR2/6 appears to act during fetal brain morphogenesis to play a role in setting the total cell number in the neocortex., Competing Interests: The authors declare a conflict of interest. PT serves on the SAB for Immunoscape and Cytoagents and has received travel support from 10X Genomics and honorarium and travel support from Illumina. The remaining authors have no conflicts of interest to disclose.

This retrospective cohort study was performed to compare clinical outcomes between patients with Staphylococcus aureus bacteremia who received an early versus late infectious disease consultation. Early consultation resulted in significantly greater adherence to quality care indicators and shorter hospital stays., Competing Interests: Potential conflicts of interest. Y. S. M. reports an honorarium paid to the author (faculty) from the Ostler Institute, a travel stipend paid to the author from the American Society for Microbiology Microbe, and a role as president (unpaid) for First Coast ID/CM Symposium. A. M. C. served on the advisory board and received honoraria from Spero Therapeutics, AbbVie, Cumberland Pharmaceuticals, and Ferring Pharmaceuticals. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding interactions between adoptively transferred immune cells and the tumor immune microenvironment (TIME) is critical for developing successful T-cell based immunotherapies. Here we investigated the impact of the TIME and chimeric antigen receptor (CAR) design on anti-glioma activity of B7-H3-specific CAR T-cells. We show that five out of six B7-H3 CARs with varying transmembrane, co-stimulatory, and activation domains, exhibit robust functionality in vitro . However, in an immunocompetent glioma model, these CAR T-cells demonstrated significantly varied levels of anti-tumor activity. We used single-cell RNA sequencing to examine the brain TIME after CAR T-cell therapy. We show that the TIME composition was influenced by CAR T-cell treatment. We also found that successful anti-tumor responses were supported by the presence and activity of macrophages and endogenous T-cells. Together, our study demonstrates that efficacy of CAR T-cell therapy in high-grade glioma is dependent on CAR structural design and its capacity to modulate the TIME., Competing Interests: Conflict of interest: GK and CD have patent applications in the field of immunotherapy. SG is a co-inventor on patent applications in the fields of cell or gene therapy for cancer, a consultant of TESSA Therapeutics, a scientific advisory board member of Be Biopharma, a member of the Data and Safety Monitoring Board (DSMB) of Immatics, and has received honoraria from Tidal, Catamaran Bio, and Sanofi within the last 2 years. Additional Declarations: There is NO Competing Interest.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4 + and CD8 + T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people., (© 2023. The Author(s).)

Background: Acute hematogenous osteomyelitis (AHO) is a serious infection in children. Pediatric Infectious Diseases Society guidelines recommend empiric methicillin-resistant Staphylococcus aureus (MRSA) therapy in regions where MRSA accounts for more than 10-20% of all staphylococcal osteomyelitis. We sought to examine factors present at the time of admission which may predict etiology and guide empiric treatment for pediatric AHO in a region with endemic MRSA., Methods: We reviewed admissions with International Classification of Diseases 9/10 codes for AHO from 2011 to 2020 in otherwise healthy children. Medical records were reviewed for clinical and laboratory parameters present on the day of admission. Logistic regression was used to determine clinical variables independently associated with (1) MRSA infection and (2) non- Staphylococcus aureus infection., Results: A total of 545 cases were included. An organism was identified in 77.1% of cases and S. aureus was the most common (66.2%); 18.9% of all AHO cases were MRSA. Organisms besides S. aureus were identified in 10.8% of cases. CRP >7 mg/dL, subperiosteal abscess, history of any prior skin or soft tissue infection (SSTI) and need for intensive care unit admission were independently associated with MRSA infection. Vancomycin was used as an empiric treatment in 57.6% of cases. If the above criteria were relied upon to predict MRSA AHO, empiric vancomycin use could have been reduced by 25%., Conclusions: Critical illness, CRP >7 mg/dL at the time of presentation, subperiosteal abscess and history of SSTI are suggestive of MRSA AHO, and could be considered when planning empiric therapy. Further work is needed to validate these findings before wider implementation., Competing Interests: J.C.McN. receives grant funding from the Agency for Healthcare Research and Quality (AHRQ R01HS026896) for work unrelated to this manuscript. He is the local PI on a multi-center clinical trial sponsored by Nabriva Therapeutics unrelated to the work under consideration; S.L.K. and J.G.V. are coinvestigators on this study. J.C.McN. has also received a donation of laboratory materials from Allergan for work unrelated to this article. S.L.K. receives research support through Pfizer for unrelated work. The other authors received no additional funding. The other authors have no funding or conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4 + /CD8 + T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response., Competing Interests: Declaration of interests The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays and NDV-based SARS-CoV-2 vaccines, which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. H.A.M. and B.Y.C. are currently consulting for Ena Respiratory. B.W.T. has received research funding from MSD, Seqirus, and Sanofi and is on the advisory board for Moderna, CSL-Behring, and Takeda., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Beginning in October 2022, we observed a substantial increase in the total number of cases of invasive GAS disease (iGAS) in the pediatric population in Houston, TX. Emm12 GAS strains were disproportionately represented but the overall proportion of iGAS infections observed during the current spike was similar to pre-pandemic years., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Many health care centers have reported an association between Staphylococcus aureus isolates bearing efflux pump genes and an elevated MIC/minimal bactericidal concentration (MBC) to chlorhexidine gluconate (CHG) and other antiseptics. The significance of these organisms is uncertain, given that their MIC/MBC is typically far lower than the CHG concentration in most commercial preparations. We sought to evaluate the relationship between carriage of the efflux pump genes qacA/B and smr in S. aureus and the efficacy of CHG-based antisepsis in a venous catheter disinfection model. S. aureus isolates with and without smr and/or qacA/B were utilized. The CHG MICs were determined. Venous catheter hubs were inoculated and exposed to CHG, isopropanol, and CHG-isopropanol combinations. The microbiocidal effect was calculated as the percent reduction in CFU following exposure to the antiseptic relative to the control. The qacA/B- and smr -positive isolates had modest elevations in the CHG MIC 90 compared to the qacA/B- and smr -negative isolates (0.125 mcg/ml vs. 0.06 mcg/ml, respectively). However, the CHG microbiocidal effect was significantly lower for qacA/B - and/or smr -positive strains than for susceptible isolates, even when the isolates were exposed to CHG concentrations up to 400 μg/mL (0.04%); this finding was most notable for isolates bearing both qacA/B and smr (89.3% versus 99.9% for the qacA/B- and smr -negative isolates; P  = 0.04). Reductions in the median microbiocidal effect were also observed when these qacA/B- and smr -positive isolates were exposed to a solution of 400 μg/mL (0.04%) CHG and 70% isopropanol (89.5% versus 100% for the qacA/B- and smr -negative isolates; P  = 0.002). qacA/B - and smr- positive S. aureus isolates have a survival advantage in the presence of CHG concentrations exceeding the MIC. These data suggest that traditional MIC/MBC testing may underestimate the ability of these organisms to resist the effects of CHG. IMPORTANCE Antiseptic agents, including chlorhexidine gluconate (CHG), are commonly utilized in the health care environment to reduce rates of health care-associated infections. A number of efflux pump genes, including smr and qacA/B , have been reported in Staphylococcus aureus isolates that are associated with higher MICs and minimum bactericidal concentrations (MBCs) to CHG. Several health care centers have reported an increase in the prevalence of these S. aureus strains following an escalation of CHG use in the hospital environment. The clinical significance of these organisms, however, is uncertain, given that the CHG MIC/MBC is far below the concentration in commercial preparations. We present the results of a novel surface disinfection assay utilizing venous catheter hubs. We found that qacA/B -positive and smr -positive S. aureus isolates resist killing by CHG at concentrations far exceeding the MIC/MBC in our model. These findings highlight that traditional MIC/MBC testing is insufficient to evaluate susceptibility to antimicrobials acting on medical devices.

Drak2-deficient (Drak2 -/- ) mice are resistant to multiple models of autoimmunity yet effectively eliminate pathogens and tumors. Thus, DRAK2 represents a potential target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Here, we demonstrate that resistance to T1D in non-obese diabetic (NOD) mice is due to the absence of Drak2 in T cells and requires the presence of regulatory T cells (T regs ). Contrary to previous hypotheses, we show that DRAK2 does not limit TCR signaling. Rather, DRAK2 regulates IL-2 signaling by inhibiting STAT5A phosphorylation. We further demonstrate that enhanced sensitivity to IL-2 in the absence of Drak2 augments thymic T reg development. Overall, our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic T reg development and by impacting the sensitivity of conventional T cells to T reg -mediated suppression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the late 1940s to 1950s, Staphylococcus aureus isolates first-gained resistance to penicillin. Recently, some centers have described an increase in the proportion of methicillin susceptible S. aureus (MSSA) which are also susceptible to penicillin (PSSA). There are little data on the frequency of PSSA infections in children. We investigated the prevalence of penicillin susceptibility among pediatric MSSA acute hematogenous osteoarticular infection (OAI) isolates. MSSA OAI isolates were obtained through surveillance studies at Texas Children's and St. Louis Children's Hospitals from January 2011 to December 2019. All isolates underwent PCR for blaZ β-lactamase, PVL genes and agr group. All blaZ negative isolates then underwent penicillin MIC determination. blaZ negative isolates with penicillin MIC ≤ 0.125 μg/mL were considered PSSA. Multilocus sequence typing (MLST) was conducted on a subset of isolates. A total of 329 unique isolates were included in the study. The median patient age was 9.2 years (IQR:5.1 to 12.2). Overall, 6.7% of isolates were penicillin susceptible. No PSSA were detected prior to 2015 but increased yearly thereafter. By the final study year, 20.4% of isolates were PSSA ( P = 0.001). PSSA were similar to penicillin-resistant MSSA (PR-MSSA) isolates in terms agr group and PVL carriage as well as clinical presentation and outcomes. PSSA were of distinct sequence types compared to PR-MSSA. PSSA appears to be increasing among OAI in U.S. children. Overall, PSSA isolates are associated with a similar clinical presentation as penicillin-resistant isolates. The potential for use of penicillin treatment in PSSA OAI warrants further study.

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF., (© 2022 by The American Society of Hematology.)

Background: There are pharmacy-related barriers to the dispensing of buprenorphine for the treatment of opioid use disorders. These include pharmacists' moral objections and mistrust of treatment regimens; the perception of a limit on the amount of buprenorphine able to be ordered and dispensed; stigma and concerns about diversion; and knowledge and communication gaps., Objectives: To document pharmacy stakeholders' awareness and interpretation of regulatory policies that may impact rural community pharmacists' willingness and ability to dispense buprenorphine. To identify factors that affect rural community pharmacists' willingness and ability to dispense buprenorphine in Appalachian North Carolina., Methods: Qualitative analysis and thematic coding of phone interviews with eight pharmacists from several rural North Carolina counties where local health departments recently began prescribing MOUD and four pharmacy industry stakeholders representing knowledge of wholesale distributors and pharmacy education., Results: Three major themes were identified: stigma and misinformation, provider-prescriber communication, and perceived and actual regulatory constraints. A number of respondents indicated a desire to better understand MOUD treatment plans and displayed a misunderstanding of evidence-based treatment guidelines. Stakeholders indicated the importance of pharmacists establishing a relationship with prescribers and described pharmacist preference for dispensing buprenorphine to established patients over new or out-of-area patients. Pharmacist stakeholders and industry/education stakeholders expressed concern over a perceived DEA 'cap' for buprenorphine ordering., Conclusions: This study provides insight on possible approaches to address rural pharmacy-related barriers patients may face when filling buprenorphine prescriptions. There is a demonstrated need for further pharmacist training on evidence-based practices for treating opioid use disorders and ordering limits, as well as a need for increased communication between prescribers and pharmacists., Competing Interests: No conflicts., (© 2022 Published by Elsevier Inc.)

Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Eosinophilic meningitis can be caused by various etiologies and is reported mostly in tropical climates. The diagnosis is rare in the continental US, presenting challenges for management. Following a case of pediatric eosinophilic meningitis, we reviewed our 11-year experience with this diagnosis at a large US children's hospital., (Copyright © 2022 Elsevier Inc. All rights reserved.)

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Ceftaroline represents an attractive therapy option for methicillin-resistant Staphylococcus aureus (MRSA). Little data is available, however, regarding the frequency of reduced susceptibility (RS) to ceftaroline among pediatric MRSA infections. We screened invasive MRSA isolates at a tertiary children's hospital for ceftaroline RS. Ceftaroline RS occurred in 2.9% of isolates and only among health care associated infections. Ceftaroline RS isolates were more often clindamycin-resistant. Sequencing data indicated the predominance of the CC5 lineage among ceftaroline RS isolates.

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL ( Mlkl FLAG/FLAG ), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8 -/- Mlkl FLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8 +/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8 -/- Mlkl FLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

The early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was temporally associated with a reduction in many childhood infections, although the impact on bacterial colonization is unknown. We longitudinally assessed Staphylococcusaureus colonization prior to and through the first year of the pandemic. We observed a decline in methicillin-resistant Staphylococcus aureus colonization associated with SARS-CoV-2 prevention mandates., (Copyright © 2022 Elsevier Inc. All rights reserved.)

While adult bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) and their extrinsic regulation is well studied, little is known about the composition, function, and extrinsic regulation of the first HSPCs to enter the BM during development. Here, we functionally interrogate murine BM HSPCs from E15.5 through P0. Our work reveals that fetal BM HSPCs are present by E15.5, but distinct from the HSPC pool seen in fetal liver, both phenotypically and functionally, until near birth. We also generate a transcriptional atlas of perinatal BM HSPCs and the BM niche in mice across ontogeny, revealing that fetal BM lacks HSPCs with robust intrinsic stem cell programs, as well as niche cells supportive of HSPCs. In contrast, stem cell programs are preserved in neonatal BM HSPCs, which reside in a niche expressing HSC supportive factors distinct from those seen in adults. Collectively, our results provide important insights into the factors shaping hematopoiesis during this understudied window of hematopoietic development., (© 2022. The Author(s).)

Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products., Significance: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Many barriers, including stocking behaviors and pharmacist attitudes, can limit access to buprenorphine in pharmacy settings., Objectives: To assess North Carolina (NC) pharmacists' (1) buprenorphine stocking behaviors, (2) awareness and interpretation of federal and state policy regarding buprenorphine, (3) perceptions about changes in buprenorphine demand, and (4) reasons for not dispensing buprenorphine, including attitudes., Methods: A convenience sample of currently practicing community pharmacists was recruited to participate in a 10-minute online survey. The survey included demographic questions and assessed pharmacists' buprenorphine ordering, stocking, and dispensing behaviors. Descriptive statistics were calculated, and logistic regressions examined associations with whether pharmacists (1) had ever refused to fill a buprenorphine prescription and (2) perceived buprenorphine dispensing limits., Results: The majority (96%) of respondents (n = 646, completion rate = 5.5%) kept buprenorphine in stock regularly or ordered it as needed, with generic formulations being stocked most often. Many pharmacists (62%) had refused to fill a buprenorphine prescription. Pharmacists with more negative buprenorphine attitudes were more likely to refuse to fill a buprenorphine prescription. Many pharmacists (31%) believed there were buprenorphine ordering limits, with wholesalers most commonly being perceived as the source. Pharmacists with more negative buprenorphine attitudes were more likely to perceive buprenorphine ordering limits, while pharmacists who worked at national chain, grocery or regional chains, and other pharmacy types were less likely to perceive ordering limits than independent pharmacies., Conclusion: Although most pharmacies stocked buprenorphine products, pharmacists' refusal to dispense and perceived ordering limits could limit patient access. Refusal and perceived ordering limits were associated with pharmacist attitudes and pharmacy type. Training that addresses logistical and attitudinal barriers to dispensing buprenorphine may equip pharmacists to address buprenorphine access barriers., (Copyright © 2022 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Objectives: The primary aim of this study was to better understand North Carolina providers' specific substance use disorder (SUD) and opioid use disorder treatment practices and buprenorphine prescribing. Furthermore, this study aimed to provide novel information regarding US South and rural providers' opioid use disorder treatment behaviors and perceptions of patient experience at community pharmacies., Methods: An online survey consisting of closed-ended and open-ended questions was used. Surveys were delivered to healthcare providers' e-mails and self-administered. Surveys were administered through an online survey platform., Results: In total, 332 healthcare providers, who were eligible to be X-waivered to prescribe buprenorphine, completed the online survey. Survey participants reported not having their X-waiver to prescribe buprenorphine or actively prescribing buprenorphine. The majority of participants were uncertain of potential barriers to filling buprenorphine prescriptions. Providers treating a mix of rural and urban patients reported being less likely to screen for SUDs. Although there were no rurality differences in SUD screening, providers who treat mostly rural patients reported a lack of SUD treatment options in their area., Conclusions: Early detection of SUDs can help prevent negative health outcomes for patients. Regardless of patient rurality, providers should screen for SUDs and familiarize themselves with the patient's experience when filling a buprenorphine prescription, along with possible barriers. Furthermore, providers should incorporate questions about their patient's ability to receive buprenorphine to help ensure that patients are receiving proper and necessary treatment.