Liver transplant for primary biliary tract neuroendocrine tumor in a nine-year-old girl.

BACKGROUND: Neuroendocrine tumors (NETs) are rare epithelial neoplasms that arise most commonly from the gastrointestinal tract. In pediatrics, the most common site of origin is in the appendix, with the liver being the most common site of metastasis. Neuroendocrine tumors arising from the biliary tract are extremely rare. METHODS: We describe a case of a nine-year-old girl who presented with obstructive cholestasis and was found to have multiple liver masses identified on biopsy as well-differentiated neuroendocrine tumor with an unknown primary tumor site. RESULT: The patient underwent extensive investigation to identify a primary tumor site, including endoscopy, endoscopic ultrasound, and capsule endoscopy. The patient ultimately underwent definitive management with liver transplant, and on explant was discovered to have multiple well-differentiated neuroendocrine tumors, WHO Grade 1, with extensive infiltration into the submucosa of bile duct, consistent with primary biliary tract neuroendocrine tumor. CONCLUSION: Identifying the site of the primary tumor in NETs found within the liver can be challenging. To determine if an extrahepatic primary tumor exists, workup should include endoscopy, EUS, and capsule endoscopy. Children with well-differentiated hepatic NETs, with no identifiable primary tumor, and an unresectable tumor, are considered favorable candidates for liver transplantation.

Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.

COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/µL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.

Pancreatic Enzyme Use Reduces Pancreatitis Frequency in Children With Acute Recurrent or Chronic Pancreatitis: A Report From INSPPIRE.

INTRODUCTION: Among children who suffer from acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP), acute pancreatitis (AP) episodes are painful, often require hospitalization, and contribute to disease complications and progression. Despite this recognition, there are currently no interventions to prevent AP episodes. In this retrospective cohort study, we assessed the impact of pancreatic enzyme therapy (PERT) use on clinical outcomes among children with pancreatic-sufficient ARP or CP. METHODS: Children with pancreatic-sufficient ARP or CP in the INSPPIRE-2 cohort were included. Clinical outcomes were compared for those receiving vs not receiving PERT, as well as frequency of AP before and after PERT. Logistic regression was used to study the association between development of AP episodes after starting PERT and response predictors. RESULTS: Among 356 pancreatic-sufficient participants, 270 (76%) had ARP, and 60 (17%) received PERT. Among those on PERT, 42% did not have a subsequent AP episode, during a mean 2.1 years of follow-up. Children with a SPINK1 mutation ( P = 0.005) and those with ARP (compared with CP, P = 0.008) were less likely to have an AP episode after starting PERT. After initiation of PERT, the mean AP annual incidence rate decreased from 3.14 down to 0.71 ( P < 0.001). DISCUSSION: In a retrospective analysis, use of PERT was associated with a reduction in the incidence rate of AP among children with pancreatic-sufficient ARP or CP. These results support the need for a clinical trial to evaluate the efficacy of PERT to improve clinical outcomes among children with ARP or CP.

Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex.

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Reversible, tunable epigenetic silencing of TCF1 generates flexibility in the T cell memory decision.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

Utilizing a Functional Lumen Imaging Probe for Evaluation of the Pediatric Airway, A Pilot Study.

OBJECTIVES: Accurate and reproducible measurements of the pediatric airway are critical for diagnostic evaluation and management of subglottic and tracheal stenosis. The endoluminal functional lumen imaging probe (EndoFLIP) is a catheter-based imaging probe which utilizes impedance planimetry to calculate luminal parameters, including cross-sectional area and compliance. Herein, we demonstrate the feasibility of this system for multidimensional evaluation of the pediatric airway. METHODS: 3D-printed pediatric laryngotracheal models were created based on computed tomography scans, then artificially deformed to simulate both circumferential and posterior subglottic stenosis. Two observers made six measurements of the minimum cross-sectional area (MCSA) and length of stenosis of each model with EndoFLIP. Agreement between observer measurements and model dimensions was evaluated using Lin's concordance correlation coefficient; inter-observer reliability was assessed using intraclass correlation. RESULTS: Four models were created: two without pathology (MCSA: 132.4, 44.3 mm2 ) and two with subglottic stenosis (MCSA: 28.7, 59.7 mm2 , stenotic length 27.8, 24.4 mm). Observer measurements of MCSA and length of stenosis demonstrated high concordance with the models (r = 0.99, 0.95, p < 0.001) with a mean error of 4.5% and 18.2% respectively. There was a low coefficient of variation (0.6%-2.8%) for measurements, indicating high precision. Interrater reliability was high for both MCSA and stenotic length (ICC: 0.99, 0.98). CONCLUSIONS: The EndoFLIP system allows for accurate and reproducible measurements of cross-sectional area and stenotic length in pediatric airway models. This method may provide further advantages in the evaluation of airway distensibility, as well as measurements of asymmetric airway pathology. LEVEL OF EVIDENCE: NA Laryngoscope, 134:108-112, 2024.

Stereotactic Body Radiation Therapy for Stage IIA to IIIA Inoperable Non-Small Cell Lung Cancer: A Phase 1 Dose-Escalation Trial.

PURPOSE: Larger tumors are underrepresented in most prospective trials on stereotactic body radiation therapy (SBRT) for inoperable non-small cell lung cancer (NSCLC). We performed this phase 1 trial to specifically study the maximum tolerated dose (MTD) of SBRT for NSCLC >3 cm. METHODS AND MATERIALS: A 3 + 3 dose-escalation design (cohort A) with an expansion cohort at the MTD (cohort B) was used. Patients with inoperable NSCLC >3 cm (T2-4) were eligible. Select ipsilateral hilar and single-station mediastinal nodes were permitted. The initial SBRT dose was 40 Gy in 5 fractions, with planned escalation to 50 and 60 Gy in 5 fractions. Adjuvant chemotherapy was mandatory for cohort A and optional for cohort B, but no patients in cohort B received chemotherapy. The primary endpoint was SBRT-related acute grade (G) 4+ or persistent G3 toxicities (Common Terminology Criteria for Adverse Events version 4.03). Secondary endpoints included local failure (LF), distant metastases, disease progression, and overall survival. RESULTS: The median age was 80 years; tumor size was >3 cm and ≤5 cm in 20 (59%) and >5 cm in 14 patients (41%). In cohort A (n = 9), 3 patients treated to 50 Gy experienced G3 radiation pneumonitis (RP), thus defining the MTD. In the larger dose-expansion cohort B (n = 25), no radiation therapy-related G4+ toxicities and no G3 RP occurred; only 2 patients experienced G2 RP. The 2-year cumulative incidence of LF was 20.2%, distant failure was 34.7%, and disease progression was 54.4%. Two-year overall survival was 53%. A biologically effective dose (BED) <100 Gy was associated with higher LF (P = .006); advanced stage and higher neutrophil/lymphocyte ratio were associated with greater disease progression (both P = .004). CONCLUSIONS: Fifty Gy in 5 fractions is the MTD for SBRT to tumors >3 cm. A higher BED is associated with fewer LFs even in larger tumors. Cohort B appears to have had less toxicity, possibly due to the omission of chemotherapy.

Campylobacter jejuni uses energy taxis and a dehydrogenase enzyme for l-fucose chemotaxis.

IMPORTANCE: In this study, we identify a separate role for the Campylobacter jejuni l-fucose dehydrogenase in l-fucose chemotaxis and demonstrate that this mechanism is not only limited to C. jejuni but is also present in Burkholderia multivorans. We now hypothesize that l-fucose energy taxis may contribute to the reduction of l-fucose-metabolizing strains of C. jejuni from the gastrointestinal tract of breastfed infants, selecting for isolates with increased colonization potential.

Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods hamper crowd-sourcing approaches toward genome-wide resolution of variants in disease-related genes. Our framework, Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these issues by offering simple and cost-effective saturation mutagenesis, as well as streamlining functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for over 99.8% of all possible coding single nucleotide variants and resolved 310 clinically reported variants of uncertain significance with high confidence, enhancing clinical variant interpretation in dystroglycanopathies. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Our approach opens new directions for enabling variant-to-function insights for disease genes in a manner that is broadly useful for crowd-sourcing implementation across standard research laboratories.

Nivolumab plus ipilimumab in advanced salivary gland cancer: a phase 2 trial.

Salivary gland cancers (SGCs) are rare, aggressive cancers without effective treatments when metastasized. We conducted a phase 2 trial evaluating nivolumab (nivo, anti-PD-1) and ipilimumab (ipi, anti-CTLA-4) in 64 patients with metastatic SGC enrolled in two histology-based cohorts (32 patients each): adenoid cystic carcinoma (ACC; cohort 1) and other SGCs (cohort 2). The primary efficacy endpoint (≥4 objective responses) was met in cohort 2 (5/32, 16%) but not in cohort 1 (2/32, 6%). Treatment safety/tolerability and progression-free survival (PFS) were secondary endpoints. Treatment-related adverse events grade ≥3 occurred in 24 of 64 (38%) patients across both cohorts, and median PFS was 4.4 months (95% confidence interval (CI): 2.4, 8.3) and 2.2 months (95% CI: 1.8, 5.3) for cohorts 1 and 2, respectively. We present whole-exome, RNA and T cell receptor (TCR) sequencing data from pre-treatment and on-treatment tumors and immune cell flow cytometry and TCR sequencing from peripheral blood at serial timepoints. Responding tumors universally demonstrated clonal expansion of pre-existing T cells and mutational contraction. Responding ACCs harbored neoantigens, including fusion-derived neoepitopes, that induced T cell responses ex vivo. This study shows that nivo+ipi has limited efficacy in ACC, albeit with infrequent, exceptional responses, and that it could be promising for non-ACC SGCs, particularly salivary duct carcinomas. ClinicalTrials.gov identifier: NCT03172624 .

Actionable wastewater surveillance: application to a university residence hall during the transition between Delta and Omicron resurgences of COVID-19.

Wastewater surveillance has gained traction during the COVID-19 pandemic as an effective and non-biased means to track community infection. While most surveillance relies on samples collected at municipal wastewater treatment plants, surveillance is more actionable when samples are collected "upstream" where mitigation of transmission is tractable. This report describes the results of wastewater surveillance for SARS-CoV-2 at residence halls on a university campus aimed at preventing outbreak escalation by mitigating community spread. Another goal was to estimate fecal shedding rates of SARS-CoV-2 in a non-clinical setting. Passive sampling devices were deployed in sewer laterals originating from residence halls at a frequency of twice weekly during fall 2021 as the Delta variant of concern continued to circulate across North America. A positive detection as part of routine sampling in late November 2021 triggered daily monitoring and further isolated the signal to a single wing of one residence hall. Detection of SARS-CoV-2 within the wastewater over a period of 3 consecutive days led to a coordinated rapid antigen testing campaign targeting the residence hall occupants and the identification and isolation of infected individuals. With knowledge of the number of individuals testing positive for COVID-19, fecal shedding rates were estimated to range from 3.70 log10 gc ‧ g feces-1 to 5.94 log10 gc ‧ g feces-1. These results reinforce the efficacy of wastewater surveillance as an early indicator of infection in congregate living settings. Detections can trigger public health measures ranging from enhanced communications to targeted coordinated testing and quarantine.

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma.

Intratumoral heterogeneity (ITH)-defined as genetic and cellular diversity within a tumor-is linked to failure of immunotherapy and an inferior anti-tumor immune response. The underlying mechanism of this association is unknown. To address this question, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population, labeled with YFP and RFP tags respectively to enable precise spatial tracking. The resulting mixed-population tumors exhibited distinct regions comprised of YFP+ (hot) cells, RFP+ (cold) cells, or a mixture. We found that tumor regions occupied by hot tumor cells (YFP+) harbored more total T cells and a higher frequency of Th1 cells and IFNγ+ CD8 T cells compared to regions occupied by cold tumor cells (RFP+), whereas immunosuppressive macrophages showed the opposite spatial pattern. We identified the chemokine CX3CL1, produced at higher levels by our cold tumors, as a mediator of intratumoral macrophage accumulation, particularly immunosuppressive CD206Hi macrophages. Furthermore, we examined the response of heterogeneous tumors to a therapeutic combination of PD-1 blockade and CD40 agonist on a region-by-region basis. While the combination successfully increases Th1 abundance in "cold" tumor regions, it fails to bring overall T cell activity to the same level as seen in "hot" regions. The presence of the "cold" cells thus ultimately leads to a failure of the therapy to induce tumor rejection. Collectively, our results demonstrate that the organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

A Child with an Esophageal Web Treated with Functional Lumen Imaging Probe.

A 14-month-old male presented to the emergency department with a 4-day history of vomiting after the intake of liquids or solids. During the admission, imaging studies revealed an esophageal web, a form of congenital esophageal stenosis. He was treated with a combination of Endoluminal Functional Lumen Imaging Probe (EndoFLIP) and controlled radial expansion (CRE) balloon dilation, followed by EndoFLIP and EsoFLIP dilation 1 month later. The patient's vomiting resolved after treatment, and he was able to gain weight. This report describes one of the first cases of applying EndoFLIP and EsoFLIP to treat an esophageal web in a pediatric patient.

Esophageal dilation with EsoFLIP is faster than CRE balloon dilation combined with EndoFLIP in children.

BACKGROUND: Controlled radial expansion (CRE) balloon dilators are traditionally used to dilate esophageal strictures during an esophagogastroduodenoscopy (EGD). EndoFLIP is a diagnostic tool used during an EGD to measure important parameters of the gastrointestinal lumen, capable of assessing treatment before and after dilation. EsoFLIP is a related device that combines a balloon dilator with high-resolution impedance planimetry to provide some of the luminal parameters in real time during dilation. We sought to compare procedure time, fluoroscopy time, and safety profile of esophageal dilation using either CRE balloon dilation combined with EndoFLIP (E + CRE) versus EsoFLIP alone. METHODS: A single-center retrospective review was performed to identify patients ≤ 21 years of age who underwent an EGD with biopsy and esophageal stricture dilation using E + CRE or EsoFLIP between October 2017 and May 2022. RESULTS: Twenty-nine EGDs with esophageal stricture dilation were performed in 23 patients (19 E + CRE and 10 EsoFLIP). The two groups did not differ in age, gender, race, chief complaint, type of esophageal stricture, or history of prior gastrointestinal procedures (all p > 0.05). The most common medical history in the E + CRE and EsoFLIP groups were eosinophilic esophagitis and epidermolysis bullosa, respectively. Median procedures times were shorter in the EsoFLIP cohort compared to E + CRE balloon dilation (40.5 min [IQR 23-57 min] for the EsoFLIP group; 64 min [IQR 51-77 min] for the E + CRE group; p < 0.01). Median fluoroscopy times were also shorter for patients who underwent EsoFLIP (0.16 min [IQR 0-0.30 min] for EsoFLIP dilation; 0.30 min [IQR 0.23-0.55] for the E + CRE group; p = 0.003). There were no complications or unplanned hospitalizations in either group. CONCLUSION: EsoFLIP dilation of esophageal strictures was faster and required less fluoroscopy than CRE balloon dilation combined with EndoFLIP in children, while being equally as safe. Prospective studies are needed to further compare the two modalities.

Corrigendum: Structure-guided design of a potent Clostridioides difficile toxin A inhibitor.

[This corrects the article DOI: 10.3389/fmicb.2023.1110541.].

Single-level posterior cervical foraminotomy associated with increased incidence of early postoperative wound infection rates relative to anterior cervical discectomy with fusion and cervical disc arthroplasty.

Background: To date, there are no studies comparing perioperative outcomes of cervical radiculopathy patients managed by anterior cervical discectomy with fusion (ACDF), cervical disc arthroplasty (CDA), or posterior cervical foraminotomy (PCF). To assess if there were differences in perioperative outcomes between cervical radiculopathy patients who can be appropriately treated with ACDF, CDA, or PCF. Methods: Patients diagnosed with cervical radiculopathy who underwent a single-level ACDF, CDA, or PCF between 2012 and 2019 were retrospectively identified from the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database using current procedural terminology (CPT) codes. Patients were subsequently stratified into those who underwent ACDF, CDA, or PCF, and propensity score-matched to adjust for differences in patient demographics/characteristics. Differences were assessed in terms of operative time, healthcare utilization metrics (reoperations, readmissions, lengths-of-stay), as well as medical and surgical complications. Results: A total of 18,614 cervical radiculopathy patients undergoing surgery were identified (ACDF: n=15,862; CDA: n=1,731; PCF: n=1,021). After 1:1 propensity score matching (n=535 each), there were no differences in characteristics in patients undergoing ACDF, CDA, or PCF (P>0.05). PCF patients had statistically higher rates of reoperation (2.1%) than ACDF (0.4%), CDA (0.6%) patients (P=0.010). PCF patients also experienced higher rates of superficial infection (P=0.001), and deep infection (P=0.007), relative to ACDF and CDA patients. There were no other significant differences in medical/surgical complications between the ACDF, CDA, or PCF patients. Conclusions: Cervical radiculopathy patients undergoing PCF are associated with higher rates of perioperative infection and overall reoperation than ACDF or CDA. Further research is required to elucidate the mechanism behind this association.

Endoscopy in Pediatric Eosinophilic Esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergen-mediated clinicopathologic condition that currently requires esophagogastroduodenoscopy with biopsies and histologic evaluation to diagnose and monitor its progress. This state-of-the art review outlines the pathophysiology of EoE, reviews the application of endoscopy as a diagnostic and therapeutic tool, and discusses potential complications related to therapeutic endoscopic interventions. It also introduces recent innovations that can enhance the endoscopist's ability to diagnose and monitor EoE with minimally invasive procedures and perform therapeutic maneuvers more safely and effectively.