Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

RNA electroelution: Comparing two electroeluter models.

RNA represents a vibrant area of research and many studies use techniques that require large amounts of purified RNA. One common purification method involves slicing a section of a polyacrylamide gel containing the RNA of interest and eluting the RNA out of the gel using electroelution. Various electroeluter models are available but sometimes a given model becomes discontinued, compelling researchers to choose a different model. Here, we have compared two electroeluters with different chamber designs for their ability to recover RNA from gel pieces. Our results show that both electroeluters are effective and recover comparable amounts of purified RNA.

Chloral Hydrate Sedation in a Dexmedetomidine Era.

Brief Overview: The use of chloral hydrate as the primary sedation agent has declined across the nation after commercial production of the liquid formulation ceased. Although alternative sedatives have gained popularity, some pharmacies have continued to provide oral chloral hydrate by compounding it from raw ingredients. Thus, oral chloral hydrate use has continued in children despite the availability of alternative effective agents. Objective: The purpose of this investigation was to evaluate institutional chloral hydrate utilization as the primary agent for procedural sedation. Design/Methods: We conducted a retrospective study of patients given chloral hydrate for procedural sedation from October 2010 to December 2016. The hospital pharmacy database of chloral hydrate use at our 2 free-standing children's hospitals was reviewed and matched to procedure billing data. Results: There were 5874 chloral hydrate administrations for procedural sedation during the study period. The highest rates of use occurred in 2014, when there were 1420 chloral hydrate orders within our hospital. The large majority of sedations were for cardiac studies/procedures (n = 4250, 72.4%). Conclusions: Despite significant declines in use of chloral hydrate for procedural sedation across the country, local utilization of oral chloral hydrate remains high. Recent declines may be due to high-use clinical sites transitioning to alternative sedatives such as intranasal dexmedetomidine.

A prospective, randomized, double-blind trial of intranasal dexmedetomidine and oral chloral hydrate for sedated auditory brainstem response (ABR) testing.

BACKGROUND: Dexmedetomidine is increasingly used by various routes for pediatric sedation. However, there are few randomized controlled trials comparing the efficacy of dexmedetomidine to other commonly used sedatives. AIM: To compare the efficacy of sedation with intranasal dexmedetomidine to oral chloral hydrate for auditory brainstem response (ABR) testing. METHODS: In this double-blind, double-dummy study, children undergoing ABR testing were randomized to receive intranasal dexmedetomidine 3 mcg · kg(-1) plus oral placebo (Group IN DEX) or oral chloral hydrate 50 mg · kg(-1) plus intranasal saline placebo (Group CH). We recorded demographic data, times from sedative administration to start and completion of testing, quality of sedation, occurrence of predefined adverse events, discharge times, and return to baseline activity on the day of testing. RESULTS: Testing completion rates with a single dose of medication were higher in the IN DEX group (89% vs 66% for CH, odds ratio with 95% confidence intervals 4.04 [1.3-12.6], P = 0.018). The median [95% CI)] time to successful testing start was shorter (25 [20-29] min vs 30 [20-49] min for IN DEX and CH, respectively, log rank test P = 0.02) and the proportion of children whose parents reported a return to baseline activity on the day of testing was greater for the IN DEX than the CH group (89% vs 64%, OR [95% CI] 4.71 [1.34-16.6], P = 0.02). There were no major adverse events in either group and no significant differences in the incidence of minor events. CONCLUSION: Intranasal dexmedetomidine is an effective alternative to oral chloral hydrate sedation for ABR testing, with the advantages of a higher incidence of testing completion with a single dose, shorter time to desired sedation level, and with significantly more patients reported to return to baseline activity on the same day.

Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer.

Background: Platinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance. Methods: TCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models. Results: In response to increased fibronectin (FN) secretion and integrin ß1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and showed a strong correlation with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/ß-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice. Conclusions: This "outside-in" signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may represent a new therapeutic strategy to eradicate OCSCs and improve patient outcomes.

Integrin-linked kinase-frizzled 7 interaction maintains cancer stem cells to drive platinum resistance in ovarian cancer.

BACKGROUND: Platinum-based chemotherapy regimens are a mainstay in the management of ovarian cancer (OC), but emergence of chemoresistance poses a significant clinical challenge. The persistence of ovarian cancer stem cells (OCSCs) at the end of primary treatment contributes to disease recurrence. Here, we hypothesized that the extracellular matrix protects CSCs during chemotherapy and supports their tumorigenic functions by activating integrin-linked kinase (ILK), a key enzyme in drug resistance. METHODS: TCGA datasets and OC models were investigated using an integrated proteomic and gene expression analysis and examined ILK for correlations with chemoresistance pathways and clinical outcomes. Canonical Wnt pathway components, pro-survival signaling, and stemness were examined using OC models. To investigate the role of ILK in the OCSC-phenotype, a novel pharmacological inhibitor of ILK in combination with carboplatin was utilized in vitro and in vivo OC models. RESULTS: In response to increased fibronectin secretion and integrin ß1 clustering, aberrant ILK activation supported the OCSC phenotype, contributing to OC spheroid proliferation and reduced response to platinum treatment. Complexes formed by ILK with the Wnt receptor frizzled 7 (Fzd7) were detected in tumors and correlated with metastatic progression. Moreover, TCGA datasets confirmed that combined expression of ILK and Fzd7 in high grade serous ovarian tumors is correlated with reduced response to chemotherapy and poor patient outcomes. Mechanistically, interaction of ILK with Fzd7 increased the response to Wnt ligands, thereby amplifying the stemness-associated Wnt/ß-catenin signaling. Notably, preclinical studies showed that the novel ILK inhibitor compound 22 (cpd-22) alone disrupted ILK interaction with Fzd7 and CSC proliferation as spheroids. Furthermore, when combined with carboplatin, this disruption led to sustained AKT inhibition, apoptotic damage in OCSCs and reduced tumorigenicity in mice. CONCLUSIONS: This "outside-in" signaling mechanism is potentially actionable, and combined targeting of ILK-Fzd7 may lead to new therapeutic approaches to eradicate OCSCs and improve patient outcomes.

Retrospective observational study of chloral hydrate use in mechanically-ventilated pediatric intensive care unit (PICU) patients 2012-2017.

Introduction: Chloral hydrate (CH) has long been utilized as a pediatric procedural sedation agent. However, very little is published describing CH use in a pediatric intensive care unit (PICU) setting. The aim of this retrospective observational cohort study was to investigate and describe the use of CH in mechanically-ventilated, critically ill children at a large pediatric tertiary referral hospital. Methods: Data were extracted from the hospital electronic medical record and a locally maintained registry of all children admitted to the PICU between 2012 and 2017. Patients admitted to the cardiovascular ICU were not included in this review. The clinical and pharmacy data for 3806 consecutive PICU admissions of mechanically-ventilated, critically ill children were examined. Results: 283 admissions received CH during their first ICU stay. CH-exposed children were younger (16 months vs. 35 months, p < 0.001), the median total dose of CH (indexed to duration of ventilation) was 11 mg/kg/day, the median time to first CH dose was 3 days and more CH doses were administered at night (1112 vs. 958, p < 0.001). We constructed a propensity score to adjust for the differences in patients with and without CH exposure using logistic regression including variables of age, sex, diagnosis, and PRISM3 score. After adjustment, the median length of mechanical ventilation was 5 days longer in the CH-exposed group (95% Confidence Interval [CI] 4-6) compared to unexposed CH patients. Similarly, the median length of ICU duration was 9.4 days longer (95% CI 7.1-11.6) and median length of hospital admission duration was 13.2 days longer (95% CI 7.8-18.6) in CH-exposed patients compared to CH-non-exposed. After adjustment, CH-exposed patients had a 9% higher median exposure to HFOV (95% CI 3.9-14.6), but did not have higher median exposures to new tracheostomy (95% CI -0.4-2.2) or ECMO (95% CI -0.2-5.0). Discussion: As part of an extended sedation regimen in mechanically-ventilated and critically ill children, CH is associated with somewhat higher complexity of illness and longer ICU durations.

Clinical Outcomes Associated With Intranasal Dexmedetomidine Sedation in Children.

OBJECTIVES: We aimed to describe the quality of sedation and additional clinically-meaningful outcomes associated with different intranasal dexmedetomidine-based regimens in children undergoing nonpainful procedures. METHODS: A multicenter prospective observational study of children aged 2 months to 17 years undergoing intranasal dexmedetomidine sedation for MRI, auditory brainstem response testing, echocardiogram, EEG, or computed tomography scan. Regimens varied by dose of dexmedetomidine and use of adjunct sedatives. Quality of sedation was assessed using the Pediatric Sedation State Scale and by determining proportion of children who achieved an acceptable sedation state. Procedure completion, time-based outcomes, and adverse events were assessed. RESULTS: We enrolled 578 children across 7 sites. Median age was 2.5 years (interquartile range 1.6-3) and 37.5% were female. The most common procedures were auditory brainstem response testing (54.3%) and MRI (22.8%). The most common dose was 3 to 3.9 mcg/kg (55%), with 25.1% and 14.2% of children receiving oral or intranasal midazolam, respectively. Acceptable sedation state and procedure completion was achieved in 81.1% and 91.3% of children, and mean time to onset of sedation and total sedation time were 32.3 and 114.8 minutes, respectively. Twelve interventions were performed in 10 patients in response to an event; no patients required a serious airway, breathing, or cardiovascular intervention. CONCLUSIONS: Intranasal dexmedetomidine-based regimens can achieve acceptable sedation states and high rates of procedure completion in children undergoing sedation for nonpainful procedures. Our findings delineate clinical outcomes associated with intranasal dexmedetomidine-based sedation that can be used to guide the implementation and optimization of such regimens.

SPAG17 mediates nuclear translocation of protamines during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Outside the Box: An Experienced Registered Nurse Orientation Redesign.

Nurse educators play an important role in implementing changes within hospital orientation, supporting a learner-focused orientation. Utilizing different learning styles and delivery methods, an orientation can promote an environment where registered nurses are active participants and guide the learning. This article discusses the process of incorporating multiple learning styles and modalities into an experienced registered nurse orientation program, creating a learner-centered environment promoting engagement, increased satisfaction, and retention of experienced staff.

Outcomes of Non-anesthesiologist-Administered Propofol in Pediatric Gastroenterology Procedures.

Background and Aims: Non-anesthesiologist-administered propofol (NAAP) has been found to have an acceptable safety profile in adult endoscopy, but its use remains controversial and pediatric data is limited. Our aim was to examine the safety and efficacy of NAAP provided by pediatric hospitalists in pediatric endoscopy. Methods: We retrospectively reviewed 929 esophagogastroduodenoscopy (EGD), colonoscopy, and combined EGD/colonoscopy cases in children aged 5-20 years between April 2015 and December 2016 at a large children's hospital. We analyzed the data for adverse events in relation to demographics and anthropometrics, American Society of Anesthesiologists physical classification score, presence of a trainee, comorbid conditions, and procedure time. Results: A total of 929 cases were included of which 496 (53%) were completed with NAAP. Seventeen (3.4%) of NAAP cases had an adverse event including the following: 12 cases of hypoxia, 2 cardiac, and 3 gastrointestinal adverse events. General anesthesia cases had 62 (14.3%) adverse events including the following: 54 cases of hypoxia, 1 cardiac, 7 gastrointestinal, and 1 urologic adverse event. No adverse events in either group required major resuscitation. NAAP vs. general anesthesia had a lower overall adverse event rate (3.4 vs. 14.3%, p < 0.0004) and respiratory adverse event rate (2.4% vs. 12.5%, p < 0.0004). Overall, cardiac and gastrointestinal adverse event rates between the two groups were comparable. When accounting for all captured factors via logistic regression, both younger age (P < 0.001) and general anesthesia (P < 0.0001) remained risk factors for an adverse event. Conclusion: The overall adverse event rate of NAAP was low (3.4%) with none requiring major resuscitation or hospitalization. This is comparable to studies of NAAP in adult endoscopy and suggests that NAAP provided by pediatric hospitalists has an acceptable safety profile.

Retrospective Comparison of Intranasal Dexmedetomidine and Oral Chloral Hydrate for Sedated Auditory Brainstem Response Exams.

OBJECTIVE: Satisfactory conditions for auditory brainstem responses (ABR) screening tests for pediatric hearing loss are usually achieved with oral chloral hydrate (CH) sedation. When the US manufacture of this drug was discontinued for business reasons, we developed an alternative sedation regimen using intranasal dexmedetomidine (IN DEX) 4 µg/kg. This institutional review board-approved retrospective study compared the efficacy and adverse effect profiles of these 2 sedative regimens. METHODS: Medical records of children receiving oral CH or IN DEX for ABR were surveyed for demographic data and times from sedative administration to start and completion of the ABR procedure and recovery times. We also noted if the examination was completed with or without interruptions, failed for inadequate sedation, and if predefined cardiorespiratory adverse events occurred. RESULTS: In the IN DEX cohort, the examination could be completed more frequently with a single dose of medication (P = .002). Satisfactory sedation in these patients permitted an earlier start of both the ABR examination and recovery to the awake status (P < .001 and < .045, respectively). Hypoxia requiring oxygen therapy was more frequent in the CH group. CONCLUSIONS: This retrospective study found that IN DEX provides effective sedation for ABR examinations, with the benefits of an ability to begin the test sooner and complete the examination with a single dose, in addition to a decreased incidence of hypoxemia. A randomized controlled trial should test the hypothesis that the IN DEX technique is superior to the well-established standard oral CH regimen.