Presence of Microscopic Hematuria Does Not Predict Clinically Important Intra-Abdominal Injury in Children.

OBJECTIVE: Screening for blunt intra-abdominal injury in children often includes directed laboratory evaluation that guides need for computed tomography. We sought to evaluate the use of urinalysis in identifying patients with clinically important intraabdominal injury ( ci -IAI). METHODS: A retrospective chart review was performed for all patients less than 18 years who presented with blunt mechanisms at a level I trauma center between 2016 and 2019. Exclusion criteria included transfer from an outside facility, physical abuse, and death within thirty minutes of arrival. Demographics, physical exam findings, serum chemistries, urinalysis, and imaging were reviewed. Clinically important intraabdominal injury was defined as injury requiring ≥2 nights admission, blood transfusion, angiography with embolization, or therapeutic surgery. RESULTS: Two hundred forty patients were identified. One hundred sixty-five had a completed urinalysis. For all patients an abnormal chemistry panel and abnormal physical exam had a sensitivity of 88.9% and a negative predictive value of 99.3%. Nine patients had a ci -IAI. Patients with a ci -IAI were more likely to have abdominal pain, tenderness on exam, and elevated hepatic enzymes. When patients were stratified by the presence of an abnormal chemistry or physical exam with or without microscopic hematuria, urinalysis did not improve the ability to identify patients with a ci -IAI. In fact, presence of microscopic hematuria increased the rate of false positives by 12%. CONCLUSIONS: Microscopic hematuria was not a useful marker for ci -IAI and may lead to falsely assuming a more serious injury.

Pediatric Train Injuries: A 10-Year Review From the Pennsylvania Trauma Outcomes Study Database.

OBJECTIVES: Trains can cause severe injuries in pediatric patients requiring significant resource utilization. We sought to review train injuries in Pennsylvania to determine the burden of these injuries on the pediatric trauma system. METHODS: We queried the Pennsylvania Trauma Outcomes Study Database to identify patients younger than 18 years injured by trains between 2007 and 2016. Demographics, hospital course, outcomes, and resource utilization were reviewed. RESULTS: Thirty-five children from 17 Pennsylvania counties were included. Three counties accounted for 48.6% of injured children. The median age was 15.0 years, and most patients were White (60.0%) and male (77.1%). The median length of stay was 8.0 days and overall mortality 8.6%. Intensive care unit admission was required for 65.7%. The median Injury Severity and Functional Status at Discharge scores were 14.0 and 18.0, respectively. Major orthopedic injuries (fracture or amputation) were the most common (57.1%) followed by traumatic brain injury (45.7%), pneumothorax (14.3%), and solid organ injury (14.3%). Operative management was common with 65.7% undergoing surgery. CONCLUSIONS: Injuries caused by trains can be severe and are most commonly orthopedic or traumatic brain injuries. Targeted safety interventions may be possible given the common mechanisms and geographic clustering of these injuries.

Adherence to PECARN criteria in children transferred to a pediatric trauma center: An opportunity for improvement?

BACKGROUND: The Pediatric Emergency Care Applied Research Network (PECARN) criteria identify children at low risk of clinically important traumatic brain injury (ciTBI) in whom CT head (CTH) is unnecessary. We assessed compliance with PECARN at outside hospitals (OSH) among children transferred to our pediatric trauma center. METHODS: Patients <18 years transferred between May 2016 and December 2018 undergoing CTH at an OSH were reviewed. A ciTBI was defined as one requiring hospitalization ≥2 midnights, intubation >24 h, neurosurgical intervention, or causing death. RESULTS: 202 children were transferred after CTH. 53 were excluded for incomplete records (16), suspected abuse (33), or penetrating injury (4). Of the 149 included children, PECARN recommended CTH in 39 (26.2%), shared decision making in 79 (53.0%), and no imaging in 31 (20.8%). 26 children (17.4%) had a radiographic traumatic brain injury (rTBI) while only 6 (4.0%) had ciTBIs. Of those with ciTBIs, PECARN recommended CTH in 4 and shared decision making in 2. No child in whom CTH was not recommended had a ciTBI. 45 (30.2%) children had isolated extracranial injuries requiring transfer and 83 (55.7%) were transferred despite normal CTHs and no associated injuries. 2 (1.3%) children underwent non-emergent surgery for ciTBI. CONCLUSIONS: Compliance with PECARN was low among referring facilities with nearly 75% of CTHs being potentially avoidable with proper adherence and parental counseling. Deferring imaging until after transfer appears safe as no child underwent emergent intervention upon arrival. Early transfer and improved compliance with PECARN may reduce the number of CTHs performed.

A Standardized Protocol for Cervical Spine Evaluation in Children Reduces Imaging Utilization: A Pilot Study of the Pediatric Cervical Spine Clearance Working Group Protocol.

BACKGROUND: Cervical spine injuries (CSI) have the potential to cause severe morbidity in children. Multiple imaging studies are used during evaluation of CSIs but come at a cost, both financially and in radiation exposure. To reduce resource utilization and radiation exposure, we implemented the Pediatric Cervical Spine Clearance Working Group (PCSCWG) standardized protocol (SP) for evaluating CSIs in children. METHODS: Children below 18 years old presenting with concern for CSI at a level 1 pediatric trauma center were reviewed before (July 2015 to May 2016) and after (November 2017 to June 2018) protocol implementation. Demographics, injuries, and imaging utilization were extracted. The primary outcomes were the proportion of patients cleared with clinical exam, and the proportion undergoing x-ray, computed tomography, or magnetic resonance image. The secondary outcome was the estimated difference in imaging charges based on the annual reduction in radiographic studies. RESULTS: During the study 359 children were evaluated for CSIs (248 pre-SP, 111 post-SP). Patients were similar with respect to age, injury severity score, and mechanism of injury. Protocol adherence was 87.4%. The prevalence of CSI was similar in the preprotocol and postprotocol cohorts (2.8% vs. 1.8%, P=0.567). Children treated after protocol implementation were significantly more likely to be cleared by clinical exam (15.3% vs. 43.2%, P<0.001). Significantly fewer children had x-rays (70.2% vs. 55.0%, P=0.005) and computed tomography scans (14.5% vs. 5.4%, P=0.013) in the postprotocol period. There was no difference in the utilization of magnetic resonance image (6.9% vs. 7.2%, P=0.904) or the proportion of children discharged with a cervical collar (10.1% vs. 12.6%, P=0.476). No patients in either group were found to have a previously undiagnosed injury at follow-up. The reduction in radiographic studies translates to an estimated annual reduction in imaging charges of $396,476. CONCLUSIONS: The PCSCWG protocol for evaluating CSIs reduced the number of radiographic studies performed and estimated imaging charges while reliably identifying CSIs.

No Surgical Innovation Without Evaluation: Evolution and Further Development of the IDEAL Framework and Recommendations.

OBJECTIVE: To update, clarify, and extend IDEAL concepts and recommendations. BACKGROUND: New surgical procedures, devices, and other complex interventions need robust evaluation for safety, efficacy, and effectiveness. Unlike new medicines, there is no internationally agreed evaluation pathway for generating and analyzing data throughout the life cycle of surgical innovations. The IDEAL Framework and Recommendations were designed to provide this pathway and they have been used increasingly since their introduction in 2009. Based on a Delphi survey, expert workshop and major discussions during IDEAL conferences held in Oxford (2016) and New York (2017), this article updates and extends the IDEAL Recommendations, identifies areas for future research, and discusses the ethical problems faced by investigators at each IDEAL stage. METHODS: The IDEAL Framework describes 5 stages of evolution for new surgical therapeutic interventions-Idea, Development, Exploration, Assessment, and Long-term Study. This comprehensive update proposes several modifications. First, a "Pre-IDEAL" stage describing preclinical studies has been added. Second we discuss potential adaptations to expand the scope of IDEAL (originally designed for surgical procedures) to accommodate therapeutic devices, through an IDEAL-D variant. Third, we explicitly recognise the value of comprehensive data collection through registries at all stages in the Framework and fourth, we examine the ethical issues that arise at each stage of IDEAL and underpin the recommendations. The Recommendations for each stage are reviewed, clarified and additional detail added. CONCLUSIONS: The intention of this article is to widen the practical use of IDEAL by clarifying the rationale for and practical details of the Recommendations. Additional research based on the experience of implementing these Recommendations is needed to further improve them.

Progress in clinical research in surgery and IDEAL.

The quality of clinical research in surgery has long attracted criticism. High-quality randomised trials have proved difficult to undertake in surgery, and many surgical treatments have therefore been adopted without adequate supporting evidence of efficacy and safety. This evidence deficit can adversely affect research funding and reimbursement decisions, lead to slow adoption of innovations, and permit widespread adoption of procedures that offer no benefit, or cause harm. Improvement in the quality of surgical evidence would therefore be valuable. The Idea, Development, Exploration, Assessment, and Long-term Follow-up (IDEAL) Framework and Recommendations specify desirable qualities for surgical studies, and outline an integrated evaluation pathway for surgery, and similar complex interventions. We used the IDEAL Recommendations to assess methodological progress in surgical research over time, assessed the uptake and influence of IDEAL, and identified the challenges to further methodological progress. Comparing studies from the periods 2000-04 and 2010-14, we noted apparent improvement in the use of standard outcome measures, adoption of Consolidated Standards of Reporting Trials (CONSORT) standards, and assessment of the quality of surgery and of learning curves, but no progress in the use of qualitative research or reporting of modifications during procedure development. Better education about research, integration of rigorous evaluation into routine practice and training, and linkage of such work to awards systems could foster further improvements in surgical evidence. IDEAL has probably contributed only slightly to the improvements described to date, but its uptake is accelerating rapidly. The need for the integrated evaluation template IDEAL offers for surgery and other complex treatments is becoming more widely accepted.

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice.

The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.

A before-after study of multidisciplinary Out-of-Hours handover: combining management and frontline efforts to create sustainable improvement.

OBJECTIVE: The importance of implementation strategy in systems improvement is increasingly recognized and both 'bottom-up' and 'top-down' approaches have significant barriers. A trial of a combined approach involving frontline and managerial staff therefore seems merited. We attempted to improve handover using a Human Factors-based approach integrated with a combined 'top and bottom' implementation strategy. DESIGN: A before-after study was conducted across 9 months. SETTING: The study was set in a 236 bed district general hospital. PARTICIPANTS: Participants included any member of staff involved in Out of Hours handover. INTERVENTION: Existing processes were analysed using Human Factors methods. Changes made were based on this analysis and developed via facilitation between management and frontline staff. These included creating a single multidisciplinary handover, changing the venue, standardizing the meeting structure, developing an standard operating procedure for identifying unwell patients for handover and creating a clinical coordinator role. MAIN OUTCOME MEASURES: Meeting attendance, duration, start time efficiency, the type of patients handed over and the transfer of important information were measured pre- and post-intervention. RESULTS: We found improvement in handover start time (P = 0.002, r = 0) and multidisciplinary participation (P = 0.002, r = -0.534). Handover of unwell patients improved, but not significantly. Communication of plan (P < 0.001, r = 0.14) and pending tasks (P < 0.001, r = 0.30) improved, but diagnosis (P = 0.233, r = -0.05), history (P = 0.482, r = -0.03) and comorbidities (P = 0.19, r = -0.05) did not. CONCLUSIONS: The changes produced greater multidisciplinary participation, a broader focus and improved communication of plans and tasks outstanding. The 'top and bottom' implementation approach appeared valuable. Management involvement was essential for significant changes, while frontline staff involvement facilitated the design of context-specific practical solutions with staff buy-in.

CD8⁺ T Cell-Independent Immune-Mediated Mechanisms of Anti-Tumor Activity.

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Cancer immunotherapy seeks to recruit an effective immune response to eliminate tumor cells. To date, cancer vaccines have shown only limited effectiveness because of our incomplete understanding of the necessary effector cells and mechanisms that yield efficient tumor clearance. CD8+ T cell cytotoxic activity has long been proposed as the primary effector function necessary for tumor regression. However, there is increasing evidence that indicates that components of the immune system other than CD8+ T cells play important roles in tumor eradication and control. The following review should provide an understanding of the mechanisms involved in an effective antitumor response to guide future therapeutic designs. The information provided suggests an alternate means of effective tumor clearance in malignant glioma to the canonical CD8+ cytotoxic T cell mechanism.

Vaccine injection site matters: qualitative and quantitative defects in CD8 T cells primed as a function of proximity to the tumor in a murine glioma model.

Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with tumor-associated Ags efficiently primes T cells to clear gliomas. In clinical trials, cancer vaccines have been less effective at priming T cells and extending survival. Generalized immune suppression in the tumor draining lymph nodes has been documented in multiple cancers. However, a systematic analysis of how vaccination at various distances from the tumor (closest to farthest) has not been reported. We investigated how the injection site chosen for vaccination dictates CD8 T cell priming and survival in an OVA-transfected murine glioma model. Glioma-bearing mice were vaccinated with Poly:ICLC plus OVA protein in the neck, hind leg, or foreleg for drainage into the cervical, inguinal, or axillary lymph nodes, respectively. OVA-specific CD8 T cell number, TCR affinity, effector function, and infiltration into the brain decreased as the vaccination site approached the tumor. These effects were dependent on the presence of the tumor, because injection site did not appreciably affect CD8 T cell priming in tumor-free mice. Our data suggest the site of vaccination can greatly impact the effectiveness of cancer vaccines. Considering that previous and ongoing clinical trials have used a variety of injection sites, vaccination site is potentially a critical aspect of study design that is being overlooked.

Minicircle DNA is superior to plasmid DNA in eliciting antigen-specific CD8+ T-cell responses.

Clinical trials reveal that plasmid DNA (pDNA)-based gene delivery must be improved to realize its potential to treat human disease. Current pDNA platforms suffer from brief transgene expression, primarily due to the spread of transcriptionally repressive chromatin initially deposited on plasmid bacterial backbone sequences. Minicircle (MC) DNA lacks plasmid backbone sequences and correspondingly confers higher levels of sustained transgene expression upon delivery, accounting for its success in preclinical gene therapy models. In this study, we show for the first time that MC DNA also functions as a vaccine platform. We used a luciferase reporter transgene to demonstrate that intradermal delivery of MC DNA, relative to pDNA, resulted in significantly higher and persistent levels of luciferase expression in mouse skin. Next, we immunized mice intradermally with DNA encoding a peptide that, when presented by the appropriate major histocompatibility complex class I molecule, was recognized by endogenous CD8(+) T cells. Finally, immunization with peptide-encoding MC DNA, but not the corresponding full-length (FL) pDNA, conferred significant protection in mice challenged with Listeria monocytogenes expressing the model peptide. Together, our results suggest intradermal delivery of MC DNA may prove more efficacious for prophylaxis than traditional pDNA vaccines.

Management and Outcomes of Pediatric Lymphatic Malformations: A Systematic Review From the APSA Outcomes and Evidence-Based Practice Committee.

BACKGROUND: Significant variation in management strategies for lymphatic malformations (LMs) in children persists. The goal of this systematic review is to summarize outcomes for medical therapy, sclerotherapy, and surgery, and to provide evidence-based recommendations regarding the treatment. METHODS: Three questions regarding LM management were generated according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Publicly available databases were queried to identify articles published from January 1, 1990, to December 31, 2021. A consensus statement of recommendations was generated in response to each question. RESULTS: The initial search identified 9326 abstracts, each reviewed by two authors. A total of 600 abstracts met selection criteria for full manuscript review with 202 subsequently utilized for extraction of data. Medical therapy, such as sirolimus, can be used as an adjunct with percutaneous treatments or surgery, or for extensive LM. Sclerotherapy can achieve partial or complete response in over 90% of patients and is most effective for macrocystic lesions. Depending on the size, extent, and location of the malformation, surgery can be considered. CONCLUSION: Evidence supporting best practices for the safety and effectiveness of management for LMs is currently of moderate quality. Many patients benefit from multi-modal treatment determined by the extent and type of LM. A multidisciplinary approach is recommended to determine the optimal individualized treatment for each patient.

Evaluation and Management of Biliary Dyskinesia in Children and Adolescents: A Systematic Review From the APSA Outcomes and Evidence-Based Committee.

INTRODUCTION: The diagnosis and management of biliary dyskinesia in children and adolescents remains variable and controversial. The American Pediatric Surgical Association Outcomes and Evidence-Based Practice Committee (APSA OEBP) performed a systematic review of the literature to develop evidence-based recommendations. METHODS: Through an iterative process, the membership of the APSA OEBP developed five a priori questions focused on diagnostic criteria, indications for cholecystectomy, short and long-term outcomes, predictors of success/benefit, and outcomes of medical management. A systematic review was conducted, and articles were selected for review following Preferred Reporting Items for Systematic Review and Meta-analyses (PRISMA) guidelines. Risk of bias was assessed using Methodologic Index for Non-Randomized Studies (MINORS) criteria. The Oxford Levels of Evidence and Grades of Recommendation were utilized. RESULTS: The diagnostic criteria for biliary dyskinesia in children and adolescents are not clearly defined. Cholecystectomy may provide long-term partial or complete relief in some patients; however, there are no reliable predictors of symptom relief. Some patients may experience resolution of symptoms with non-operative management. CONCLUSIONS: Pediatric biliary dyskinesia remains an ill-defined clinical entity. Pediatric-specific guidelines are necessary to better characterize the condition, guide work-up, and provide management recommendations. Prospective studies are necessary to more reliably identify patients who may benefit from cholecystectomy. LEVEL OF EVIDENCE: Level 3-4. TYPE OF STUDY: Systematic Review of Level 3-4 Studies.

Obesity is not associated with increased resource utilization or morbidity in patients undergoing appendectomy.

BACKGROUND: Obesity is a growing public health concern that places patients at risk of morbidity and mortality following surgery. We sought to determine whether obesity influences our resource utilization and postoperative outcomes for patients who present with appendicitis. METHODS: Charts were reviewed for patients age 1-18 years identified from a prospective registry who presented with a diagnosis of appendicitis from 2017 to 2020. Patients who underwent appendectomy were eligible. Charts were reviewed for demographics, imaging studies, laboratory studies, length of stay, operative times and thirty-day postoperative adverse events defined as return to the emergency room, re-admission, postoperative abscess or return to the operating room. A multivariate logistic regression analysis was performed to identify differences in resource utilization and outcome. RESULTS: A total of 451 patients were identified. There were 126 obese patients (27.9%). Obese patients were not more likely to present with perforated appendicitis and were not more likely to undergo computed tomography scans. All patients underwent laparoscopic appendectomy. Although intraoperative times were significantly longer for Black patients and older patients, BMI did not influence length of surgery. Length of stay was significantly higher for younger patients (p = 0.019). Adverse events were seen in 38 patients (8.4%). There was no association between BMI and adverse events. CONCLUSIONS: Within our standardized management pathway, obesity does not influence management or patient outcomes for the treatment of appendicitis. Furthermore, obese patients did not require additional resource utilization. LEVEL OF EVIDENCE: III.

Derivation and Validation of a Machine Learning Algorithm for Predicting Venous Thromboembolism in Injured Children.

BACKGROUND: Venous thromboembolism (VTE) causes significant morbidity in pediatric trauma patients. We applied machine learning algorithms to the Trauma Quality Improvement Program (TQIP) database to develop and validate a risk prediction model for VTE in injured children. METHODS: Patients ≤18 years were identified from TQIP (2017-2019, n = 383,814). Those administered VTE prophylaxis ≤24 h and missing the outcome (VTE) were removed (n = 347,576). Feature selection identified 15 predictors: intubation, need for supplemental oxygen, spinal injury, pelvic fractures, multiple long bone fractures, major surgery (neurosurgery, thoracic, orthopedic, vascular), age, transfusion requirement, intracranial pressure monitor or external ventricular drain placement, and low Glasgow Coma Scale score. Data was split into training (n = 251,409) and testing (n = 118,175) subsets. Machine learning algorithms were trained, tested, and compared. RESULTS: Low-risk prediction: For the testing subset, all models outperformed the baseline rate of VTE (0.15%) with a predicted rate of 0.01-0.02% (p < 2.2e-16). 88.4-89.4% of patients were classified as low risk by the models. HIGH-RISK PREDICTION: All models outperformed baseline with a predicted rate of VTE ranging from 1.13 to 1.32% (p < 2.2e-16). The performance of the 3 models was not significantly different. CONCLUSION: We developed a predictive model that differentiates injured children for development of VTE with high discrimination and can guide prophylaxis use. LEVEL OF EVIDENCE: Prognostic, Level II. TYPE OF STUDY: Retrospective, Cross-sectional.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor contains an immunoreceptor tyrosine-based inhibitory motif that activates Shp2.

The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a constitutively active, highly angiogenic homologue of the interleukin-8 (IL-8) receptors that signals in part via the cytoplasmic protein tyrosine phosphatase Shp2. We show that vGPCR contains a bona fide immunoreceptor tyrosine-based inhibitory motif (ITIM) that binds and constitutively activates Shp2.

A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses.

How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

Human CD19-specific switchable CAR T-cells are efficacious as constitutively active CAR T-cells but cause less morbidity in a mouse model of human CD19+ malignancy.

Current Food and Drug Administration (FDA)-approved CD19-specific chimeric antigen receptor (CAR) T-cell therapies for B-cell malignancies are constitutively active and while efficacious, can cause morbidity and mortality. Their toxicities might be reduced if CAR T-cell activity was regulatable rather than constitutive. To test this, we compared the efficacies and morbidities of constitutively active (conventional) and regulatable (switchable) CAR (sCAR) T-cells specific for human CD19 (huCD19) in an immune-competent huCD19+ transgenic mouse model.Conventional CAR (CAR19) and sCAR T-cells were generated by retrovirally transducing C57BL/6 (B6) congenic T-cells with constructs encoding antibody-derived single chain Fv (sFv) fragments specific for huCD19 or a peptide neoepitope (PNE), respectively. Transduced T-cells were adoptively transferred into huCD19 transgenic hemizygous (huCD19Tg/0 ) B6 mice; healthy B-cells in these mice expressed huCD19Tg Prior to transfer, recipients were treated with a lymphodepleting dose of cyclophosphamide to enhance T-cell engraftment. In tumor therapy experiments, CAR19 or sCAR T-cells were adoptively transferred into huCD19Tg/0 mice bearing a syngeneic B-cell lymphoma engineered to express huCD19. To regulate sCAR T cell function, a switch protein was generated that contained the sCAR-specific PNE genetically fused to an anti-huCD19 Fab fragment. Recipients of sCAR T-cells were injected with the switch to link sCAR effector with huCD19+ target cells. Mice were monitored for survival, tumor burden (where appropriate), morbidity (as measured by weight loss and clinical scores), and peripheral blood lymphocyte frequency.CAR19 and sCAR T-cells functioned comparably regarding in vivo expansion and B-cell depletion. However, sCAR T-cells were better tolerated as evidenced by the recipients' enhanced survival, reduced weight loss, and improved clinical scores. Discontinuing switch administration allowed healthy B-cell frequencies to return to pretreatment levels.In our mouse model, sCAR T-cells killed huCD19+ healthy and malignant B-cells and were better tolerated than CAR19 cells. Our data suggest sCAR might be clinically superior to the current FDA-approved therapies for B-cell lymphomas due to the reduced acute and chronic morbidities and mortality, lower incidence and severity of side effects, and B-cell reconstitution on cessation of switch administration.

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.

Multidisciplinary approach to the management of an extracranial internal carotid artery aneurysm approaching the base of the skull in a young patient.

Internal carotid artery aneurysms are rare in young patients, especially those without connective tissue disorders or vessel trauma. We present the case of a 29-year-old man who was referred for an asymptomatic pulsatile mass. Computed tomographic angiography identified a true aneurysm between the carotid bifurcation and the base of the skull. This location combined with extensive perineural fibrosis made dissection difficult. Collaboration with an otolaryngologist provided enough distal internal carotid artery exposure for a saphenous vein interposition graft to be placed. Despite initial cranial nerve dysfunction, which later resolved completely, the patient tolerated the procedure well and remains asymptomatic 2 years postoperatively.