Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency.

Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish bone fide DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet- phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells in the bone marrow and spleen compartments. Ex vivo stimulation of CD4+ T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.

Predicting flow cytometry crossmatch results from single-antigen bead testing.

The aim of this study was to devise an algorithm that would predict flow cytometry crossmatch (FCXM) results using single-antigen bead (SAB) mean fluorescent intensity (MFI) levels using samples received through the National External Quality Assurance Scheme (NEQAS) 2B external proficiency testing scheme between 2019 and 2023. A total of 159 serum samples were retrospectively screened using LABScreen Single Antigen Class I and II (SAB), and 40 peripheral blood samples were human leucocyte antigen (HLA) typed with LABType SSO. Donor-specific antibodies were identified for each cell-serum combination tested, and cumulative MFI values were calculated for each test before correlating the screening result with the consensus crossmatch results for this scheme. HLA Class I MFIs were combined to predict the T cell crossmatch. For the B cell crossmatch prediction, two options were considered: (i) HLA Class II MFI values alone and (ii) HLA Class I + Class II MFIs. Receiver operating characteristic analysis was carried out to identify the combined MFI threshold that predicted NEQAS consensus results with the greatest sensitivity and specificity. HLA Class I combined MFI >5000 predicted T cell crossmatch results with 96% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 92% negative predictive value (NPV). For B cell results, HLA Class I + Class II combined MFIs >11,000 gave the best model, showing 97% sensitivity, 82% specificity, 96% PPV and 85% NPV. However, for samples with only HLA Class II sensitization, combined MFIs >13,000 improved the B cell crossmatch predictions: 92% sensitivity, 95% specificity, 96% PPV and 91% NPV. Using this model, combined MFI can be used to predict the immunological risk posed by donor-specific antibodies when it is not possible to carry out an FCXM.

Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency.

Dendritic cells (DC) are mediators of adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for leukemia patients. To date, there is little information on the effects of FLT3-ITD in DC biology. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with acute myeloid leukemia (AML) to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet - phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells. Furthermore, co-culture of AML mouse- derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes.

Annexin A2 Loss After Cardiopulmonary Bypass and Development of Acute Postoperative Respiratory Dysfunction in Children.

The primary objective of this study was to determine whether expression of the multifunctional and adherens junction-regulating protein, annexin A2 (A2), is altered following cardiopulmonary bypass (CPB). A secondary objective was to determine whether depletion of A2 is associated with post-CPB organ dysfunction in children. DESIGN: In a prospective, observational study conducted over a 1-year period in children undergoing cardiac surgery requiring CPB, we analyzed A2 expression in peripheral blood mononuclear cells at different time points. We then assessed the relationship of A2 expression with organ function at each time point in the early postoperative period. SETTING: Twenty-three-bed mixed PICU in a tertiary academic center. PARTICIPANTS: Patients 1 month to 18 years old undergoing cardiac surgery requiring CPB. MEAN OUTCOME MEASUREMENTS AND RESULTS: We analyzed A2 expression in 22 enrolled subjects (n = 9, 1-23 mo old; n = 13, 2-18 yr old) and found a proteolysis-mediated decline in intact A2 immediately after bypass (p = 0.0009), reaching a median of 4% of baseline at 6 hours after bypass (p < 0.0001), and recovery by postoperative day 1. The degree of A2 depletion immediately after bypass in 1-23-month-olds correlated strongly with the extent of organ dysfunction, as measured by PICU admission Vasoactive-Ventilation-Renal (p = 0.004) and PEdiatric Logistic Organ Dysfunction-2 (p = 0.039) scores on postoperative day 1. A2 depletion immediately after bypass also correlated with more protracted requirement for both respiratory support (p = 0.007) and invasive ventilation (p = 0.013) in the 1-23-month-olds. CONCLUSIONS AND RELEVANCE: The degree of depletion of A2 following CPB correlates with more severe organ dysfunction, especially acute respiratory compromise in children under 2 years. These findings suggest that loss of A2 may contribute to pulmonary microvascular leak in young children following CPB.

BET inhibitors rescue anti-PD1 resistance by enhancing TCF7 accessibility in leukemia-derived terminally exhausted CD8+ T cells.

Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid-derived suppressor cells, suppressive regulatory T cells and dysfunctional T cells. Similarly, we have identified the same immune-related features, including exhausted CD8+ T cells (TEx) in a mouse model of AML. Here we show that inhibitors that target bromodomain and extra-terminal domain (BET) proteins affect tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor-exhausted T cells. This reversal was enhanced by combined BETi and anti-PD1 treatment. BETi synergized with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increase in expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. Finally, we profiled the epigenomes of in vivo JQ1-treated AML-derived CD8+ T cells by single-cell ATAC-seq and found that JQ1 increases Tcf7 accessibility specifically in Tex cells, suggesting that BETi likely acts mechanistically by relieving repression of progenitor programs in Tex CD8+ T cells and maintaining a pool of anti-PD1 responsive CD8+ T cells.

Coronary Artery Dilation in an Asymptomatic Pediatric Patient with COVID19 Antibodies.

We describe a 16-year-old asymptomatic male who presented with coronary artery dilation (z score + 2.3) identified on echo performed solely for presence of COVID-19 antibodies. This case raises the question of whether cardiac screening should be considered for all patients with a history of COVID-19.

The Deconditioning Effect of the COVID-19 Pandemic on Unaffected Healthy Children.

The COVID-19 pandemic has had devastating direct consequences on the health of affected patients. It has also had a significant impact on the ability of unaffected children to be physically active. We evaluated the effect of deconditioning from social distancing and school shutdowns implemented during the COVID-19 pandemic on the cardiovascular fitness of healthy unaffected children. This is a single-center, retrospective case-control study performed in an urban tertiary referral center. A cohort of 10 healthy children that underwent cardiopulmonary exercise testing after COVID-19 hospital restrictions were lifted was compared to a matched cohort before COVID-19-related shutdowns on school and after-school activities. Comparisons of oxygen uptake (VO2) max and VO2 at anaerobic threshold between the pre- and post-COVID-19 cohorts were done. The VO2 max in the post-COVID cohort was significantly lower than in the pre-COVID cohort (39.1 vs. 44.7, p = 0.031). Only one out of ten patients had a higher VO2 max when compared to their matched pre-COVID control and was also the only patient with a documented history of participation in varsity-type athletics. The percentile of predicted VO2 was significantly lower in the post-COVID cohort (95% vs. 105%, p = 0.042). This study for the first time documented a significant measurable decline in physical fitness of healthy children as a result of the COVID-19 pandemic and its associated restrictions. Measures need to be identified that encourage and facilitate regular exercise in children in a way that are not solely dependent on school and organized after-school activities.

Epipericardial Fat Pad Necrosis-A Rare Cause of Chest Pain in an Adolescent.

ABSTRACT: Epipericardial fat necrosis is a rare cause of acute pleuritic chest pain reported in approximately 40 cases. This diagnosis mimics a myocardial infarction, pulmonary embolism, or pericarditis; however, the cardiac enzymes and electrocardiogram are usually normal. We present the first reported case of epipericardial fat necrosis in an adolescent.

Effectiveness of echocardiography simulation training for paediatric cardiology fellows in CHD.

Simulation is used in many aspects of medical training but less so for echocardiography instruction in paediatric cardiology. We report our experience with the introduction of simulator-based echocardiography training at Weill Cornell Medicine for paediatric cardiology fellows of the New York-Presbyterian Hospital of Columbia University and Weill Cornell Medicine. Knowledge of CHD and echocardiographic performance improved following simulation-based training. Simulator training in echocardiography can be an effective addition to standard training for paediatric cardiology trainees.

A novel HSV-2 subunit vaccine induces GLA-dependent CD4 and CD8 T cell responses and protective immunity in mice and guinea pigs.

BACKGROUND/OBJECTIVES: There is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection. METHODS: We developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination. RESULTS: Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score. CONCLUSIONS: Taken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.

Virological and preclinical characterization of a dendritic cell targeting, integration-deficient lentiviral vector for cancer immunotherapy.

Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.

Cardiac troponin I after cardiopulmonary bypass in infants in comparison with older children.

OBJECTIVE: At the present time, there is a trend towards performing open heart surgery at a younger age. Myocardium of infants has been thought to be more vulnerable to cardiopulmonary bypass in comparison with adults. For this study, we evaluated the degree of myocardial injury by measurement of cardiac troponin levels in infants in comparison with older children for similar surgeries. METHODS: Serum was collected before bypass, after bypass, and daily after surgery and serum cardiac troponin I level (micrograms per litre). The demographic data, cardiac diagnoses, types of surgery performed, and peri-operative parameters were collected. RESULTS: Of the 21 children enrolled consecutively, five were infants. Among the 21 patients, four patients had post-operative peak troponin values greater than 100 (three were infants) and all four patients survived and had normal left ventricular systolic function upon discharge echocardiogram. The five infants had peak troponin levels of 222.3, 202, 129, 26.7, and 82.3. The post-operative peak troponin levels were significantly higher in infants (mean 132.5 with a standard deviation of 81.6) than in the older children (mean 40.3 with a standard deviation of 33.4), although there was no significant difference in bypass time, bypass temperature, cross-clamp time, or the length of stay in the intensive care unit between the two age groups. CONCLUSIONS: Higher troponin release is seen in infants in comparison with older children after bypass for similar surgeries. A troponin level greater than 100 after bypass does not necessarily predict death or a severe cardiovascular event in the very young.

A Rev-Independent gag/pol Eliminates Detectable psi-gag Recombination in Lentiviral Vectors.

Lentiviral vectors (LVs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. A safety concern for use of LVs in humans is the generation of replication-competent lentivirus (RCL), which may arise due to recombination between the split genomes of third-generation LVs. Although no RCL has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may further reduce the risk of RCL formation. Here we describe design elements introduced to the gag/pol plasmid with the intention of eliminating psi-gag recombination between the vector genome and gag/pol. These design changes, consisting of codon optimization of the gag/pol sequence and the deletion of the Rev-responsive element, abrogate the requirement for Rev in expression of Gag protein, thus the resulting gag/pol construct being Rev independent (RI gag/pol). We show that generating vector using the RI gag/pol construct has no effect on particle production or transduction titers. The RI and wild-type gag/pol vectors function equivalently as antigen-specific immunotherapy, potently inducing antigen-specific CD8 T cells that protect against challenge with vaccinia virus. Most importantly, the designed RI gag/pol eliminated detectable psi-gag recombination. Interestingly, we detected recombination between the vector genome and gag/pol from regions without sequence homology. Our findings imply that although unpredictable recombination events may still occur, the RI gag/pol design is sufficient to prevent psi-gag recombination.

Early pleural effusions related to the myocardial injury after open-heart surgery for congenital heart disease.

OBJECTIVES: The degree of effusion immediately after cardiopulmonary bypass (CPB) can vary and may reflect several factors including the degree of myocardial injury. We compared the degree of pleural effusions after CPB to the overall myocardial injury as determined by serum cardiac troponin I (cTnI) levels after elective repair of a variety of congenital heart defects, including univentricular surgeries via cavopulmonary shunts. METHODS: Serum was collected pre-CPB, post-CPB, and daily after that and cTnI level measured. The postoperative pleural effusion was measured each day until the chest tube was removed. Results. The 21 study patients were of average age of 5.5 years (+/-5.6). The duration of chest-tube drainage after open-heart surgery was 4.3 days (+/-3.5) and the amount was 2.4 mL/kg/hour (+/-2.9). For the biventricular repairs, cTnI levels on the postoperative day (POD) 1 best correlated with amount of effusion (n = 16, r = 0.5, P = 0.02) and the average (POD 0-3) cTnI levels with the total duration (n = 16, r = 0.4, P = 0.01) and also the amount (n = 16, r = 0.5, P = 0.02) of effusions. For the cavopulmonary shunts, the post-CBP cTnI level best correlated with the duration (n = 5, r = 0.8, P = 0.02) and amount (n = 5, r = 0.9, P = 0.02) of effusions. A cTnI level on the first postoperative day >or=15 microg/L was associated with effusions >2 days (sensitivity of 81% and specificity of 80%). CONCLUSION: We found that higher the cTnI released, especially >or=15 microg/L, longer the duration and greater the amount of early pleural effusions for a variety of congenital heart surgeries including cavopulmonary shunts. A number of factors may lead to excessive pleural effusions and the degree of myocardial injury may be one of them.

Atherosclerosis in survivors of Kawasaki disease.

OBJECTIVES: To test the hypothesis that long-term survivors of low-risk Kawasaki disease (KD) have ongoing vascular inflammation and dysfunction and a higher risk of accelerated atherosclerosis than healthy control subjects. STUDY DESIGN: Twenty-eight patients with KD (7-20 years after acute illness) and 27 age-matched healthy control subjects were examined for medical and dietary history, serum markers of atherosclerotic risk and inflammation, carotid intimal-medial thickness (CIMT) with vascular ultrasound scanning and arterial stiffness with applanation tonometry. RESULTS: Patients and control subjects were similar in age, sex, body mass index, waist-to-hip ratio, blood pressure, cigarette smoking, family history, diet, high-density lipoprotein cholesterol level, lipoprotein (a) level, homocysteine level, glucose level, insulin level, CIMT, arterial stiffness, C-reactive protein level, and inflammatory cytokine level. Levels of total cholesterol and apolipoprotein B were significantly higher in patients with KD than in control subjects. CONCLUSIONS: There was no evidence of increased atherosclerosis. Small but significant differences in cholesterol and apolipoprotein B levels could suggest increased future risk for atherosclerosis and warrant further study.

Longitudinal changes of brain-type natriuretic peptide in preterm neonates.

OBJECTIVE: To determine age-related concentrations of brain-type natriuretic peptide in preterm infants using bedside Triage brain-type natriuretic peptide test and correlate it to the presence or absence of the patent ductus arteriosus and ventilatory support. METHODS: Serum brain-type natriuretic peptide levels were measured in infants who were born at <32 weeks' gestation from birth to 2 months of age. Serial echocardiograms were performed, until closure of the patent ductus arteriosus, or until discharge. Brain-type natriuretic peptide levels were correlated to the day of life, gestational age, presence or absence of the patent ductus arteriosus, and the degree of ventilatory support. Nineteen preterm infants (gestational age: 24-31 weeks; birth weight: 645-1670 g) were enrolled prospectively during the first 2 weeks of life. Serum brain-type natriuretic peptide levels (pg/mL) were determined in 177 blood samples, and 87 paired echocardiograms were performed. RESULTS: Significant negative correlation was found between brain-type natriuretic peptide levels and the day of life and remained significant when the patients were stratified by gestational age (< or =28 weeks and >28 weeks). Higher brain-type natriuretic peptide levels correlated with increasing grade of the patent ductus arteriosus. Significant differences in brain-type natriuretic peptide levels were seen with increasing ventilatory support. Comparisons between the size of patent ductus arteriosus and the degree of ventilatory support to brain-type natriuretic peptide levels revealed that the size of the patent ductus arteriosus was the major determinant of both brain-type natriuretic peptide levels and the degree of ventilatory support. CONCLUSIONS: Similar to term infants, brain-type natriuretic peptide levels of preterm infants are related to the chronological age and decline during the first month of life. Rapid bedside Triage brain-type natriuretic peptide is a potentially valuable and practical assay in determining the hemodynamic changes in preterm infants.

The use of a bedside assay for plasma B-type natriuretic peptide as a biomarker in the management of patent ductus arteriosus in premature neonates.

OBJECTIVE: To test the utility of the bedside plasma concentration of B-type natriuretic peptide (BNP) assay as a screen for patent ductus arteriosus (PDA) in premature neonates. STUDY DESIGN: Newborn infants admitted to the neonatal intensive care unit (NICU) had paired echocardiography and BNP measurements at enrollment and every 4 to 5 days. RESULTS: Twenty neonates (gestational age approximately 28.6 weeks and birth weight approximately 1161 g) had 81 paired echocardiography and BNP determinations. BNP ranged from 5 to 3900 pg/mL. Fifty-six of 81 echocardiograms showed PDA. Significant correlations were found between BNP and ductal size and degree of shunting. Correlation was greater in infants >2 days of age. BNP >300 pg/mL predicted significant PDA, whereas BNP <105 pg/mL predicted absence of significant PDA. CONCLUSION: Bedside measurement of BNP correlates with magnitude of PDA in premature newborns, particularly beyond day 2, and may be useful in guiding diagnostic and management strategies.