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Introduction: The Greulich & Pyle (G&P) Radiographic Atlas of Skeletal Development uses hand x-rays obtained between 1926 and 1942 on children of Caucasian ancestry. Our study uses modern Caucasian, Black, Hispanic, and Asian children to investigate patterns of development as a function of percent final height (PFH)., Methods: A retrospective review, at a single institution, was conducted using children who received a hand x-ray, a height measurement taken within 60 days of that x-ray, and a final height. BA and CA were compared between races. PFH was calculated by dividing height at the time of the x-ray by their final height. To further evaluate differences between races in CA or BA, PFH was then modeled as a function of CA or BA using a fifth-degree polynomial regression, and mean ages at the 85th PFH were compared. Patients were then divided into Sanders stages 1, 2-4, and 5-8 and the mean PFH, CA, and BA of the Asian, Black, and Hispanic children were compared with the White children using Student t test. P values less than 0.05 were considered significant., Results: We studied 498 patients, including 53 Asian, 83 Black, 190 Hispanic, and 172 White patients. Mean BA was significantly greater than CA in Black males (1.27 y) and females (1.36 y), Hispanic males (1.12 y) and females (1.29 y), and White females (0.74 y). Hispanic and Black patients were significantly more advanced in BA than White patients ( P <0.001). At the 85th PFH, White and Hispanic males were older than Black males by at least 7 months ( P <0.001), and White females were significantly older than Hispanic females by 6.4 months ( P <0.001). At 85th PFH for males, Hispanic and Black males had greater BA than White males by at least 5 months ( P <0.001), and Asian females had a greater BA than Black females by at least 5 months ( P <0.001). Compared with White children, Hispanic children were significantly younger at Sanders 2-4 than White children, and Black children were skeletally older at Sanders 5-8., Conclusions: BA was greater than CA by ≥1 year in Black and Hispanic children, and that these children had a significantly greater BA than their White counterparts. Black males and Hispanic females reached their 85th PFH at younger ages, and Hispanic males and Asian females were the most skeletally mature at their 85th PFH. Our results suggest that BA and CA may vary as a function of race, and further studies evaluating growth via the 85th PFH may be necessary., Level of Evidence: Therapeutic Study - Level IV., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Hip dysplasia reportedly occurs in up to 12% of the general population and may be higher in patients with adolescent idiopathic scoliosis (AIS). When using lateral center edge angle (LCEA) measurements to help identify hip dysplasia, it is uncertain if this measurement can be performed reliably on scoliosis radiographs due to parallax. The purpose of this study is to evaluate the reliability of measuring LCEA on scoliosis radiographs compared with standard pelvis radiographs., Methods: This study evaluated 40 hips on 20 patients (mean age 12.5 years ± 3.1; 50% Female) who received PA scoliosis radiographs and AP pelvis radiographs within 1 year of each other. Review was performed by 4 pediatric radiologists (2 general, 2 MSK), 2 pediatric orthopaedic surgeons, and 1 research fellow. Radiographic measurement of the LCEA for each hip was first performed on the scoliosis radiograph. After a minimum of 3 days, the LCEA was measured on a pelvis radiograph of the same individual obtained within 1 year of the scoliosis radiograph. Pearson coefficient was used to measure agreement between scoliosis and pelvis radiograph measurements. Intraclass correlation coefficient (ICC) was used to evaluate intraobserver and interobserver agreement. ICC values <0.5 were classified as poor reliability, 0.5 to 0.75 were classified moderate, 0.75 to 0.90 were classified good, and >0.9 indicated excellent reliability., Results: The mean difference between scoliosis and pelvis radiographs was 54 ± 79 days. There was good-to-excellent interobserver agreement on LCEA measurements made between readers on scoliosis radiographs (ICC: 0.94, 95% CI: 0.90-0.96, P <0.001) and pelvis radiographs (ICC: 0.91, 95% CI: 0.83-0.95, P <0.001), and moderate-to-excellent intraobserver agreement for scoliosis radiographs (ICC range: 0.68 to 0.98; P <0.001) and pelvis radiographs (ICC range: 0.62 to 0.96; P <0.001). There was a strong correlation between LCEA measurements made on scoliosis and pelvis radiographs ( r2 =0.66, P <0.001), and the intermodality agreement between scoliosis and pelvis radiograph LCEA measurements were moderate to good (ICC range: 0.68 to 0.89, P <0.001)., Conclusions: Overall, there was good-to-excellent agreement between readers on scoliosis and pelvis radiographs, respectively, and moderate-to-excellent intraobserver agreement between LCEA measurements made on scoliosis radiographs and pelvis radiographs, respectively. LCEA measurements made on scoliosis radiographs strongly correlated to the measurements made on pelvis radiographs, and the intermodality ICC was also considered moderate to good. Dedicated pelvis radiographs may not be necessary during scoliosis workup and follow-up surveillance, thereby decreasing radiation exposure, cost, and improving patient care workflow., Level of Evidence: Level IV-diagnostic study., Competing Interests: No conflicts of interest were declared by all authors. This study was internally funded., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The modified Gartland classification is the most widely accepted grading method of supracondylar humeral fractures among orthopedic surgeons and is relevant to identifying fractures that may require surgery., Objective: To assess the interobserver reliability of the modified Gartland classification among pediatric radiologists, pediatric orthopedic surgeons, and pediatric emergency medicine physicians., Materials and Methods: Elbow radiographs for 100 children with supracondylar humeral fractures were retrospectively independently graded by two pediatric radiologists, two pediatric orthopedic surgeons, and two pediatric emergency medicine physicians using the modified Gartland classification. A third grader of the same subspecialty served as a tie-breaker as needed to reach consensus. Readers were blinded to one another and to the medical record. The modified Gartland grade documented in the medical record by the treating orthopedic provider was used as the reference standard. Interobserver agreement was assessed using kappa statistics., Results: There was substantial interobserver agreement (kappa = 0.77 [95% CI, 0.69-0.85]) on consensus fracture grade between the three subspecialties. Similarly, when discriminating between Gartland type I and higher fracture grades, there was substantial interobserver agreement between specialties (kappa = 0.77 [95% CI, 0.66-0.89]). The grade assigned by pediatric radiologists differed from the reference standard on 15 occasions, pediatric emergency medicine differed on 19 occasions, and pediatric orthopedics differed on 9 occasions., Conclusion: The modified Gartland classification for supracondylar humeral fractures is reproducible among pediatric emergency medicine physicians, radiologists, and orthopedic surgeons., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The modified Gartland classification is an important tool for evaluation of pediatric supracondylar humerus fractures (SCHF) because it can direct treatment decisions. Gartland type I can be managed outpatient, while emergent surgical consult occurs with type II and III. This study assesses the interobserver reliability of the Gartland classification between pediatric radiologists and orthopedic providers., Methods: A retrospective review of 320 children diagnosed with a SCHF at a single tertiary children's hospital during 2022 was conducted. The Gartland classification documented in the radiographic report by a pediatric radiologist and the classification documented in the first encounter with an orthopedic provider was collected. Kappa value was used to assess interobserver reliability of classifications between radiologists and orthopedic providers. A second group of 76 Gartland type I SCHF from 2015, prior to our institution's implementation of structured reporting, was reviewed for comparison of unnecessary orthopedic consults at initial presentation., Results: The Gartland classification has excellent interobserver reliability between radiologists and orthopedic providers with 90 % (289/320) agreement and kappa of 0.854 (confidence interval: 0.805-0.903). The most frequent disagreement that occurred was fractures classified as type II by radiology and type III by orthopedics. There were similar rates of consults for the 2015 and 2022 cohorts (p = 0.26)., Conclusion: The Gartland classification system is a reliable and effective tool for communication between radiologists and orthopedic providers. Implementing a structured reporting system has the potential to improve triage efficiency for SCHF., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: When acute compartment syndrome (ACS) occurs in pediatric patients requiring venoarterial extracorporeal membrane oxygen (VA ECMO) support, there is little data to guide surgeons on appropriate management. The purpose of this study is to characterize the presentation, diagnosis, timeline, and outcomes of patients who developed this complication., Methods: This is a single-center retrospective case series of children below 19 years old on VA ECMO support who subsequently developed extremity ACS between January 2016 and December 2022. Outcomes included fasciotomy findings, amputation, mortality, and documented function at the last follow-up., Results: Of 343 patients on VA ECMO support, 18 (5.2%) were diagnosed with ACS a median 29 hours after starting ECMO. Initial cannulation sites included 8 femoral, 6 neck, and 4 central. Femoral artery cannulation was associated with an increased risk of ACS [odds ratio=6.0 (CI: 2.2 to 15), P <0.0001]. In the hospital, the mortality rate was 56% (10/18). Fourteen (78%) patients received fasciotomies a median of 1.2 hours after ACS diagnosis. Only 4 (29%) patients had all healthy muscles at initial fasciotomy, while 9 (64%) had poor muscular findings in at least 1 compartment. Patients with worse findings at fasciotomy had a significantly longer duration between ischemia onset and ACS diagnosis. Patients required a median of 1.5 additional procedures after fasciotomy, and only 1 (7%) developed a surgical site infection. Of the 7 surviving fasciotomy patients, 2 required amputations, 3 developed an equinus contracture, 1 developed foot drop, and 3 had no ACS-related deficits. Four patients did not receive fasciotomies: 3 were deemed too ill and later died, and 1 was diagnosed too late to benefit. The only surviving nonfasciotomy patient required bilateral amputations., Conclusions: Pediatric ECMO-associated ACS is not exclusive to patients with femoral artery cannulation. The majority of fasciotomy patients were diagnosed with ACS after muscle necrosis had already started. We were unable to definitively conclude whether fasciotomies provide better outcomes. There is a need for increased awareness and earlier recognition of this rare yet potentially devastating complication., Level of Evidence: Level IV-retrospective case series., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Adolescent idiopathic scoliosis radiographic screening will often include the hips. The purpose of this study is to evaluate the prevalence of hip dysplasia by lateral center edge angle (LCEA) on scoliosis radiographs in patients presenting with severe range (>45 deg) idiopathic curves., Methods: We reviewed 200 consecutive patients (400 hips) with idiopathic scoliosis with major coronal curves ≥45 degrees between October 2009 and October 2022. The presenting scoliosis radiograph PA were reviewed for the following variables: major coronal curve direction, coronal balance, and lateral center edge angles. An LCEA value ≤18 was considered dysplastic. To assess potential measurement variability related to parallax of the scoliosis radiographs, the intermodality intraclass correlation coefficient (ICC) was measured from a subset of 20 hip LCEA values from patients with both scoliosis and pelvis radiographs., Results: A total of 200 patients [mean age: 13.6 (1.8) years; Female: 79%] were reviewed. The mean major curve (SD) was 58.6 degrees (11.4 deg). Overall 19% (37/200) patients or 11% (43/400) of hips had hip dysplasia. Of the dysplastic hips, 16% (6/43, P <0.001) were bilateral and 57% (21/43, P =0.04) were right sided. There was no association between dysplastic hip laterality and ipsilateral or contralateral coronal balance ( P =0.26) or curve direction ( P =0.49). The interobserver ICC for assessment of LCEA on scoliosis radiographs was 0.85, and the intermodality ICC between pelvic and scoliosis radiographs was 0.78., Conclusion: Hip dysplasia was present in 19% of patients presenting with large major curves, and many of these patients had right sided hip dysplasia. There was no association between hip dysplasia laterality and the patient's major curve direction or coronal balance. There was good intermodality reliability (ICC=0.78) between LCEA values in patients who had both scoliosis and pelvis radiographs, and good inter-rater reliability between raters. Radiographic interpretation of adolescent idiopathic scoliosis should focus not only on the description of the curve and ruling out underlying segmentation anomalies but should also include critical assessment of the hips to exclude co-existing hip dysplasia., Level of Evidence: Therapeutic study-level IV., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Pediatric patients with leg length discrepancies and complex deformities may require multiplanar external fixators for correction. We have encountered 4 cases of half-pin breakage with the Orthex hexapod frame. The purpose of this study is to report factors associated with half-pin breakage and compare various deformity correction characteristics between 2 hexapod frames - Taylor Spatial Frame (TSF) and Orthex., Methods: Pediatric patients with lower extremity deformities treated with an Orthex or TSF at a single tertiary children's hospital between 2012 and 2022 were included for retrospective review. Variables compared between frame groups include frame configuration, half-pin/wire fixation, length achieved, angular correction, and frame time., Results: There were 23 Orthex frames (23 patients) and 36 TSF (33 patients) included. Four Orthex and zero TSF had proximal half-pin breakage. The Orthex group was younger on average (10 vs. 12 y, P =.04*) at the time of frame placement. The majority (52%) of Orthex frames were used for simultaneous lengthening and angular correction, while the majority (61%) of TSF was used for only angular correction. Orthex had more half-pins used for proximal fixation (median 3 vs. 2, P <0.0001*) and more frames with nonstandard configuration (7 (30%) vs. 1 (3%), P =0.004*). Orthex group had a longer total frame time (median 189 vs. 146 days, P =0.012*) and longer time required for regenerate healing (117 vs. 89 d, P =0.02*). There were no significant differences in length gained, angular correction, or healing index between Orthex and TSF. Nonstandard configuration, increased number of proximal half-pins, younger age at index surgery, and increased lengthening were associated with pin breakage., Conclusions: This is the first study to report half-pin breakage while using multiplanar frames in pediatric lower extremity deformity correction. The Orthex and TSF groups consisted of significantly different patients and frame configurations, making it difficult to identify any specific cause for pin breakage. This study shows that pin breakage is likely caused by multiple factors and is associated with the increased complexity of deformity correction., Level of Evidence: Level III-Retrospective Comparison Study., Competing Interests: J.H. gives paid lectures for Orthopediatrics and is a paid consultant for Stryker Orthopedics. The remaining authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Lateral humeral condyle fractures (LHCF) are the second most frequent type of pediatric elbow fracture. The purpose of this study is to characterize infections and nonunions of LHCF treated at a single institution. This is the largest series on infections and nonunions of LHCF to date., Methods: Pediatric patients undergoing surgical treatment for a LHCF from 2012 to 2022 at a single children's hospital were identified for a retrospective review. Patients who were diagnosed with an active infection or nonunion after surgery were included. Data collected included demographics, original treatment course, presentation, effective treatments, outcomes, and timing of events., Results: Out of 863 surgical patients, 12 (1.4%) patients developed 13 infections: 8 were diagnosed with superficial cellulitis, 3 with soft tissue infections, and 2 with osteomyelitis. Eleven fractures were stabilized with percutaneous pins and 1 with buried pins. The average time to infection diagnosis was 29 days and the most common presenting symptom was increased or new onset of pain. All 12 patients received antibiotics for an average 18 days, 6 required hospital admission, 3 required surgical incision and drainage, and 2 required intravenous antibiotics without admission. One patient that developed osteomyelitis developed a nonunion. Ten (1.2%) surgical patients developed nonunion. There were 3 Weiss type II fractures and 7 type III fractures. On average, nonunions were diagnosed 12 weeks after initial treatment. Nine patients underwent nonunion surgery, and all went on to union. Patient's elbows were immobilized for an average 16 weeks and at least 5 patients required an average of 10 physical therapy sessions to regain their range of motion., Conclusion: Infection and nonunion are rare complications of LHCF, but greatly change the timeline and number of healthcare interactions required for healing. Infectious complications typically require admission, additional surgery, or emergency department visits. Nonunions require extensive cast time, additional surgery, and rehabilitation., Level of Evidence: Level IV - case series., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Neuromuscular scoliosis in Rett syndrome (RS) is common, progressive, and often requires posterior spinal fusion (PSF). While PSF is associated with improved overall outcomes, there is a paucity of information describing complications. We aimed to report the postoperative complications, readmissions, and reoperations for patients with RS undergoing PSF., Methods: Female pediatric patients with RS treated by PSF with segmental instrumentation, with or without concurrent pelvis fixation, during January 2012 to August 2022 were included. Preoperative patient characteristics, intraoperative data (estimated blood loss, cell saver, packed red blood cells transfused), postoperative complications according to the Modified Clavien-Dindo-Sink classification within 90 days, unplanned readmissions within 30 days, and unplanned reoperations within 90 days were recorded., Results: A total of 25 females were included. The mean (SD) age at surgery was 12.9 (1.8) years and the mean follow-up of 38.6 (24.9) months. The mean preoperative major coronal curve was 79 degrees (23 degrees) which decreased to 32 degrees (15 degrees) by the last follow-up ( P <0.001). The median estimated blood loss was 600 mL and length of stay was 7 days. There were 81 total postoperative complications (3.2 complications/patient). Eight (32%) had grade IVa complications (disseminated intravascular coagulopathy, hypotensive shock, respiratory failure, chronic urosepsis). Five (20%) patients experienced seizures, 48% had pulmonary complications, and 56% had gastrointestinal complications. There were 3 readmissions (12%) within 30 days for pneumonia and 2 (8%) reoperations (an incision and drainage and C2-T2 fusion for significant kyphosis) within 90 days. One patient also had their fusion extended to the pelvis 1 year later. There were more nonambulatory patients in the group fused to the pelvis, but otherwise no differences between those fused and unfused to the pelvis., Conclusions: This is the largest review of early postoperative complications for patients with RS who underwent PSF. PSF effectively reduced the major coronal curve, but surgeons and families should be aware of a high postoperative seizure and respiratory complication rate, as well as 8% having reoperations within 90 days and 12% being readmitted within 30 days., Level of Evidence: Level IV-therapeutic study., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

The mitogen-activated protein kinase signaling cascade is conserved across eukaryotes, where it plays a critical role in the regulation of activities including proliferation, differentiation, and stress responses. This pathway propagates external stimuli through a series of phosphorylation events, which allows external signals to influence metabolic and transcriptional activities. Within the cascade, MEK, or MAP2K, enzymes occupy a molecular crossroads immediately upstream to significant signal divergence and cross-talk. One such kinase, MAP2K7, also known as MEK7 and MKK7, is a protein of great interest in the molecular pathophysiology underlying pediatric T cell acute lymphoblastic leukemia (T-ALL). Herein, we describe the rational design, synthesis, evaluation, and optimization of a novel class of irreversible MAP2K7 inhibitors. With a streamlined one-pot synthesis, favorable in vitro potency and selectivity, and promising cellular activity, this novel class of compounds wields promise as a powerful tool in the study of pediatric T-ALL., Competing Interests: The authors declare the following competing financial interest(s): Intellectual property filings., (© 2023 American Chemical Society.)

Novel drugs are needed to increase treatment response in children with high-risk T-cell acute lymphoblastic leukemia (T-ALL). Following up on our previous report on the activation of the MAP2K7-JNK pathway in pediatric T-ALL, here we demonstrate that OTSSP167, recently shown to inhibit MAP2K7, has antileukemic capacity in T-ALL. OTSSP167 exhibited dose-dependent cytotoxicity against a panel of T-ALL cell lines with IC50 in the nanomolar range (10-50 nM). OTSSP167 induces apoptosis and cell cycle arrest in T-ALL cell lines, associated at least partially with the inhibition of MAP2K7 kinase activity and lower activation of its downstream substrate, JNK. Other leukemic T-cell survival pathways, such as mTOR and NOTCH1 were also inhibited. Daily intraperitoneal administration of 10 mg/kg OTSSP167 was well tolerated, with mice showing no hematological toxicity, and effective at reducing the expansion of human T-ALL cells in a cell-based xenograft model. The same dosage of OTSSP167 efficiently controlled the leukemia burden in the blood, bone marrow, and spleen of 3 patient-derived xenografts, which resulted in prolonged survival. OTSSP167 exhibited synergistic interactions when combined with dexamethasone, L-asparaginase, vincristine, and etoposide. Our findings reveal novel antileukemic properties of OTSSP167 in T-ALL and support the use of OTSSP167 as an adjuvant drug to increase treatment response and reduce relapses in pediatric T-ALL., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric leukemia with a worse prognosis than most frequent B-cell ALL due to a high incidence of treatment failures and relapse. Our previous work showed that loss of the pioneer factor KLF4 in a NOTCH1-induced T-ALL mouse model accelerated the development of leukemia through expansion of leukemia-initiating cells and activation of the MAP2K7 pathway. Similarly, epigenetic silencing of the KLF4 gene in children with T-ALL was associated with MAP2K7 activation. Here, we showed the small molecule 5Z-7-oxozeaenol (5Z7O) induces dose-dependent cytotoxicity in a panel of T-ALL cell lines mainly through inhibition of the MAP2K7-JNK pathway, which further validates MAP2K7 as a therapeutic target. Mechanistically, 5Z7O-mediated apoptosis was caused by the downregulation of regulators of the G2/M checkpoint and the inhibition of survival pathways. The anti-leukemic capacity of 5Z7O was evaluated using leukemic cells from two mouse models of T-ALL and patient-derived xenograft cells generated using lymphoblasts from pediatric T-ALL patients. Finally, a combination of 5Z7O with dexamethasone, a drug used in frontline therapy, showed synergistic induction of cytotoxicity. In sum, we report here that MAP2K7 inhibition thwarts survival mechanisms in T-ALL cells and warrants future pre-clinical studies for high-risk and relapsed patients., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Chen et al.)

Acute myeloid leukemia (AML) is an aggressive hematological malignancy of the bone marrow that affects mostly elderly adults. Alternative therapies are needed for AML patients because the overall prognosis with current standard of care, high dose chemotherapy and allogeneic transplantation, remains poor due to the emergence of refractory and relapsed disease. Here, we found expression of the transcription factor KLF4 in AML cell lines is not silenced through KLF4 gene methylation nor via proteasomal degradation. The deletion of KLF4 by CRISPR-CAS9 technology reduced cell growth and increased apoptosis in both NB4 and MonoMac-6 cell lines. Chemical induced differentiation of gene edited NB4 and MonoMac6 cells with ATRA and PMA respectively increased apoptosis and altered expression of differentiating markers CD11b and CD14. Transplantation of NB4 and MonoMac-6 cells lacking KLF4 into NSG mice resulted in improved overall survival compared to the transplantation of parental cell lines. Finally, loss-of-KLF4 did not alter sensitivity of leukemic cells to the chemotherapeutic drugs daunorubicin and cytarabine. These results suggest that KLF4 expression supports AML cell growth and survival, and the identification and disruption of KLF4-regulated pathways could represent an adjuvant therapeutic approach to increase response., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Lewis et al.)

Background: Measuring S100A12 concentrations in serum and feces is a sensitive and specific marker of inflammation, such as seen with chronic gastrointestinal inflammation in people and dogs. Biomarkers of inflammation in cats are currently lacking., Objectives: We aimed to analytically cross-validate the canine S100A12-ELISA for the measurement of S100A12 in feline specimens., Methods: The ELISA was analytically validated by assessing dilutional linearity, spiking/recovery, intra- and inter-assay variability. Reference intervals for serum and fecal feline S100A12 concentrations were calculated using samples from healthy cats, and the short-term biological variation of fecal S100A12 was assessed., Results: Observed-to-expected ratios (O/E) for serial dilutions of serum and fecal extracts ranged from 91%-159% (mean, 120%) and 100%-128% (mean, 114%), and for the spiking/recovery method ranged from 106%-263% (mean, 154%) and 52%-171% (mean, 112%). Intra- and inter-assay CV% for serum were ≤5.6% and ≤14.0%, and for fecal extracts were ≤3.8% and ≤19.1%, repsectively. RIs for feline serum and fecal S100A12 concentrations were <43 µg/L and < 20 ng/g, respectively. A mild short-term biologic variation, but large individuality were detected when measuring fecal S100A12 concentrations in healthy cats., Conclusions: The canine S100A12-ELISA is accurate, reproducible, and sufficiently linear and precise for the measurement of S100A12 in feline serum and fecal samples. The use of this assay is a reasonable option for the measurement of S100A12 concentrations in feline specimens and provides a basis for the further evaluation of  S100A12 in cats with gastrointestinal disease. Using a population-based RI for fecal feline S100A12 appears to be of limited value., (© 2019 American Society for Veterinary Clinical Pathology.)

Leukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Krüppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)-induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc-mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules., (© 2019 by The American Society of Hematology.)

Calprotectin is a useful biomarker of inflammation in dogs. However, the biological variation of serum canine calprotectin is unknown. Indices of biological variation were determined in serial serum samples (n=147) from 11 healthy dogs (males/females: 4/7, median age: 5 years): analytical (3.0%), intra-individual (29.9%), and inter-individual variation (33.2%), reciprocal index of individuality (1.1), and index of heterogeneity (4.9). Serum calprotectin concentrations measured by ELISA and by the previous radioimmunoassay were highly correlated, but a constant and proportional bias exists between both assays. A de novo ELISA-reference interval (RI) for serum calprotectin concentration was established (0.6-11.8mg/L). Moderate changes in serum calprotectin (minimum critical difference: 6.4mg/L) between sequential measurements are needed to be considered relevant, and a population-based RI may or may not be appropriate for serum calprotectin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Measurement of serum trypsin-like immunoreactivity (TLI) is used to assess exocrine pancreatic function in dogs and cats. Ferrets ( Mustela putorius furo) serve as valuable animal models for human diseases such as cystic fibrosis and other pulmonary diseases, and may be a useful model of other diseases including pancreatitis. We developed and analytically validated a competitive radioimmunoassay (RIA) for measurement of TLI in ferret serum by determination of analytical sensitivity, assay linearity, accuracy of spiking recovery, precision, and reproducibility. Analytical sensitivity of the assay was 0.55 μg/L. Observed-to-expected (O/E) ratio for dilutional parallelism was 90.2-127.9% (mean: 108.1 ± 11.9%). The O/E ratio for spiking recovery was 94.5-113.0% (mean: 103.9 ± 7.2%). The intra- and inter-assay coefficients of variation (CVs) were 2.7-5.7% and 3.5-8.2%, respectively. The reference interval (RI) for serum TLI derived from 31 healthy ferrets was 28-115 μg/L; the 90% confidence interval for the lower and upper limits of the RI were 10.0-32.1 μg/L and 103-126 μg/L, respectively. This TLI RIA is analytically sensitive, sufficiently linear, accurate, precise, and reproducible for the measurement of TLI in ferret serum samples.

Background: Folate and cobalamin are essential cofactors for homocysteine (HCY) metabolism. Hyperhomocysteinemia, a multifactorial condition, may reflect B vitamin deficiency and is associated with increased risk of cardiovascular disease, thrombosis, and neurodegenerative and chronic gastrointestinal diseases in humans. Hyperhomocysteinemia has been reported in Greyhounds with suspected chronic enteropathy., Objectives: To evaluate the frequencies of and the association between hypofolatemia and hyperhomocysteinemia in Greyhounds., Animals: Data and serum samples from 559 Greyhounds., Methods: Nested case-control study. The frequency of hypofolatemia in Greyhounds was determined by a laboratory database search. The relationship between hyperhomocysteinemia (measured by gas chromatography-mass spectrometry) and hypocobalaminemia and hypofolatemia was evaluated, and its frequency compared between healthy Greyhounds and Greyhounds with thrombosis or chronic diarrhea., Results: Hypofolatemia was identified in 172 of 423 (41%) Greyhounds and was more common in hypo- than in normocobalaminemic dogs (49% vs. 35%; P = .0064). Hyperhomocysteinemia was detected in 53 of 78 (68%) of Greyhounds, being more common in hypo- than in normofolatemic dogs (88% vs. 59%; P = .0175). All healthy Greyhounds, 21 of 30 (70%) of dogs with chronic diarrhea and 6 of 8 (75%) of those with thrombosis, were hyperhomocysteinemic. Serum HCY concentrations were inversely correlated with serum folate concentration (ρ = -0.28; P = .0386) and were positively associated with serum albumin concentration (ρ = 0.66; P = .0022)., Conclusions and Clinical Relevance: Hyperhomocysteinemia occurs frequently in the Greyhound population. Its association with hypofolatemia suggests decreased intracellular availability of B vitamins, but the functional implications warrant further investigation. Hyperhomocysteinemia in Greyhounds potentially may serve as a spontaneous canine model to further investigate hyperhomocysteinemia in humans., (Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Fecal canine alpha1-proteinase inhibitor (cα1-PI) concentration has been reported to be increased in dogs with protein-losing enteropathy due to the loss of cα1-PI into the gastrointestinal tract. A chronic loss of cα1-PI may theoretically deplete serum cα1-PI, potentially altering the proteinase-to-proteinase inhibitor balance. Protein-losing enteropathy has been reported to occur frequently in certain dog breeds such as Yorkshire Terriers and to be associated with hypocobalaminemia. The objective was to compare serum cα1-PI concentrations in Yorkshire Terriers with and without cobalamin (COB) deficiency. Serum samples from 52 COB-deficient and 69 normocobalaminemic Yorkshire Terriers, which had been submitted to the Gastrointestinal Laboratory (2008-2011; College Station, TX), were included retrospectively. Serum cα1-PI concentrations were measured using an in-house radioimmunoassay and compared between Yorkshire Terriers with and without COB deficiency using a Mann-Whitney U test. A Fisher exact test was used to evaluate whether a decreased serum cα1-PI concentration is associated with COB deficiency in Yorkshire Terriers. Serum cα1-PI concentrations were significantly lower in COB-deficient Yorkshire Terriers (median: 1,016 mg/l, range: 315-3,945 mg/l) than in normocobalaminemic Yorkshire Terriers (median: 1,665 mg/l, range: 900-2,970 mg/l; P < 0.0001). One-fourth (n = 13) of the COB-deficient Yorkshire Terriers had a serum cα1-PI concentration below the lower limit of the reference interval (<732 mg/l), and COB deficiency was associated with decreased serum cα1-PI concentrations (P < 0.0001). In the current study, serum cα1-PI concentrations are significantly lower in COB-deficient Yorkshire Terriers when compared to normocobalaminemic Yorkshire Terriers. Further studies are needed to determine the functional and potential prognostic implications of serum cα1-PI concentrations in dogs with gastrointestinal disease.