Immersion in Raloxifene does not significantly improve bone toughness or screw pull-out strength in multiple in vitro models.

BACKGROUND: Failure of surgical fixation in orthopaedic fractures occurs at a significantly higher rate in osteoporotic patients due to weakened osteoporotic bone. A therapy to acutely improve the mechanical properties of bone during fracture repair would have profound clinical impact. A previous study has demonstrated an increase in mechanical properties of acellular cortical canine bone after immersion in raloxifene. The goal of this study was to determine if similar treatment yields the same results in cancellous fetal bovine bone and whether this translates into a difference in screw pull-out strength in human cadaveric tissue. METHODS: Cancellous bone from fetal bovine distal femora underwent quasi-static four-point bending tests after being immersed in either raloxifene (20 µM) or phosphate-buffered saline as a control for 7 days (n = 10). Separately, 5 matched pairs of human osteoporotic cadaveric humeral heads underwent the same procedure. Five 3.5 mm unicortical cancellous screws were then inserted at standard surgical fixation locations to a depth of 30 mm and quasi-static screw pull-out tests were performed. RESULTS: In the four-point bending tests, there were no significant differences between the raloxifene and control groups for any of the mechanical properties - including stiffness (p = 0.333) and toughness (p = 0.546). In the screw pull-out tests, the raloxifene soaked samples and control samples had pullout strengths of 122 ± 74.3 N and 89.5 ± 63.8 N, respectively. CONCLUSIONS: Results from this study indicate that cancellous fetal bovine samples did not demonstrate an increase in toughness with raloxifene treatment, which is in contrast to previously published data that studied canine cortical bone. In vivo experiments are likely required to determine whether raloxifene will improve implant fixation.

Eosinophilic esophagitis (EoE) genetic susceptibility is mediated by synergistic interactions between EoE-specific and general atopic disease loci.

BACKGROUND: Eosinophilic esophagitis (EoE) is an esophageal inflammatory disease associated with atopic diseases. Thymic stromal lymphopoietin (TSLP) and calpain 14 (CAPN14) genetic variations contribute to EoE, but how this relates to atopy is unclear. OBJECTIVE: The purpose of this study was to explore the relationship between EoE, atopy, and genetic risk. METHODS: EoE-atopy enrichment was tested by using 700 patients with EoE and 801 community control subjects. Probing 372 single nucleotide polymorphisms (SNPs) in 63 atopy genes, we evaluated EoE associations using 412 nonatopic and 868 atopic disease control subjects. Interaction and stratified analyses of EoE-specific and atopy-related SNPs were performed. RESULTS: Atopic disease was enriched in patients with EoE (P < .0001). Comparing patients with EoE and nonatopic control subjects, EoE associated strongly with IL-4/kinesin family member 3A (IL4/KIF3A) (P = 2.8 × 10-6; odds ratio [OR], 1.87), moderately with TSLP (P = 1.5 × 10-4; OR, 1.43), and nominally with CAPN14 (P = .029; OR, 1.35). Comparing patients with EoE with atopic disease control subjects, EoE associated strongly with ST2 (P = 3.5 × 10-6; OR, 1.77) and nominally with IL4/KIF3A (P = .019; OR, 1.25); TSLP's association persisted (P = 4.7 × 10-5; OR, 1.37), and CAPN14's association strengthened (P = .0001; OR, 1.71). Notably, there was gene-gene interaction between TSLP and IL4 SNPs (P = .0074). Children with risk alleles for both genes were at higher risk for EoE (P = 2.0 × 10-10; OR, 3.67). CONCLUSIONS: EoE genetic susceptibility is mediated by EoE-specific and general atopic disease loci, which can have synergistic effects. These results might aid in identifying potential therapeutics and predicting EoE susceptibility.

Alignment of parent- and child-reported outcomes and histology in eosinophilic esophagitis across multiple CEGIR sites.

BACKGROUND: Patient-reported outcome metrics for eosinophilic esophagitis (EoE) have been developed and validated but not used in a multicenter pediatric population or systematically aligned with histology. OBJECTIVE: We sought to understand (1) the potential of caregiver report to predict patient self-reported symptoms and (2) the correlation of patient-reported outcome domains with histology. METHODS: Patients with EoE (n = 310) and their parents participating in the Consortium of Gastrointestinal Eosinophilic Disease Researchers (CEGIR) observational clinical trial were queried for baseline patient symptoms and quality of life (QOL) by using the Pediatric Eosinophilic Esophagitis Symptom Score, version 2 (PEESSv2.0), and the Pediatric QOL EoE module (PedsQL-EoE), and biopsy specimens were analyzed by using the EoE Histology Scoring System. RESULTS: PEESSv2.0 parental and child reports aligned across all domains (r = 0.68-0.73, P < .001). PedsQL-EoE reports correlated between parents and children across ages and multiple domains (r = 0.48-0.79, P < .001). There was a tight correlation between symptoms on PEESSv2.0 and their effects on QOL both on self-report and parental report (P < .001). Self-reported symptoms on PEESSv2.0 (positively) and PedsQL-EoE (inversely) showed a weak correlation with proximal, but not distal, peak eosinophil counts and features and architectural tissue changes on the EoE Histology Scoring System (P < .05). CONCLUSIONS: Parents of children with EoE aged 3 to 18 years accurately reflected their children's disease symptoms and QOL. Self- and parent-reported symptoms correlate with proximal esophageal histology. Our data suggest that parental report in young children can function as an adequate marker for self-reported symptoms and that self-reported symptoms can reflect changes in tissue histology in the proximal esophagus. These findings should be considered during clinical trials for drug development.

Pediatric Eosinophilic Esophagitis Symptom Scores (PEESS v2.0) identify histologic and molecular correlates of the key clinical features of disease.

BACKGROUND: The Pediatric Eosinophilic Esophagitis Symptom Score (PEESS v2.0) measures patient-relevant outcomes. However, whether patient-identified domains (dysphagia, gastroesophageal reflux disease [GERD], nausea/vomiting, and pain) align with clinical symptomology and histopathologic and molecular features of eosinophilic esophagitis (EoE) is unclear. OBJECTIVE: The purpose of this study was to determine whether clinical features of EoE, measured through PEESS v2.0, associate with histopathologic and molecular features of EoE. This represents a novel approach for analysis of allergic diseases, given the availability of allergic tissue biopsy specimens. METHODS: We systematically recruited treated and untreated pediatric patients with EoE (aged 2-18 years) and examined parent proxy-reported symptoms using the PEESS v2.0. Clinical symptomology was collected by questionnaire. Esophageal biopsy samples were quantified for levels of eosinophils, eosinophil peroxidase (EPX) immunohistochemical staining, and mast cells. Molecular features were assessed by using the EoE Diagnostic Panel (94 EoE-related gene transcripts). Associations between domain scores and clinical symptoms and biological features were analyzed with Wilcoxon rank sum and Spearman correlation. RESULTS: The PEESS v2.0 domains correlated to specific parent-reported symptoms: dysphagia (P = .0012), GERD (P = .0001), and nausea/vomiting (P < .0001). Pain correlated with multiple symptoms (P < .0005). Dysphagia correlated most strongly with overall histopathology, particularly in the proximal esophagus (P ≤ .0049). Markers of esophageal activity (EPX) were significantly associated with dysphagia (strongest r = 0.37, P = .02). Eosinophil levels were more associated with pain (r = 0.27, P = .06) than dysphagia (r = 0.24, P = .13). The dysphagia domain correlated most with esophageal gene transcript levels, predominantly with mast cell-specific genes. CONCLUSION: We have (1) established a validated, parent proxy-reported measure for pediatric EoE, the PEESS v2.0; (2) verified that the parent proxy effectively captures symptoms; (3) determined that the dysphagia domain most closely aligns with symptoms and tissue-based molecular biomarkers; (4) established that symptoms correlate with EPX staining; and (5) observed association between mast cells and dysphagia.

Twin and family studies reveal strong environmental and weaker genetic cues explaining heritability of eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic antigen-driven allergic inflammatory disease, likely involving the interplay of genetic and environmental factors, yet their respective contributions to heritability are unknown. OBJECTIVE: To quantify the risk associated with genes and environment on familial clustering of EoE. METHODS: Family history was obtained from a hospital-based cohort of 914 EoE probands (n = 2192 first-degree "Nuclear-Family" relatives) and an international registry of monozygotic and dizygotic twins/triplets (n = 63 EoE "Twins" probands). Frequencies, recurrence risk ratios (RRRs), heritability, and twin concordance were estimated. Environmental exposures were preliminarily examined. RESULTS: Analysis of the Nuclear-Family-based cohort revealed that the rate of EoE, in first-degree relatives of a proband, was 1.8% (unadjusted) and 2.3% (sex-adjusted). RRRs ranged from 10 to 64, depending on the family relationship, and were higher in brothers (64.0; P = .04), fathers (42.9; P = .004), and males (50.7; P < .001) than in sisters, mothers, and females, respectively. The risk of EoE for other siblings was 2.4%. In the Nuclear-Family cohort, combined gene and common environment heritability was 72.0% ± 2.7% (P < .001). In the Twins cohort, genetic heritability was 14.5% ± 4.0% (P < .001), and common family environment contributed 81.0% ± 4% (P < .001) to phenotypic variance. Probandwise concordance in monozygotic co-twins was 57.9% ± 9.5% compared with 36.4% ± 9.3% in dizygotic co-twins (P = .11). Greater birth weight difference between twins (P = .01), breast-feeding (P = .15), and fall birth season (P = .02) were associated with twin discordance in disease status. CONCLUSIONS: EoE RRRs are increased 10- to 64-fold compared with the general population. EoE in relatives is 1.8% to 2.4%, depending on relationship and sex. Nuclear-Family heritability appeared to be high (72.0%). However, the Twins cohort analysis revealed a powerful role for common environment (81.0%) compared with additive genetic heritability (14.5%).

PedsQL eosinophilic esophagitis module: feasibility, reliability, and validity.

OBJECTIVE: Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory condition with a paucity of information on health-related quality of life (HRQOL). The objective of the study was to report on the measurement properties of the PedsQL EoE Module. METHODS: The PedsQL EoE Module was completed in a multisite study by 196 pediatric patients with EoE and 262 parents of patients with EoE. RESULTS: The PedsQL EoE Module scales evidenced excellent feasibility (0.6%-3.1% missing), excellent group comparison reliability across total scale scores (patient α 0.93; parent proxy α 0.94), good reliability for the 7 individual scales (patient α 0.75-0.87; parent proxy α 0.81-0.92), excellent test-retest reliability (patient intraclass correlation coefficient 0.88; parent intraclass correlation coefficient 0.82), demonstrated no floor effects and low ceiling effects, and demonstrated a high percentage of scaling success for most scales. Intercorrelations with the PedsQL Generic Core Scales were in the medium (0.30) to large (0.50) range. PedsQL EoE Module scores were worse among patients with active histologic disease (≥ 5 eos/hpf) compared with those in remission (patient self-report: 63.3 vs 69.9 [P < 0.05]; parent proxy report: 65.1 vs 72.3 [P < 0.01]), and those treated with dietary restrictions compared with those with no restrictions (patient self-report: 61.6 vs 74.3 [P < 0.01]; parent proxy report: 65.5 vs 74.7 [P < 0.01]). CONCLUSIONS: The results demonstrate excellent measurement properties of the PedsQL EoE Module. Patients with active histologic disease and those treated with dietary restrictions demonstrated worse PedsQL scores. The PedsQL EoE Module may be used in the evaluation of pediatric EoE disease-specific HRQOL in clinical research and practice.

MicroRNA signature in patients with eosinophilic esophagitis, reversibility with glucocorticoids, and assessment as disease biomarkers.

BACKGROUND: The role of microRNAs (miRNAs), a key class of regulators of mRNA expression and translation, in patients with eosinophilic esophagitis (EoE) has not been explored. OBJECTIVE: We aimed to identify miRNAs dysregulated in patients with EoE and assess the potential of these miRNAs as disease biomarkers. METHODS: Esophageal miRNA expression was profiled in patients with active EoE and those with glucocorticoid-induced disease remission. Expression profiles were compared with those of healthy control subjects and patients with chronic (noneosinophilic) esophagitis. Expression levels of the top differentially expressed miRNAs from the plasma of patients with active EoE and patients with EoE remission were compared with those of healthy control subjects. RESULTS: EoE was associated with 32 differentially regulated miRNAs and was distinguished from noneosinophilic forms of esophagitis. The expression levels of the most upregulated miRNAs (miR-21 and miR-223) and the most downregulated miRNA (miR-375) strongly correlated with esophageal eosinophil levels. Bioinformatic analysis predicted interplay of miR-21 and miR-223 with key roles in the polarization of adaptive immunity and regulation of eosinophilia, and indeed, these miRNAs correlated with key elements of the EoE transcriptome. The differentially expressed miRNAs were largely reversible in patients who responded to glucocorticoid treatment. EoE remission induced a single miRNA (miR-675) likely to be involved in DNA methylation. Plasma analysis of the most upregulated esophageal miRNAs identified miR-146a, miR-146b, and miR-223 as the most differentially expressed miRNAs in the plasma. CONCLUSIONS: We have identified a marked dysregulated expression of a select group of miRNAs in patients with EoE and defined their reversibility with glucocorticoid treatment and their potential value as invasive and noninvasive biomarkers.

Do Prophylactic Inferior Vena Cava Filters Affect the Rates of Venous Thromboembolism and Pulmonary Embolism in Patients Undergoing Major Spine Surgery?

STUDY DESIGN: Retrospective Analysis. BACKGROUND: Venous thromboembolism (VTE) represents a significant cause of morbidity and mortality in major spine surgery. Placement of prophylactic inferior vena cava filters (IVCF) in patients undergoing major spine surgery was previously adopted at our institution. This study reports our experience and compares VTE rates between patients with and without preoperative IVCF placement. METHODS: A Retrospective comparative study was conducted on adult patients who underwent IVCF placement and those who did not prior to their spinal fusion procedure, between 2013 and 2016. Thoracolumbar fusions (anterior and/or posterior) of 7 or more levels, spinal osteotomies, and a minimum of a 3-month follow-up were included. Traumatic, oncologic, and cervical pathology were excluded. Primary outcomes measured included the incidence of overall VTE (DVT/PE), death, IVCF related complications, and IVCF retrieval. RESULTS: 386 patients who underwent major spine surgery, 258 met the eligibility criteria. Of those patients, 105 patients (40.7%) had prophylactic IVCF placement. All patients had postoperative SCDs and chemoprophylaxis. The presence of an IVCF was associated with an increased rate of overall VTE (14.3% vs 6.5%, P ≤ .05) and DVT episodes (8.6% vs 2.6%, P = .04). The rate of PE for the IVCF group and non-IVCF group was 8.6% and 4.6%, respectively, which was not statistically significant (P = .32). The all-cause mortality rate overall of 2.3% was statistically similar between both groups (P = 1.0). The IVCF group had higher rates of hematoma/seroma vs the non-IVCF group (12.4% vs 3.9%, P ≤ .05). 99 IVCFs were retrievable designs, and 85% were successfully retrieved. Overall IVCF-related complication rate was 11%. CONCLUSIONS: No statistical difference in PE or mortality rates existed between the IVCF and the control group. Patients with IVCF placement experienced approximately twice the rate of VTE and three times the rate of DVT compared to those without IVCF. The IVCF-related complication rate was 11%. Based on the results of this study, the authors recommend against the routine use of prophylactic IVCFs in adults undergoing major spine surgery. LEVEL OF EVIDENCE: III.

Setting cumulative emissions targets to reduce the risk of dangerous climate change.

Avoiding "dangerous anthropogenic interference with the climate system" requires stabilization of atmospheric greenhouse gas concentrations and substantial reductions in anthropogenic emissions. Here, we present an inverse approach to coupled climate-carbon cycle modeling, which allows us to estimate the probability that any given level of carbon dioxide (CO2) emissions will exceed specified long-term global mean temperature targets for "dangerous anthropogenic interference," taking into consideration uncertainties in climate sensitivity and the carbon cycle response to climate change. We show that to stabilize global mean temperature increase at 2 degrees C above preindustrial levels with a probability of at least 0.66, cumulative CO2 emissions from 2000 to 2500 must not exceed a median estimate of 590 petagrams of carbon (PgC) (range, 200 to 950 PgC). If the 2 degrees C temperature stabilization target is to be met with a probability of at least 0.9, median total allowable CO2 emissions are 170 PgC (range, -220 to 700 PgC). Furthermore, these estimates of cumulative CO2 emissions, compatible with a specified temperature stabilization target, are independent of the path taken to stabilization. Our analysis therefore supports an international policy framework aimed at avoiding dangerous anthropogenic interference formulated on the basis of total allowable greenhouse gas emissions.

Resorbable Pins to Enhance Scaffold Retention in a Porcine Chondral Defect Model.

OBJECTIVE: Cartilage repair strategies have seen improvement in recent years, especially with the use of scaffolds that serve as a template for cartilage formation. However, current fixation strategies are inconsistent with regards to retention, may be technically challenging, or may damage adjacent tissues or the implant itself. Therefore, the goal of this study was to evaluate the retention and repair potential of cartilage scaffolds fixed with an easy-to-implement bioresorbable pin. DESIGN: Electrospun hyaluronic acid scaffolds were implanted into trochlear groove defects in 3 juvenile and 3 adult pigs to evaluate short-term retention (2 weeks; pin fixation vs. press-fit and fibrin fixation) and long-term repair (8 months; scaffold vs. microfracture), respectively. RESULTS: For the retention study, press-fit and fibrin fixation resulted in short-term scaffold dislodgment (n = 2 each), whereas pin fixation retained all scaffolds that were implanted (n = 6). Pin fixation did not cause any damage to the opposing patellar surface, and only minor changes in the subchondral bone were observed. For long-term repair, no differences were observed between microfracture and scaffold groups, in terms of second-look arthroscopy and indentation testing. On closer visualization with micro computed tomography and histology, a high degree of variability was observed between animals with regard to subchondral bone changes and cartilage repair quality, yet each Scaffold repair displayed similar properties to its matched microfracture control. CONCLUSIONS: In this study, pin fixation did not cause adverse events in either the short- or the long-term relative to controls, indicating that pin fixation successfully retained scaffolds within defects without inhibiting repair.

Nanofibrous hyaluronic acid scaffolds delivering TGF-ß3 and SDF-1α for articular cartilage repair in a large animal model.

Focal cartilage injuries have poor intrinsic healing potential and often progress to osteoarthritis, a costly disease affecting almost a third of adults in the United States. To treat these patients, cartilage repair therapies often use cell-seeded scaffolds, which are limited by donor site morbidity, high costs, and poor efficacy. To address these limitations, we developed an electrospun cell-free fibrous hyaluronic acid (HA) scaffold that delivers factors specifically designed to enhance cartilage repair: Stromal Cell-Derived Factor-1α (SDF-1α; SDF) to increase the recruitment and infiltration of mesenchymal stem cells (MSCs) and Transforming Growth Factor-ß3 (TGF-ß3; TGF) to enhance cartilage tissue formation. Scaffolds were characterized in vitro and then deployed in a large animal model of full-thickness cartilage defect repair. The bioactivity of both factors was verified in vitro, with both SDF and TGF increasing cell migration, and TGF increasing matrix formation by MSCs. In vivo, however, scaffolds releasing SDF resulted in an inferior cartilage healing response (lower mechanics, lower ICRS II histology score) compared to scaffolds releasing TGF alone. These results highlight the importance of translation into large animal models to appropriately screen scaffolds and therapies, and will guide investigators towards alternative growth factor combinations. STATEMENT OF SIGNIFICANCE: This study addresses an area of orthopaedic medicine in which treatment options are limited and new biomaterials stand to improve patient outcomes. Those suffering from articular cartilage injuries are often destined to have early onset osteoarthritis. We have created a cell-free nanofibrous hyaluronic acid (HA) scaffold that delivers factors specifically designed to enhance cartilage repair: Stromal Cell-Derived Factor-1α (SDF-1α; SDF) to increase the recruitment and infiltration of mesenchymal stem cells (MSCs) and Transforming Growth Factor-ß3 (TGF-ß3; TGF) to enhance cartilage tissue formation. To our knowledge, this study is the first to evaluate such a bioactive scaffold in a large animal model and demonstrates the capacity for dual growth factor release.

Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14-mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.

Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO.

The NF-kappaB family of transcription factors is activated in response to many stimuli, including pro-inflammatory cytokines, environmental stresses and, in the case of B and T lymphocytes, by antigenic stimulation. Bcl10 is essential for NF-kappaB activation by T- and B-cell receptors. T and B lymphocytes from Bcl10-deficient mice fail to activate NF-kappaB in response to antigen-receptor stimulation and, as a consequence, are unable to proliferate. Bcl10 overexpression is sufficient to activate NF-kappaB, a process that requires the NF-kappaB essential modulator NEMO (also known as IKK-gamma), which is the regulatory subunit of the IkappaB kinase complex. However, the cellular mechanism by which Bcl10 activates the NF-kappaB pathway remains unclear. Here we show that Bcl10 targets NEMO for lysine-63-linked ubiquitination. Notably, a mutant form of NEMO that cannot be ubiquitinated inhibited Bcl10-induced NF-kappaB activation. Paracaspase and a ubiquitin-conjugating enzyme (UBC13) were both required for Bcl10-induced NEMO ubiquitination and subsequent NF-kappaB activation. Furthermore, short interfering RNAs that reduced the expression of paracaspase and UBC13 abrogated the effects of Bcl10. Thus, the adaptor protein Bcl10 promotes activation of NF-kappaB transcription factors through paracaspase- and UBC13-dependent ubiquitination of NEMO.

De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling.

NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta. Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.

Transection of the medial meniscus anterior horn results in cartilage degeneration and meniscus remodeling in a large animal model.

The meniscus plays a central load-bearing role in the knee joint. Unfortunately, meniscus injury is common and can lead to joint degeneration and osteoarthritis (OA). In small animal models, progressive degenerative changes occur with the unloading of the meniscus via destabilization of the medial meniscus (DMM). However, few large animal models of DMM exist and the joint-wide initiation of the disease has not yet been defined in these models. Thus, the goal of this study is to develop and validate a large animal model of surgically induced DMM and to use multimodal (mechanical, histological, and magnetic resonance imaging) and multiscale (joint to tissue level) quantitative measures to evaluate degeneration in both the meniscus and cartilage. DMM was achieved using an arthroscopic approach in 13 Yucatan minipigs. One month after DMM, joint contact area decreased and peak pressure increased, indicating altered load transmission as a result of meniscus destabilization. By 3 months, the joint had adapted to the injury and load transmission patterns were restored to baseline, likely due to the formation and maturation of a fibrovascular scar at the anterior aspect of the meniscus. Despite this, we found a decrease in the indentation modulus of the tibial cartilage and an increase in cartilage histopathology scores at 1 month compared to sham-operated animals; these deleterious changes persisted through 3 months. Over this same time course, meniscus remodeling was evident through decreased proteoglycan staining in DMM compared to sham menisci at both 1 and 3 months. These findings support that arthroscopic DMM results in joint degeneration in the Yucatan minipig and provide a new large animal testbed in which to evaluate therapeutics and interventions to treat post-traumatic OA that originates from a meniscal injury.

Pediatric Hypereosinophilia: Characteristics, Clinical Manifestations, and Diagnoses.

BACKGROUND: Eosinophilia is associated with various conditions, including allergic, infectious, and neoplastic disorders. The diagnostic differential is broad, and data on hypereosinophilia in pediatric patients are limited. OBJECTIVE: The objectives of this study were to identify cases of hypereosinophilia in a tertiary pediatric medical center, determine clinical characteristics and disease associations, and estimate the incidence of hypereosinophilia in the hospital and geographic populations. METHODS: A retrospective chart review included patients younger than 18 years presenting to a tertiary pediatric medical center (January 1, 2008, to May 31, 2017) with absolute eosinophil counts (AECs) greater than or equal to 1.50 thousand eosinophils/microliter (K/µL) recorded on at least 2 occasions at least 4 weeks apart (N = 176). Clinical characteristics, laboratory values, treatment course, and associated diagnoses were evaluated. RESULTS: The most common cause of hypereosinophilia in this cohort was secondary hypereosinophilia. Atopic dermatitis, graft-versus-host disease, sickle cell disease, and parasitic infections were the most common conditions associated with hypereosinophilia. Median age at diagnosis was 4.6 (interquartile range, 1.5-10.5) years. Median peak AEC was 3.16 (2.46-4.78) K/µL. Hypereosinophilia occurred most frequently in patients aged between 6 and 11 years (24.4%) and younger than 1 year (18.2%). Patients with neoplasms and immune deficiencies had significantly higher peak AECs than did patients with overlap hypereosinophilic syndrome and atopic diseases (P < .0001). CONCLUSIONS: Pediatric hypereosinophilia has an incidence of 54.4 per 100,000 persons per year, with children younger than 1 year and aged 6 to 11 years accounting for most affected patients. Pediatric hypereosinophilia is not uncommon and remains underrecognized, highlighting a need for clinicians to identify patients who meet criteria for hypereosinophilia and to pursue a thorough evaluation.

Oncogenic BRAF is required for tumor growth and maintenance in melanoma models.

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.

Eosinophilic oesophagitis endotype classification by molecular, clinical, and histopathological analyses: a cross-sectional study.

BACKGROUND: Eosinophilic oesophagitis is understood in terms of quantifiable histological, endoscopic, and molecular features. Data are scant for inter-relations of these features and their potential to identify distinct disease endotypes. We aimed to identify clinical-pathological correlations between endoscopic and histological disease variables by transcription profiling of the oesophagus of patients with eosinophilic oesophagitis of varying severity and disease activity states. METHODS: We did a cross-sectional study across ten hospital sites in the USA associated with the Consortium of Eosinophilic Gastrointestinal Disease Researchers. We analysed oesophageal biopsy specimens taken from paediatric and adult patients with eosinophilic oesophagitis (discovery cohort), using the eosinophilic oesophagitis diagnostic panel (EDP), a set of 96 informative transcripts. Histological and endoscopic features were assessed by quantification of oesophageal eosinophils and use of the eosinophilic oesophagitis histology scoring system (HSS) and the eosinophilic oesophagitis endoscopic reference score (EREFS). Associations among the various histological, endoscopic, and molecular features were analysed by Spearman correlation. Results were replicated in a biologically independent, single-centre, validation cohort of patients with active eosinophilic oesophagitis. FINDINGS: The discovery cohort contained 185 samples and the validation cohort comprised 100 specimens. In the discovery cohort, EDP showed intersite consistency, significant correlation with oesophageal eosinophils (p<0·0001), and similar findings between paediatric and adult patients. Of eight HSS domains, basal zone hyperplasia correlated with the EDP (median Spearman ρ 0·47 [IQR 0·36-0·60]). Of five EREFS features, distal furrows correlated with the EDP (median Spearman ρ 0·42 [0·32-0·50]). By analysing active eosinophilic oesophagitis in the discovery cohort, the EDP identified three clusters associated with distinct endotypes (termed EoEe1-3) despite similar eosinophil levels. EoEe1 was associated with a normal-appearing oesophagus (risk ratio [RR] 3·27, 95% CI 1·04-10·27; p=0·0443), an inverse association with a history of oesophageal dilation (0·27, 0·09-0·82; p=0·0105) and showed relatively mild histological, endoscopic, and molecular changes. EoEe2 showed an inflammatory and steroid-refractory phenotype (RR 2·77, 95% CI 1·11-6·95; p=0·0376) and had the highest expression of inflammatory cytokines and steroid-responding genes. EoEe3 was associated with a narrow-calibre oesophagus (RR 7·98, 95% CI 1·84-34·64; p=0·0013) and adult onset (2·22, 1·19-4·12; p=0·0155), and showed the highest degree of endoscopic and histological severity and the lowest expression of epithelial differentiation genes. These endotypes were replicated in the validation cohort by clustering and with an eosinophilic oesophagitis endotype-prediction algorithm. INTERPRETATION: Our new disease classification stratifies patients with eosinophilic oesophagitis into subgroups with potential clinical and therapeutic significance and provides a framework for a precision medicine approach to eosinophilic oesophagitis. FUNDING: National Institutes of Health.

Regulation of ERK3/MAPK6 expression by BRAF.

Several forms of cancer are characterized by frequent activating mutations in the serine/threonine kinase, BRAF. Substitution of glutamic acid for valine at codon 600 (V600E) accounts for approximately 90% of all BRAF activating mutations and leads to stimulation of kinase activity, downstream signaling, and cell transformation. To better understand the molecular pathogenesis induced by oncogenic BRAF signaling, we used microarray gene expression profiling to comprehensively analyze the BRAF-directed transcriptional program of cells expressing a conditionally active form of BRAFV600E. Several novel genes that affect proliferation, cell survival, angiogenesis and immune surveillance were identified as possible mediators of BRAF-induced oncogenic signaling. Moreover, we show that a MAPK family member, extracellular signal-regulated kinase-3 (ERK3/MAPK6) is highly expressed in response to BRAF signaling in this system. Cellular ERK3 protein is highly unstable and pharmacological inhibition of BRAF activity resulted in rapid ERK3 degradation. In melanoma cells, RNAi-mediated knockdown of endogenous BRAF or treatment with MEK inhibitors that prevent ERK1/2 activation led to a reduction in ERK3 levels, indicating that elevated ERK3 expression is mediated through MEK1/2 signaling. These results provide strong evidence for another mode by which BRAF can regulate the ERK protein kinase family and suggest ERK3 to be a potential pharmacodynamic marker for targeting BRAF signaling in melanoma.