Patient-derived colon epithelial organoids reveal lipid-related metabolic dysfunction in pediatric ulcerative colitis.

Ulcerative colitis (UC) is associated with epithelial metabolic derangements which exacerbate gut inflammation. Patient-derived organoids recapitulate complexities of the parent tissue in health and disease; however, whether colon organoids (colonoids) model the metabolic impairments in the pediatric UC epithelium is unclear. Here, we developed colonoid lines from pediatric patients with endoscopically active UC, inactive UC, and those without endoscopic or histologic evidence of colon inflammation (non-IBD controls) to interrogate functional metabolic differences in the colon epithelia. We demonstrate that colonoids from active UC patients exhibit hypermetabolic features and cellular stress, specifically during differentiation. Hypermetabolism in differentiating active UC colonoids was driven, in part, by increased proton leak, and supported by enhanced glycolytic capacity and dysregulated neutral lipid accumulation. Transcriptomic and pathway analyses indicated a role for PPAR-α in lipid-induced hypermetabolism in aUC colonoids, which was validated by PPAR-α activation in non-IBD colonoids. Accordingly, limiting neutral lipid accumulation in active UC colonoids through pharmacological inhibition of PPAR-α induced a metabolic shift towards glucose utilization, suppressed hypermetabolism and chemokine secretion, and improved markers of cellular stress and epithelial differentiation. Taken together, we reveal a role for lipid-related metabolic dysfunction in the pediatric UC epithelium and support the advancement of colonoids as a preclinical human model for testing epithelial-directed therapies against such metabolic dysfunction.

Clinical validation of a blood-based liquid biopsy test integrating cell-free DNA quantification and next-generation sequencing for cancer screening in dogs.

OBJECTIVE: To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis. SAMPLE: Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets. METHODS: Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected). RESULTS: Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%. CLINICAL RELEVANCE: This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs.

Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2-3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

OBJECTIVE: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory. SAMPLE: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022. PROCEDURES: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022. RESULTS: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases. CLINICAL RELEVANCE: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

Age at cancer diagnosis by breed, weight, sex, and cancer type in a cohort of more than 3,000 dogs: Determining the optimal age to initiate cancer screening in canine patients.

The goal of cancer screening is to detect disease at an early stage when treatment may be more effective. Cancer screening in dogs has relied upon annual physical examinations and routine laboratory tests, which are largely inadequate for detecting preclinical disease. With the introduction of non-invasive liquid biopsy cancer detection methods, the discussion is shifting from how to screen dogs for cancer to when to screen dogs for cancer. To address this question, we analyzed data from 3,452 cancer-diagnosed dogs to determine the age at which dogs of certain breeds and weights are typically diagnosed with cancer. In our study population, the median age at cancer diagnosis was 8.8 years, with males diagnosed at younger ages than females, and neutered dogs diagnosed at significantly later ages than intact dogs. Overall, weight was inversely correlated with age at cancer diagnosis, and purebred dogs were diagnosed at significantly younger ages than mixed-breed dogs. For breeds represented by ≥10 dogs, a breed-based median age at diagnosis was calculated. A weight-based linear regression model was developed to predict the median age at diagnosis for breeds represented by ≤10 dogs and for mixed-breed dogs. Our findings, combined with findings from previous studies which established a long duration of the preclinical phase of cancer development in dogs, suggest that it might be reasonable to consider annual cancer screening starting 2 years prior to the median age at cancer diagnosis for dogs of similar breed or weight. This logic would support a general recommendation to start cancer screening for all dogs at the age of 7, and as early as age 4 for breeds with a lower median age at cancer diagnosis, in order to increase the likelihood of early detection and treatment.

Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs.

BACKGROUND: Guidelines-driven screening protocols for early cancer detection in dogs are lacking, and cancer often is detected at advanced stages. HYPOTHESIS/OBJECTIVES: To examine how cancer typically is detected in dogs and whether the addition of a next-generation sequencing-based "liquid biopsy" test to a wellness visit has the potential to enhance cancer detection. ANIMALS: Client-owned dogs with definitive cancer diagnoses enrolled in a clinical validation study for a novel blood-based multicancer early detection test. METHODS: Retrospective medical record review was performed to establish the history and presenting complaint that ultimately led to a definitive cancer diagnosis. Blood samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. RESULTS: In an unselected cohort of 359 cancer-diagnosed dogs, 4% of cases were detected during a wellness visit, 8% were detected incidentally, and 88% were detected after the owner reported clinical signs suggestive of cancer. Liquid biopsy detected disease in 54.7% (95% confidence interval [CI], 49.5%-59.8%) of patients, including 32% of dogs with early-stage cancer, 48% of preclinical dogs, and 84% of dogs with advanced-stage disease. CONCLUSIONS/CLINICAL IMPORTANCE: Most cases of cancer were diagnosed after the onset of clinical signs; only 4% of dogs had cancer detected using the current standard of care (i.e., wellness visit). Liquid biopsy has the potential to increase detection of cancer when added to a dog's wellness visit.

Multi-Family Pediatric Pain Group Therapy: Capturing Acceptance and Cultivating Change.

Behavioral health interventions for pediatric chronic pain include cognitive-behavioral (CBT), acceptance and commitment (ACT), and family-based therapies, though literature regarding multi-family therapy (MFT) is sparse. This investigation examined the utility and outcomes of the Courage to Act with Pain: Teens Identifying Values, Acceptance, and Treatment Effects (CAPTIVATE) program, which included all three modalities (CBT, ACT, MFT) for youth with chronic pain and their parents. Program utility, engagement, and satisfaction were evaluated via quantitative and qualitative feedback. Pain-specific psychological, behavioral, and interpersonal processes were examined along with outcomes related to disability, quality of life, pain interference, fatigue, anxiety, and depressive symptoms. Participants indicated that CAPTIVATE was constructive, engaging, and helpful for social and family systems. Clinical and statistical improvements with large effect sizes were captured for pain catastrophizing, acceptance, and protective parenting but not family functioning. Similar effects were found for functional disability, pain interference, fatigue, anxiety, and depression. Given the importance of targeting multiple systems in the management of pediatric chronic pain, preliminary findings suggest a potential new group-based treatment option for youth and families. Next steps involve evaluating the differential effect of the program over treatment as usual, as well as specific CBT, ACT, and MFT components and processes that may affect outcomes.

The Effect of Pain Catastrophizing on Outcomes: A Developmental Perspective Across Children, Adolescents, and Young Adults With Chronic Pain.

Pain catastrophizing is one of the most powerful predictors of poor outcomes in youth and adults with pain; however, little is known about differential effects of pain catastrophizing on outcomes as a function of age. The current study examined the predictive value of pain catastrophizing on pain interference and pain intensity across children, adolescents, and 2 age groups of young adults with chronic pain. Cross-sectional data are presented from the adult and pediatric Collaborative Health Outcomes Information Registry (CHOIR), including measures of pain catastrophizing, pain intensity, pain interference, and emotional distress from 1,028 individuals with chronic pain. Results revealed that age moderated the relation between pain catastrophizing and pain interference, with the strength of these effects declining with age. The effect of pain catastrophizing on pain interference was strongest in adolescents and relatively weak in all 3 other groups. Emotional distress was the strongest predictor of pain interference for children, whereas pain intensity was the strongest predictor for both adult groups. Pain catastrophizing was found to predict pain intensity and, although age was a significant moderator, statistical findings were weak. Developmental considerations and clinical implications regarding the utility of the construct of pain catastrophizing across age groups are discussed. PERSPECTIVE: This article explores differences in pain catastrophizing as predictors of pain interference and pain intensity across cohorts of children, adolescents, and 2 age groups of young adults. This work may stimulate further research on chronic pain from a developmental perceptive and inform developmentally tailored treatment interventions that target catastrophizing, emotional distress, and pain intensity.

Survey of external cephalic version for breech presentation and neuraxial blockade use.

STUDY OBJECTIVE: Neuraxial blockade may increase external cephalic version (ECV) success rates. This survey aimed to assess the frequency and characteristics of neuraxial blockade used to facilitate ECV. SETTING AND DESIGN: We surveyed Society for Obstetric Anesthesia and Perinatology members regarding ECV practice using a 15-item survey developed by 3 obstetric anesthesiologists and tested for face validity. The survey was e-mailed in January 2015 and again in February 2015 to the 1056 Society of Obstetric Anesthesiology and Perinatology members. We present descriptive statistics of responses. PARTICIPANTS: Our survey response rate was 322 of 1056 (30.5%). MAIN RESULT: Neuraxial blockade was used for ECV always by 18 (5.6%), often by 52 (16.1%), sometimes by 98 (30.4%), rarely by 78 (24.2%), and never by 46 (14.3%) of respondents. An anesthetic sensory block target was selected by 141 (43.8%) respondents, and analgesic by 102 (31.7%) respondents. Epidural drug doses ranged widely, including sufentanil 5-25 µg; lidocaine 1% or 2% 10-20 mL, bupivacaine 0.0625% to 0.5% 6-15 mL, and ropivacaine 0.2% 20 mL. Intrathecal bupivacaine was used by 182 (56.5%) respondents; the most frequent doses were 2.5 mg used by 24 (7.5%), 7.5 mg used by 35 (10.9%), and 12 mg used by 30 (9.3%). CONCLUSIONS: Neuraxial blockade is not universally offered to facilitate ECV, and there is wide variability in neuraxial blockade techniques, in drugs and doses administered, and in the sensory blockade (anesthetic or analgesic) targeted. Future studies need to evaluate and remove barriers to allow for more widespread use of neuraxial blockade for pain relief and to optimize ECV success rates.

Pediatric-Collaborative Health Outcomes Information Registry (Peds-CHOIR): a learning health system to guide pediatric pain research and treatment.

The pediatric adaptation of the Collaborative Health Outcomes Information Registry (Peds-CHOIR) is a free, open-source, flexible learning health care system (LHS) that meets the call by the Institute of Medicine for the development of national registries to guide research and precision pain medicine. This report is a technical account of the first application of Peds-CHOIR with 3 aims: (1) to describe the design and implementation process of the LHS; (2) to highlight how the clinical system concurrently cultivates a research platform rich in breadth (eg, clinic characteristics) and depth (eg, unique patient- and caregiver-reporting patterns); and (3) to demonstrate the utility of capturing patient-caregiver dyad data in real time, with dynamic outcomes tracking that informs clinical decisions and delivery of treatments. Technical, financial, and systems-based considerations of Peds-CHOIR are discussed. Cross-sectional retrospective data from patients with chronic pain (N = 352; range, 8-17 years; mean, 13.9 years) and their caregivers are reported, including National Institutes of Health Patient-Reported Outcomes Measurement Information System (PROMIS) domains (mobility, pain interference, fatigue, peer relations, anxiety, and depression) and the Pain Catastrophizing Scale. Consistent with the literature, analyses of initial visits revealed impairments across physical, psychological, and social domains. Patients and caregivers evidenced agreement in observable variables (mobility); however, caregivers consistently endorsed greater impairment regarding internal experiences (pain interference, fatigue, peer relations, anxiety, and depression) than patients' self-report. A platform like Peds-CHOIR highlights predictors of chronic pain outcomes on a group level and facilitates individually tailored treatment(s). Challenges of implementation and future directions are discussed.

Secreted HoxA3 Promotes Epidermal Proliferation and Angiogenesis in Genetically Modified Three-Dimensional Composite Skin Constructs.

Objective: Homeobox (HOX) transcription factors coordinate gene expression in wound repair and angiogenesis. Previous studies have shown that gene transfer of HoxA3 to wounds of diabetic mice accelerates wound healing, increasing angiogenesis and keratinocyte migration. In this study, we examined whether HoxA3 can also improve angiogenesis, epidermal integrity, and viability of composite skin grafts. Approach: To determine the effects of HoxA3 on composite skin grafts, we constructed bilayered composite grafts incorporating fibroblasts engineered to constitutively secrete HoxA3. We then transplanted these composite grafts in vivo. Results: The composite grafts produced a stratified epidermal layer after seventeen days in culture and following transplantation in vivo, these grafts exhibit normal epidermal differentiation and reduced contraction compared to controls. In addition, HoxA3 grafts showed increased angiogenesis. Quantitative polymerase chain reaction (PCR) analyses of HoxA3 graft tissue reveal an increase in the downstream HoxA3 target genes MMP-14 and uPAR expression, as well as a reduction in CCL-2 and CxCl-12. Innovation: Expression of secreted HoxA3 in composite grafts represents a comprehensive approach that targets both keratinocytes and endothelial cells to promote epidermal proliferation and angiogenesis. Conclusion: Secreted HoxA3 improves angiogenesis, reduces expression of inflammatory mediators, and prolongs composite skin graft integrity.

Temporal changes in Hox gene expression accompany endothelial cell differentiation of embryonic stem cells.

In pluripotent embryonic stem cells (ESCs), expression of the Hox master regulatory transcription factors that play essential roles in organogenesis, angiogenesis, and maintenance of differentiated tissues, is globally suppressed. We investigated whether differentiation of endothelial cells (ECs) from mouse ESCs was accompanied by activation of distinct Hox gene expression profiles. Differentiation was observed within 3 days, as indicated by the appearance of cells expressing specific endothelial marker genes (Flk-1+ /VE-Cadherin+ ). Expression of HoxA3 and HoxD3, which drive adult endothelial cell invasion and angiogenesis, peaked at day 3 and declined thereafter, whereas expression of HoxA5 and HoxD10, which maintain a mature quiescent EC phenotype, was low at day 3, but increased over time. The temporal and reciprocal changes in HoxD3 and HoxA5 expression were accompanied by corresponding changes in expression of established downstream target genes including integrin ß3 and Thrombospondin-2. Our results indicate that differentiation and maturation of ECs derived from cultured ESCs mimic changes in Hox gene expression that accompany maturation of immature angiogenic endothelium into differentiated quiescent endothelium in vivo.

Single-stage reconstruction of a devastating antebrachial injury with brachial artery, median nerve, and soft tissue deficit: a case report and review of the literature.

OBJECTIVE: We present a case of a 31-year-old man who fell through a skylight sustaining a deep laceration injury to his dominant arm. A single-stage radial artery flow-through free flap and cabled sural nerve graft for reconstruction of a complex antebrachial defect involving skin, soft tissue, muscle, brachial artery, and median nerve was performed. A technical description of the case and review of the literature are described. METHODS: Traumatic injuries to the arm and antecubital fossa often lead to devastating outcomes. Advances in microsurgical technique as well as improved skin and dermal substitutes have allowed improved outcomes as well as shorter hospital stays. In this case, surgical treatment involved microsurgical reconstruction of the brachial artery with a radial artery flow-through flap and a single-stage donor-site closure with an Integra dermal matrix template and split-thickness skin graft. RESULTS: Successful vascular flow and soft tissue coverage were performed with successful salvage of the limb. CONCLUSION: A single-stage reconstruction versus a multistage, delayed reconstruction of a devastating arm injury with a radial forearm flow-through flap and single-stage closure with Integra and autologous skin graft can provide a safe, effective, and clinically satisfactory outcome.

Surgical treatment options for subacute ischemia of the hand: case report and literature review.

PURPOSE: The most effective surgical approach to the treatment of digital ischemia has not yet been established. The purpose of this study is to review currently accepted options for revascularization in acute and chronic settings of digital ischemia, and to augment this discussion by describing the approach of our surgical team in a unique case of subacute ischemia. OPERATIVE TECHNIQUE: To restore blood flow to a patient's ischemic hand, we performed a microvascular reconstruction, using a reverse interpositional vein graft with 3 anastomoses: the ulnar artery was used for inflow and the superficial palmar arch and the common digital artery were used for outflow. RESULTS: The patient experienced immediate postoperative pain relief. Blood flow was restored, which prevented digital amputation. The graft remained patent at 18 months' follow-up and the patient exhibited normal motor and sensory function. CONCLUSIONS: Surgical reconstruction of the hand is a viable treatment option for carefully selected patients presenting with subacute digital ischemia. Other medical and surgical techniques have been described in the recent literature, but further study is needed to determine the long-term success of newer microsurgical interventions.

Mice lacking both G-CSF and IL-6 are more susceptible to Candida albicans infection: critical role of neutrophils in defense against Candida albicans.

Neutrophils play an important role in the host's defense against infection with various pathogenic organisms. Granulocyte colony stimulating factor (G-CSF) is regarded as a major regulator of neutrophil production and function. Mice lacking G-CSF or its receptor are neutropenic. IL-6 is another cytokine that has been shown to promote neutrophil production and modulate the function of many types of immune cells. We have analyzed G-CSF/IL-6 double deficient (G-CSF(- / - )/IL-6(- / - )) mice to gain an insight into the possible contribution of IL-6 to the residual granulopoiesis in G-CSF-deficient (G-CSF(- / - )) mice. Furthermore, we have evaluated the ability of G-CSF(- / - )/IL-6(- / - ) mice to combat an experimental infection with Candida albicans. Our data shows that IL-6 plays a role in granulopoiesis during early post natal period but it is dispensable for steady-state granulopoiesis in adult mice. However, adult G-CSF(- / - )/IL-6(- / - ) mice are more susceptible to Candida infection than similarly infected G-CSF(- / - ) mice. Although, the candidacidal function of neutrophils of G-CSF(- / - )/IL-6(- / - ) mice is deficient, the ability to produce IFN-gamma and TNF-alpha in response to Candida infection is not compromised. Similarly, nitric oxide production by peritoneal macrophages from G-CSF(- / - )/IL-6(- / - ) mice in response to Candida is comparable to G-CSF(- / - ) mice.

Macrophage-colony stimulating factor is required for the production of neutrophil-promoting activity by mouse embryo fibroblasts deficient in G-CSF and GM-CSF.

G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an "in vitro" technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF(-/-)/GM-CSF(-/-) mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF(-/-)/GM-CSF(-/-) MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF(-/-)/GM-CSF(-/-) MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF(-/-)/GM-CSF(-/-)/M-CSF(-/-) fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.