The regenerative capacity of neonatal tissues.

It is well established that humans and other mammals are minimally regenerative compared with organisms such as zebrafish, salamander or amphibians. In recent years, however, the identification of regenerative potential in neonatal mouse tissues that normally heal poorly in adults has transformed our understanding of regenerative capacity in mammals. In this Review, we survey the mammalian tissues for which regenerative or improved neonatal healing has been established, including the heart, cochlear hair cells, the brain and spinal cord, and dense connective tissues. We also highlight common and/or tissue-specific mechanisms of neonatal regeneration, which involve cells, signaling pathways, extracellular matrix, immune cells and other factors. The identification of such common features across neonatal tissues may direct therapeutic strategies that will be broadly applicable to multiple adult tissues.

Antibody response to a third SARS-CoV-2 vaccine dose in recipients of an allogeneic haematopoietic cell transplantation.

Allogeneic haematopoietic cell transplantation (allo-HCT) recipients show impaired antibody (Ab) response to a standard two-dose vaccination against severe acute respiratory syndrome coronavirus-2 and currently a third dose is recommended as part of the primary vaccination regimen. By assessing Ab titres 1 month after a third mRNA vaccine dose in 74 allo-HCT recipients we show sufficient neutralisation activity in 77% of the patients. Discontinuation of immunosuppression before the third vaccine led to serological responses in 50% of low responders to two vaccinations. Identifying factors that might contribute to better vaccine responses in allo-HCT recipients is critical to optimise current vaccination strategies.

Subjective emotion trajectories in couple therapy and associations with improvement in relationship satisfaction.

Existing couple therapies are generally effective for reducing romantic relationship distress and divorce, but therapy outcomes remain poor for many. Outcomes can be improved through greater understanding of session-by-session therapeutic processes, particularly in real-world treatment settings. Modern couple therapy models commonly emphasize the importance of emotional experiences as key change processes, yet few empirical studies have tested the merits of this focus. The present study addresses this limitation by examining trajectories of subjective emotions and their association with change in a key relationship outcome, relationship satisfaction, among military veterans and their partners at a VA Medical Center. Partners rated their relationship satisfaction prior to couple therapy sessions and subjective emotions immediately after sessions. Consistent with hypotheses, both hard (e.g., anger) and soft (e.g., sadness) negative emotions decreased significantly over the course of therapy. Those couples with greater decreases in hard negative, but not soft negative, emotions showed significantly more improvement in relationship satisfaction. Positive emotions did not significantly change across couples in general, but those couples whose positive emotions did increase also showed more improvement in relationship satisfaction. These results suggest change in subjective emotions may be one process underlying improvement in couple therapy and lend empirical support to the emphasis on emotion-based change processes underlying acceptance-based and emotion-focused couple therapies.

Loss of Smad4 in the scleraxis cell lineage results in postnatal joint contracture.

Growth of the musculoskeletal system requires precise coordination between bone, muscle, and tendon during development. Insufficient elongation of the muscle-tendon unit relative to bone growth results in joint contracture, a condition characterized by reduction or complete loss of joint range of motion. Here we establish a novel murine model of joint contracture by targeting Smad4 for deletion in the tendon cell lineage using Scleraxis-Cre (ScxCre). Smad4ScxCre mutants develop a joint contracture shortly after birth. The contracture is stochastic in direction and increases in severity with age. Smad4ScxCre mutant tendons exhibited a stable reduction in cellularity and a progressive reduction in extracellular matrix volume. Collagen fibril diameters were reduced in the Smad4ScxCre mutants, suggesting a role for Smad4 signaling in the regulation of matrix accumulation. Although ScxCre also has sporadic activity in both cartilage and muscle, we demonstrate an essential role for Smad4 loss in tendons for the development of joint contractures. Disrupting the canonical TGFß-pathway in Smad2;3ScxCre mutants did not result in joint contractures. Conversely, disrupting the BMP pathway by targeting BMP receptors (Alk3ScxCre/Alk6null) recapitulated many features of the Smad4ScxCre contracture phenotype, suggesting that joint contracture in Smad4ScxCre mutants is caused by disruption of BMP signaling. Overall, these results establish a model of murine postnatal joint contracture and a role for BMP signaling in tendon elongation and extracellular matrix accumulation.

Requirement for scleraxis in the recruitment of mesenchymal progenitors during embryonic tendon elongation.

The transcription factor scleraxis (Scx) is required for tendon development; however, the function of Scx is not fully understood. Although Scx is expressed by all tendon progenitors and cells, only long tendons are disrupted in the Scx-/- mutant; short tendons appear normal and the ability of muscle to attach to skeleton is not affected. We recently demonstrated that long tendons are formed in two stages: first, by muscle anchoring to skeleton via a short tendon anlage; and second, by rapid elongation of the tendon in parallel with skeletal growth. Through lineage tracing, we extend these observations to all long tendons and show that tendon elongation is fueled by recruitment of new mesenchymal progenitors. Conditional loss of Scx in mesenchymal progenitors did not affect the first stage of anchoring; however, new cells were not recruited during elongation and long tendon formation was impaired. Interestingly, for tenocyte recruitment, Scx expression was required only in the recruited cells and not in the recruiting tendon. The phenotype of Scx mutants can thus be understood as a failure of tendon cell recruitment during tendon elongation.

Macrophage depletion impairs neonatal tendon regeneration.

Tendons are dense connective tissues that transmit muscle forces to the skeleton. After adult injury, healing potential is generally poor and dominated by scar formation. Although the immune response is a key feature of healing, the specific immune cells and signals that drive tendon healing have not been fully defined. In particular, the immune regulators underlying tendon regeneration are almost completely unknown due to a paucity of tendon regeneration models. Using a mouse model of neonatal tendon regeneration, we screened for immune-related markers and identified upregulation of several genes associated with inflammation, macrophage chemotaxis, and TGFß signaling after injury. Depletion of macrophages using AP20187 treatment of MaFIA mice resulted in impaired functional healing, reduced cell proliferation, reduced ScxGFP+ neo-tendon formation, and altered tendon gene expression. Collectively, these results show that inflammation is a key component of neonatal tendon regeneration and demonstrate a requirement for macrophages in effective functional healing.

Outcomes after liver transplantation in MPV17 deficiency (Navajo neurohepatopathy): A single-center case series.

BACKGROUND: MPV17-related mitochondrial DNA maintenance defect (MPV17 deficiency) is a rare, autosomal recessive mitochondrial DNA depletion syndrome with a high mortality rate in infancy and early childhood due to progression to liver failure. Liver transplantation for children with MPV17 deficiency has been considered controversial due to uncertainty about the potential progression of extrahepatic manifestations following liver transplantation. METHODS: We describe our institution's experience for two infants diagnosed with infantile MPV17 deficiency who presented in acute on chronic liver failure, but with normal development and normal neurological status who successfully underwent liver transplantation. RESULTS: Both patients underwent successful liver transplantation with normal development and neurological status at 3 years and 16 months post-transplant, respectively. CONCLUSIONS: In this rare disease population, we describe two infants with MPV17 deficiency who underwent liver transplantation for acute on chronic liver failure who continue to have normal development, without progression of neurological disease. MPV17 deficiency should not be considered a contraindication to liver transplantation.

Tendon progenitor cells as biological augmentation improve functional gait and reduce scar formation after rotator cuff repair.

BACKGROUND: High rates of structural failure are reported after rotator cuff repairs due to inability to recreate the native enthesis during healing. The development of biological augmentation methods that mitigate scar formation and regenerate the enthesis is still an unmet need. Since neonatal enthesis is capable of regeneration after injury, this study tested whether delivery of neonatal tendon progenitor cells (TPCs) into the adult injured environment can enhance functional and structural supraspinatus enthesis and tendon healing. METHODS: TPCs were isolated from Ai14 Rosa26-TdTomato mouse Achilles tendons and labeled using adenovirus-Cre. Fifty-two CB57BL/6J mice underwent detachment and acute repair of the supraspinatus tendon and received either a fibrin-only or TPC-fibrin gel. Immunofluorescence analysis was carried out to determine cellularity (DAPI), fibrocartilage (SOX9), macrophages (F4/80), myofibroblasts (α-smooth muscle actin), and scar (laminin). Assays for function (gait and biomechanical testing) and structure (micro-computed tomography imaging, picrosirius red/Alcian Blue staining, type I and III collagen staining) were carried out. RESULTS: Analysis of TdTomato cells after injury showed minimal retention of TPCs by day 7 and day 14, with detected cells localized near the bursa and deltoid rather than the enthesis/tendon. However, TPC delivery led to significantly increased %Sox9+ cells in the enthesis at day 7 after injury and decreased laminin intensity across almost all time points compared to fibrin-only treatment. Similarly, TPC-treated mice showed gait recovery by day 14 (paw area and stride length) and day 28 (stance time), while fibrin-treated mice failed to recover gait parameters. Despite improved gait, biomechanical testing showed no differences between groups. Structural analysis by micro-computed tomography suggests that TPC application improves cortical thickness after surgery compared to fibrin. Superior collagen alignment at the neo-enthesis was also observed in the TPC-augmented group at day 28, but no difference was detected in type I and III collagen intensity. CONCLUSION: We found that neonatal TPCs improved and restored functional gait by reducing overall scar formation, improving enthesis collagen alignment, and altering bony composition response after supraspinatus tendon repair. TPCs did not appear to integrate into the healing tissue, suggesting improved healing may be due to paracrine effects at early stages. Future work will determine the factors secreted by TPCs to develop translational targets.

Prolonged myosin binding increases muscle stiffness in Drosophila models of Freeman-Sheldon syndrome.

Freeman-Sheldon syndrome (FSS) is characterized by congenital contractures resulting from dominant point mutations in the embryonic isoform of muscle myosin. To investigate its disease mechanism, we used Drosophila models expressing FSS myosin mutations Y583S or T178I in their flight and jump muscles. We isolated these muscles from heterozygous mutant Drosophila and performed skinned fiber mechanics. The most striking mechanical alteration was an increase in active muscle stiffness. Y583S/+ and T178I/+ fibers' elastic moduli increased 70 and 77%, respectively. Increased stiffness contributed to decreased power generation, 49 and 66%, as a result of increased work absorbed during the lengthening portion of the contractile cycle. Slower muscle kinetics also contributed to the mutant phenotype, as shown by 17 and 32% decreases in optimal frequency for power generation, and 27 and 41% slower muscle apparent rate constant 2πb. Combined with previous measurements of slower in vitro actin motility, our results suggest a rate reduction of at least one strongly bound cross-bridge cycle transition that increases the time myosin spends strongly bound to actin, ton. Increased ton was further supported by decreased ATP affinity and a 16% slowing of jump muscle relaxation rate in T178I heterozygotes. Impaired muscle function caused diminished flight and jump ability of Y583S/+ and T178I/+ Drosophila. Based on our results, assuming that our model system mimics human skeletal muscle, we propose that one mechanism driving FSS is elevated muscle stiffness arising from prolonged ton in developing muscle fibers.

CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane.

The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo, and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation.

The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production.

KEY POINTS: Hypertrophic cardiomyopathy (HCM) is a genetic disease that causes thickening of the heart's ventricular walls and is a leading cause of sudden cardiac death. HCM is caused by missense mutations in muscle proteins including myosin, but how these mutations alter muscle mechanical performance in largely unknown. We investigated the disease mechanism for HCM myosin mutation R249Q by expressing it in the indirect flight muscle of Drosophila melanogaster and measuring alterations to muscle and flight performance. Muscle mechanical analysis revealed R249Q decreased muscle power production due to slower muscle kinetics and decreased force production; force production was reduced because fewer mutant myosin cross-bridges were bound simultaneously to actin. This work does not support the commonly proposed hypothesis that myosin HCM mutations increase muscle contractility, or causes a gain in function; instead, it suggests that for some myosin HCM mutations, hypertrophy is a compensation for decreased contractility. ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, we expressed an HCM myosin mutation, R249Q, in Drosophila indirect flight muscle (IFM) and assessed myofibril structure, skinned fibre mechanical properties, and flight ability. Mechanics experiments were performed on fibres dissected from 2-h-old adult flies, prior to degradation of IFM myofilament structure, which started at 2 days old and increased with age. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. The muscle apparent rate constant 2πb decreased 33% for homozygous but increased for heterozygous fibres. The apparent rate constant 2πc was greater for homozygous fibres. This indicates that R249Q myosin is slowing attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, our results do not support the increased contractility hypothesis. Instead, our results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output.

Same- vs Different-Hospital Readmissions in Patients With Cirrhosis After Hospital Discharge.

INTRODUCTION: There is a lack of data on the impact of readmission to the same vs a different hospital following an index hospital discharge in cirrhosis patients. METHODS: We sought to describe rates and predictors of different-hospital readmissions (DHRs) among patients with cirrhosis and also determine the impact on cirrhosis outcomes including all-cause inpatient mortality and hospital costs. Using the national readmissions database, we identified cirrhosis hospitalizations in 2013. Regression analysis was used to determine the predictors of DHRs. A time-to-event analysis was performed to assess the impact on subsequent readmissions and all-cause inpatient mortality. RESULTS: In 2013, there were 109,039 cirrhosis readmissions with 67% of these being same-hospital readmissions and 33% being DHRs (P < 0.001). Two percent of readmitted patients were treated at ≥4 different hospitals. The 30-day readmission rate was 29.1%. Predictors of DHR included Medicaid payer (adjusted odds ratio [OR] 1.07, 95% confidence interval [95% CI] 1.01-1.14), age (OR 0.98, 95% CI 0.978-0.982), elective admission (OR 1.09, 95% CI 1.01-1.17), hepatic encephalopathy (OR 1.20, 95% CI 1.16-1.25), hepatorenal syndrome (OR 1.09, 95% CI 1.03-1.16), and low socioeconomic status (OR 1.15, 95% CI 1.06-1.25). No difference was observed in 30-day readmission risk following a DHR (adjusted hazard ratio 1.044, 95% CI 0.975-1.118). In addition, there was no increased risk of inpatient death observed during a DHR within 30 days (adjusted hazard ratio 1.08, 95% CI 0.94-1.23). However, patients with DHR had significantly higher hospital costs and length of stay. CONCLUSIONS: Majority of cirrhosis readmissions are same-hospital readmissions. Different-hospital readmissions do not increase the risk of 30-day readmissions and inpatient mortality but are associated with higher hospital costs.

Neonatal mouse intervertebral discs heal with restored function following herniation injury.

Adult intervertebral discs (IVDs) have poor endogenous healing capacity, because of their challenging microenvironment and complex mechanical demands, which can result in painful IVD herniation. There are no regenerative strategies available to improve IVD healing and restore its function. Neonatal mice are excellent models of mammalian regeneration, but there are no studies of the regenerative capacity of neonatal IVDs. In this study, we developed a neonatal model of improved IVD healing to inform repair strategies after herniation. In vivo puncture injuries were performed to simulate herniation with complete annulus fibrosus (AF) tears in caudal IVDs of neonatal (postnatal d 5) and adult (4-6 mo) Scleraxis green fluorescent protein ( ScxGFP) mice. Acute and long-term healing responses were assessed with histologic, radiologic, and biomechanical measurements. Neonates underwent accelerated IVD healing compared to adults with functional restoration and enhanced structural repair after herniation. A population of ScxGFP- cells identified in the neonatal repair site may be associated with this improved healing and warrants future investigation. In summary, function of neonatal IVDs was restored after herniation injury, whereas that of adult discs was not. This improved healing response is likely driven by multiple mechanisms that may include differences in mechanical loading and available repair cells during growth.-Torre, O. M., Das, R., Berenblum, R. E., Huang, A. H., Iatridis, J. C. Neonatal mouse intervertebral discs heal with restored function following herniation injury.

Coordinated development of the limb musculoskeletal system: Tendon and muscle patterning and integration with the skeleton.

Functional movement and stability of the limb depends on an organized and fully integrated musculoskeletal system composed of skeleton, muscle, and tendon. Much of our current understanding of musculoskeletal development is based on studies that focused on the development and differentiation of individual tissues. Likewise, research on patterning events have been largely limited to the primary skeletal elements and the mechanisms that regulate soft tissue patterning, the development of the connections between tissues, and their interdependent development are only beginning to be elucidated. This review will therefore highlight recent exciting discoveries in this field, with an emphasis on tendon and muscle patterning and their integrated development with the skeleton and skeletal attachments.

Musculoskeletal integration at the wrist underlies the modular development of limb tendons.

The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.

Optimizing a 3D model system for molecular manipulation of tenogenesis.

PURPOSE: Tendon injuries are clinically challenging due to poor healing. A better understanding of the molecular events that regulate tendon differentiation would improve current strategies for repair. The mouse model system has been instrumental to tendon studies and several key molecules were initially established in mouse. However, the study of gene function has been limited by the absence of a standard in vitro tendon system for efficiently testing multiple mutations, physical manipulations, and mis-expression. The purpose of this study is therefore to establish such a system. METHODS: We adapted an existing design for generating three-dimensional (3D) tendon constructs for use with mouse progenitor cells harboring the ScxGFP tendon reporter and the Rosa26-TdTomato Cre reporter. Using these cells, we optimized the parameters for construct formation, inducing tenogenesis via transforming growth factor-ß2 (TGFß2), and genetic recombination via an adenovirus encoding Cre recombinase. Finally, for proof of concept, we used Smad4 floxed cells and tested the robustness of the system for gene knockdown. RESULTS: We found that TGFß2 treatment induced a tenogenic phenotype depending on the timing of initiation. Addition of TGFß2 after 3D "tensioning" enhanced tendon differentiation. Interestingly, while TGFß2-induced proliferation depended on Smad4, tenogenic parameters such as ScxGFP expression and fibril diameter were independent of Smad4. CONCLUSIONS: Our results demonstrate the feasibility of this optimized system for harnessing the power of mouse genetics for in vitro applications.

Consistency of Renal Stone Volume Measurements Across CT Scanner Model and Reconstruction Algorithm Configurations.

OBJECTIVE: The objective of this prospective study is to evaluate the consistency of renal stone volume estimation using dual-energy CT across scanner model and reconstruction algorithm configurations. SUBJECTS AND METHODS: Patients underwent scanning with routine kidney stone composition protocols on both second- and third-generation dual-source CT scanners. Images were reconstructed using filtered back projection and iterative reconstruction (IR). In addition, a modified IR kernel on the third-generation CT scanner was evaluated. Individual kidney stone volumes were determined and compared. RESULTS: No significant difference was noted in measured volumes between filtered back-projection data, IR data from the second-generation scanner, and the modified IR kernel data (p > 0.05). The third-generation commercially available IR kernel yielded lower volumes than did the other configurations (p < 0.0001). CONCLUSION: With the use of a modified kernel for the third-generation scanner, patients being monitored for changes in kidney stone volume can undergo scanning performed with second- or third-generation dual-energy CT scanners, and the images obtained can be reconstructed with either filtered back projection or IR without the introduction of bias into kidney stone volume measurements.

The Oral Health of Preschool Children of Refugee and Immigrant Families in Manitoba.

INTRODUCTION: Children of newcomers to Canada have been identified as at moderately high risk for developing early childhood caries (ECC). The purpose of this study was to investigate the oral health of preschool children of refugee and immigrant families in Winnipeg. METHODS: Children < 72 months of age and their parent or primary caregiver were recruited through several newcomer settlement agencies, dental clinics and community programs. Parents and caregivers completed a short questionnaire with the assistance of a research team member. Children underwent a dental examination. Results of the questionnaire were combined with those of the clinical examination and subjected to statistical analysis. RESULTS: We recruited 211 children. Their mean age was 40.2 ± 15.4 months, and 54.0% were boys. Overall, 45.5% of the children had ECC and 31.8% had severe ECC (S-ECC). The mean decayed, missing and filled teeth (dmft) score was 2.2 ± 3.8 (range 0-19), while the mean decayed, missing, filled surfaces (dmfs) score was 4.8 ± 11.0 (range 0-63). Infant dental enucleation was observed in 6 children. Logistic regression analyses showed that increasing age, the presence of debris on teeth, parents believing their child has dental problems and the presence of enamel hypoplasia were significantly and independently associated with ECC and S-ECC (p ≤ 0.05). CONCLUSIONS: ECC is prevalent in children of newcomer families in Manitoba. These data will inform advocacy efforts to improve access to dental care and tailor early childhood oral health promotion and ECC prevention activities for refugees and recent immigrants.