The Arabidopsis transcription factor TCP9 modulates root architectural plasticity, reactive oxygen species-mediated processes, and tolerance to cyst nematode infections.

Infections by root-feeding nematodes have profound effects on root system architecture and consequently shoot growth of host plants. Plants harbor intraspecific variation in their growth responses to belowground biotic stresses by nematodes, but the underlying mechanisms are not well understood. Here, we show that the transcription factor TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR-9 (TCP9) modulates root system architectural plasticity in Arabidopsis thaliana in response to infections by the endoparasitic cyst nematode Heterodera schachtii. Young seedlings of tcp9 knock-out mutants display a significantly weaker primary root growth inhibition response to cyst nematodes than wild-type Arabidopsis. In older plants, tcp9 reduces the impact of nematode infections on the emergence and growth of secondary roots. Importantly, the altered growth responses by tcp9 are most likely not caused by less biotic stress on the root system, because TCP9 does not affect the number of infections, nematode development, and size of the nematode-induced feeding structures. RNA-sequencing of nematode-infected roots of the tcp9 mutants revealed differential regulation of enzymes involved in reactive oxygen species (ROS) homeostasis and responses to oxidative stress. We also found that root and shoot growth of tcp9 mutants is less sensitive to exogenous hydrogen peroxide and that ROS accumulation in nematode infection sites in these mutants is reduced. Altogether, these observations demonstrate that TCP9 modulates the root system architectural plasticity to nematode infections via ROS-mediated processes. Our study further points at a novel regulatory mechanism contributing to the tolerance of plants to root-feeding nematodes by mitigating the impact of belowground biotic stresses.

Single-Surgery Co-Implantation of Autologous Primary Articular Chondrocytes with Bone Marrow Cells in the Treatment of Articular Cartilage Lesions: Observations from the Operating Room on Tissue Input and Cell Output.

OBJECTIVE: Single-surgery variants of autologous chondrocyte implantation to repair cartilage are emerging, but practical data on such procedures are scarce. We set out to describe a 1-stage autologous chondrocyte co-implantation procedure and include quantitative characteristics of the biopsy tissues collected and of the cells obtained from those tissues in the operating room. DESIGN: Data concerning patient age, articular cartilage lesion size and location, as well as measurements of cartilage biopsy mass, bone marrow aspirate volume, and the cell yields harvested from those biopsies were intraoperatively collected for 141 patients. RESULTS: The mean patient age was 35.7 ± 9.8 years, and the mean total lesion size was 4.0 ± 3.1 cm2. Cartilage biopsy mass ranged from 0.19 to 2.0 gram and provided a mean yield of 1.23 × 106 chondrocytes/gram. Bone marrow aspirate volume ranged from 7.2 to 62 milliliters and provided a mean yield of 7.18 × 106 mononuclear cells/mL. The cell yields did not correlate with patient age, which ranged from 12 to 57 years. The mean primary chondrocyte supply was 272 thousand per cm2 of lesion, ranging from 10.4 thousand to 1.07 million per cm2. The total cell supply was kept at 9 million cells per cm2 of lesion by adding mononuclear cells to the chondrocytes. The mean tissue processing time was 100 ± 19 minutes, which was frequently used to perform concurrent interventions. CONCLUSION: Single-surgery co-implantation of clinically relevant numbers of autologous primary articular chondrocytes and bone marrow cells is feasible for a wide range of ages and lesion sizes.

Single-Stage Autologous Chondrocyte-Based Treatment for the Repair of Knee Cartilage Lesions: Two-Year Follow-up of a Prospective Single-Arm Multicenter Study.

BACKGROUND: There is an unmet need for a single-stage cartilage repair treatment that is cost-effective and chondrocyte-based. PURPOSE: To evaluate the safety and preliminary efficacy of autologous freshly isolated primary chondrocytes and bone marrow mononucleated cells (MNCs) seeded into a PolyActive scaffold in patients with symptomatic cartilage lesions of the knee. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 40 patients with symptomatic knee cartilage lesions were treated with freshly isolated autologous chondrocytes combined with bone marrow MNCs delivered in a biodegradable load-bearing scaffold. The treatment requires only 1 surgical intervention and is potentially a cost-effective alternative to autologous chondrocyte implantation. The primary chondrocytes and bone marrow MNCs were isolated, washed, counted, mixed, and seeded into a load-bearing scaffold in the operating room. Patients were followed up at 3, 6, 12, 18, and 24 months. Primary endpoints were treatment-related adverse events up to 3 months, adverse implant effects between 3 and 24 months, and the implant success rate at 3 months as measured by lesion filling. RESULTS: Successful lesion filling (≥67% on magnetic resonance imaging) was found in 40 patients at 3 months and in 32 of the 32 patients analyzed at 24 months. Significant improvement over baseline was found for visual analog scale for pain from 3 months onward; Knee injury and Osteoarthritis Outcome Score (KOOS)-Pain and KOOS-Activities of Daily Living from 6 months onward; for KOOS-Symptoms and Stiffness, KOOS-Quality of Life and International Knee Documentation Committee from 12 months onward; and for KOOS-Sport and Recreation from 18 months onward. Hyaline-like repair tissue was found in 22 of 31 patients available for biopsy. Arthralgia and joint effusion were the most common adverse events. Scaffold delamination and adhesions led to removal of the implant in 2 patients. CONCLUSION: The treatment of knee cartilage lesions with autologous primary chondrocytes and bone marrow MNCs, both isolated and seeded into a load-bearing PolyActive scaffold within a single surgical intervention, is safe and clinically effective. Good lesion fill and sustained clinically important and statistically significant improvement in all patient-reported outcome scores were found throughout the 24-month study. Hyaline-like cartilage was observed on biopsy specimen in at least 22 of the 40 patients. REGISTRATION: NCT01041885 (ClinicalTrials.gov identifier).

Hedgehog proteins and parathyroid hormone-related protein are involved in intervertebral disc maturation, degeneration, and calcification.

Parathyroid hormone-related protein (PTHrP) and hedgehog signaling play an important role in chondrocyte development, (hypertrophic) differentiation, and/or calcification, but their role in intervertebral disc (IVD) degeneration is unknown. Better understanding their involvement may provide therapeutic clues for low back pain due to IVD degeneration. Therefore, this study aimed to explore the role of PTHrP and hedgehog proteins in postnatal canine and human IVDs during the aging/degenerative process. The expression of PTHrP, hedgehog proteins and related receptors was studied during the natural loss of the notochordal cell (NC) phenotype during IVD maturation using tissue samples and de-differentiation in vitro and degeneration by real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Correlations between their expression and calcification levels (Alizarin Red S staining) were determined. In addition, the effect of PTHrP and hedgehog proteins on canine and human chondrocyte-like cells (CLCs) was determined in vitro focusing on the propensity to induce calcification. The expression of PTHrP, its receptor (PTHR1) and hedgehog receptors decreased during loss of the NC phenotype. N-terminal (active) hedgehog (Indian hedgehog/Sonic hedgehog) protein expression did not change during maturation or degeneration, whereas expression of PTHrP, PTHR1 and hedgehog receptors increased during IVD degeneration. Hedgehog and PTHR1 immunopositivity were increased in nucleus pulposus tissue with abundant vs no/low calcification. In vitro, hedgehog proteins facilitated calcification in CLCs, whereas PTHrP did not affect calcification levels. In conclusion, hedgehog and PTHrP expression is present in healthy and degenerated IVDs. Hedgehog proteins had the propensity to induce calcification in CLCs from degenerated IVDs, indicating that in the future, inhibiting hedgehog signaling could be an approach to inhibit calcification during IVD degeneration.

Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside.

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as 'instructive matrix', by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR.

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo.

Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 µm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses.

External validation of the Blunt Abdominal Trauma in Children (BATiC) score: ruling out significant abdominal injury in children.

BACKGROUND: The aim of this study was to validate the use of the Blunt Abdominal Trauma in Children (BATiC) score. The BATiC score uses only readily available laboratory parameters, ultrasound results, and results from physical examination and does therefore not carry any risk of additional radiation exposure. METHODS: Data of pediatric trauma patients admitted to the shock room between 2006 and 2010 were retrospectively analyzed. Blunt abdominal trauma was defined radiologically or surgically. The BATiC score was computed using 10 parameters as follows: abnormal abdominal ultrasound finding, abdominal pain, peritoneal irritation, hemodynamic instability, aspartate aminotransferase greater than 60 U/L, alanine aminotransferase greater than 25 U/L, white blood cell count greater than 10 × 10/L, lactate dehydrogenase greater than 330 U/L, amylase greater than 100 U/L, and creatinine greater than 110 µmol/L. Sensitivity, specificity, negative predictive value, and positive predictive value were computed. Missing values were replaced using multiple imputation, and BATiC scores were calculated based on imputed values. RESULTS: Included were 216 patients, with 144 males, 72 females, and a median age of 12 years. Eighteen patients (8%) sustained abdominal injury. Median BATiC scores of patients with and without intra-abdominal injury were 9.2 (range, 6.6-15.4) and 2.2 (range, 0.0-10.6) respectively (p < 0.001). When the BATiC score is used with a cutoff point of 6, the test showed a sensitivity of 100% and a specificity of 87%. Negative and positive predictive values were 100% and 41% respectively. The area under the curve was 0.98. CONCLUSION: The BATiC score can be a useful adjunct in the assessment of the presence of abdominal trauma in children and can help determine which patients might benefit from a computed tomographic scan and/or further treatment and which might not. LEVEL OF EVIDENCE: Prognostic study, level II.

External validation of the Emergency Trauma Score for early prediction of mortality in trauma patients.

OBJECTIVES: The Emergency Trauma Score has been developed for early estimation of mortality risk in adult trauma patients with an Injury Severity Score of 16 or higher. Emergency Trauma Score combines four early predictors available at the trauma resuscitation room: age, Glasgow Coma Scale, base excess, and prothrombin time. Our goal was to validate the Emergency Trauma Score in two large external cohorts. As the Injury Severity Score is not accurately known at the time patients present at the resuscitation room, we evaluated the performance of Emergency Trauma Score in all trauma patients. DESIGN: External validation study using data from two prospectively collected trauma registries. SETTING: Two academic level 1 trauma centers. PATIENTS: Adult patients admitted to the hospital after treatment at the trauma resuscitation room. INTERVENTION: Calibration and discrimination of the original Emergency Trauma Score were assessed within each cohort separately. MEASUREMENT AND MAIN RESULTS: A total of 4,418 consecutive patients were evaluated. Discrimination was good in both validation cohorts, with areas under the receiver-operating curve curves that were even higher (0.94 and 0.92, respectively) than that in the original cohort (0.83). Predicted mortality was systematically too high compared with actual mortality in patients with low-to-medium expected risk (< 25%). Calibration improved in the lower expected risk range after exclusion of patients with Injury Severity Score less than 16. CONCLUSIONS: The Emergency Trauma Score model performs well in discriminating between trauma patients who will survive and who will not. If applied to all trauma patients, predicted mortality risks are too high in the low-risk category.