Reduction of mortality, cardiac damage, and cerebral damage by IL-1 inhibition in a murine model of TTP.

ABSTRACT: Thrombotic thrombocytopenic purpura (TTP), a rare but fatal disease if untreated, is due to alteration in von Willebrand factor cleavage resulting in capillary microthrombus formation and ischemic organ damage. Interleukin-1 (IL-1) has been shown to drive sterile inflammation after ischemia and could play an essential contribution to postischemic organ damage in TTP. Our objectives were to evaluate IL-1 involvement during TTP and to test the efficacy of the recombinant IL-1 receptor antagonist, anakinra, in a murine TTP model. We retrospectively measured plasma IL-1 concentrations in patients with TTP and controls. Patients with TTP exhibited elevated plasma IL-1α and -1ß concentrations, which correlated with disease course and survival. In a mouse model of TTP, we administered anakinra (IL-1 inhibitor) or placebo for 5 days and evaluated the efficacy of this treatment. Anakinra significantly reduced mortality of mice (P < .001). Anakinra significantly decreased TTP-induced cardiac damage as assessed by blood troponin concentrations, evaluation of left ventricular function by echocardiography, [18F]fluorodeoxyglucose positron emission tomography of myocardial glucose metabolism, and cardiac histology. Anakinra also significantly reduced brain TTP-induced damage evaluated through blood PS100b concentrations, nuclear imaging, and histology. We finally showed that IL-1α and -1ß trigger endothelial degranulation in vitro, leading to the release of von Willebrand factor. In conclusion, anakinra significantly reduced TTP mortality in a preclinical model of the disease by inhibiting both endothelial degranulation and postischemic inflammation, supporting further evaluations in humans.

Characterization of the mechanical properties of the mouse Achilles tendon enthesis by microindentation. Effects of unloading and subsequent reloading.

The fibrocartilaginous tendon enthesis, i.e. the site where a tendon is attached to bone through a fibrocartilaginous tissue, is considered as a functionally graded interface. However, at local scale, a very limited number of studies have characterized micromechanical properties of this transitional tissue. The first goal of this work was to characterize the micromechanical properties of the mineralized part of the healthy Achilles tendon enthesis (ATE) through microindentation testing and to assess the degree of mineralization and of carbonation of mineral crystals by Raman spectroscopy. Since little is known about enthesis biological plasticity, our second objective was to examine the effects of unloading and reloading, using a mouse hindlimb-unloading model, on both the micromechanical properties and the mineral phase of the ATE. Elastic modulus, hardness, degree of mineralization, and degree of carbonation were assessed after 14 days of hindlimb suspension and again after a subsequent 6 days of reloading. The elastic modulus gradually increased along the mineralized part of the ATE from the tidemark to the subchondral bone, with the same trend being found for hardness. Whereas the degree of carbonation did not differ according to zone of measurement, the degree of mineralization increased by >70 % from tidemark to subchondral bone. Thus, the gradient in micromechanical properties is in part explained by a mineralization gradient. A 14-day unloading period did not appear to affect the gradient of micromechanical properties of the ATE, nor the degree of mineralization or carbonation. However, contrary to a short period of unloading, early return to normal mechanical load reduced the micromechanical properties gradient, regardless of carbonate-to-phosphate ratios, likely due to the more homogeneous degree of mineralization. These findings provide valuable data not only for tissue bioengineering, but also for musculoskeletal clinical studies and microgravity studies focusing on long-term space travel by astronauts.

Soluble CD146, a biomarker and a target for preventing resistance to anti-angiogenic therapy in glioblastoma.

RATIONALE: Glioblastoma multiforme (GBM) is a primary brain tumor with poor prognosis. The U.S. food and drug administration approved the use of the anti-VEGF antibody bevacizumab in recurrent GBM. However, resistance to this treatment is frequent and fails to enhance the overall survival of patients. In this study, we aimed to identify novel mechanism(s) responsible for bevacizumab-resistance in CD146-positive glioblastoma. METHODS: The study was performed using sera from GBM patients and human GBM cell lines in culture or xenografted in nude mice. RESULTS: We found that an increase in sCD146 concentration in sera of GBM patients after the first cycle of bevacizumab treatment was significantly associated with poor progression free survival and shorter overall survival. Accordingly, in vitro treatment of CD146-positive glioblastoma cells with bevacizumab led to a high sCD146 secretion, inducing cell invasion. These effects were mediated through integrin αvß3 and were blocked by mucizumab, a novel humanized anti-sCD146 antibody. In vivo, the combination of bevacizumab with mucizumab impeded CD146 + glioblastoma growth and reduced tumor cell dissemination to an extent significantly higher than that observed with bevacizumab alone. CONCLUSION: We propose sCD146 to be 1/ an early biomarker to predict and 2/ a potential target to prevent bevacizumab resistance in patients with glioblastoma.

Mechanical Characterization at the Microscale of Mineralized Bone Callus after Bone Lengthening.

Distraction osteogenesis (DO) involves several processes to form an organized distracted callus. While bone regeneration during DO has been widely described, no study has yet focused on the evolution profile of mechanical properties of mineralized tissues in the distracted callus. The aim of this study was therefore to measure the elastic modulus and hardness of calcified cartilage and trabecular and cortical bone within the distracted callus during the consolidation phase. We used a microindentation assay to measure the mechanical properties of periosteal and endosteal calluses; each was subdivided into two regions. Histological sections were used to localize the tissues. The results revealed that the mechanical properties of calcified cartilage did not evolve over time. However, trabecular bone showed temporal variation. For elastic modulus, in three out of four regions, a similar evolution profile was observed with an increase and decrease over time. Concerning hardness, this evolves differently depending on the location in the distracted callus. We also observed spatial changes in between regions. A first duality was apparent between regions close to the native cortices and the central area, while latter differences were seen between periosteal and endosteal calluses. Data showed a heterogeneity of mechanical properties in the distracted callus with a specific mineralization profile.

Effects of hindlimb unloading and subsequent reloading on the structure and mechanical properties of Achilles tendon-to-bone attachment.

While muscle and bone adaptations to deconditioning have been widely described, few studies have focused on the tendon enthesis. Our study examined the effects of mechanical loading on the structure and mechanical properties of the Achilles tendon enthesis. We assessed the fibrocartilage surface area, the organization of collagen, the expression of collagen II, the presence of osteoclasts, and the tensile properties of the mouse enthesis both after 14 days of hindlimb suspension (HU) and after a subsequent 6 days of reloading. Although soleus atrophy was severe after HU, calcified fibrocartilage (CFc) was a little affected. In contrast, we observed a decrease in non-calcified fibrocartilage (UFc) surface area, collagen fiber disorganization, modification of morphological characteristics of the fibrocartilage cells, and altered collagen II distribution. Compared to the control group, restoring normal loads increased both UFc surface area and expression of collagen II, and led to a crimp pattern in collagen. Reloading induced an increase in CFc surface area, probably due to the mineralization front advancing toward the tendon. Functionally, unloading resulted in decreased enthesis stiffness and a shift in site of failure from the osteochondral interface to the bone, whereas 6 days of reloading restored the original elastic properties and site of failure. In the context of spaceflight, our results suggest that care must be taken when performing countermeasure exercises both during missions and during the return to Earth.

Oral manifestations associated with inherited hyperhomocysteinemia: A first case description.

Hyperhomocysteinemia is a rare disease caused by nutritional deficiencies or genetic impairment of cysteine metabolism. To date, no oral manifestations of hyperhomocysteinemia have been described in humans. Therefore, to our knowledge, the present case report is the first description of a hyperhomocysteinemic patient showing oral tissue alterations leading to both early tooth loss and failed implant osseointegration. The patient presented with a methylenetetrahydrofolate reductase gene mutation (677T polymorphism) leading to mild hyperhomocysteinemia. The radiologic analysis showed hyperdense lesions scattered in the maxillae. The histologic observations indicated alterations in both collagen and elastic networks in the gingiva and dermis. Interestingly, the presence of ectopic mineralized inclusions was noted in both periodontal ligament and gingiva. Strong osteoclastic activity was associated with abnormal calcification of trabecular spaces. Uneven oral tissue remodeling due to high tissue levels of homocysteine could explain the pathologic manifestations observed in this case.

Systemic Administration of G-CSF Accelerates Bone Regeneration and Modulates Mobilization of Progenitor Cells in a Rat Model of Distraction Osteogenesis.

Granulocyte colony-stimulating factor (G-CSF) was shown to promote bone regeneration and mobilization of vascular and osteogenic progenitor cells. In this study, we investigated the effects of a systemic low dose of G-CSF on both bone consolidation and mobilization of hematopoietic stem/progenitor cells (HSPCs), endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) in a rat model of distraction osteogenesis (DO). Neovascularization and mineralization were longitudinally monitored using positron emission tomography and planar scintigraphy. Histological analysis was performed and the number of circulating HSPCs, EPCs and MSCs was studied by flow cytometry. Contrary to control group, in the early phase of consolidation, a bony bridge with lower osteoclast activity and a trend of an increase in osteoblast activity were observed in the distracted callus in the G-CSF group, whereas, at the late phase of consolidation, a significantly lower neovascularization was observed. While no difference was observed in the number of circulating EPCs between control and G-CSF groups, the number of MSCs was significantly lower at the end of the latency phase and that of HSPCs was significantly higher 4 days after the bone lengthening. Our results indicate that G-CSF accelerates bone regeneration and modulates mobilization of progenitor cells during DO.

Early-stage knee OA induced by MIA and MMT compared in the murine model via histological and topographical approaches.

Osteoarthritis (OA) is a common degenerative disease whose early management includes promising mechanical treatments. New treatments are initially validated using an animal model in which OA is induced. The MMT (mechanical induction) and MIA (chemical induction) models of OA induction are widespread, but their use to generate early OA is poorly documented. We analyzed and compared early-stage knee OA-induction via these two methods in 16 rats divided into two groups. After 4 weeks of induction, the knees were sampled and studied using both histology (Toluidine Blue and Sirius Red) and surface topology, an innovative technique for characterizing osteoarthritic cartilage. The Mankin-modified score confirms that the two OA-induction models evolved at the same speed. At this early stage, the two models can be differentiated morphologically, although no significant differences were revealed by either cellularity or birefringence analysis. However, the topological analysis generated two forms of quantitative data, the deformation ratio and the cohesion index, that differentiated between the two groups. Thus, the early-stage OA induced by these two models is revealed to differ. The patterns of cartilage damage induced point to MMT as the better choice to assess mechanical approaches to clinical OA treatment.

Total Knee Arthroplasty with a Ti6Al4V/PEEK Prosthesis on an Osteoarthritis Rat Model: Behavioral and Neurophysiological Analysis.

Arthroplasty is a surgical procedure to restore the function of the joint of patient suffering from knee osteoarthritis. However, postoperative functional deficits are reported even after a rehabilitation program. In order to determine the origin of functional deficits of patient suffering from knee osteoarthritis and total knee arthroplasty, we developed a rodent model including a chemically-induced-osteoarthritis and designed a knee prosthesis (Ti6Al4V/PEEK) biomechanically and anatomically adapted to rat knee joint. Dynamic Weight-Bearing, gait kinematics, H-reflex from vastus medialis muscle and activities from metabosensitive III and IV afferent fibers in femoral nerve were assessed at 1 and 3 months post-surgery. Results indicate that knee osteoarthritis altered considerably the responses of afferent fibers to their known activators (i.e., lactic acid and potassium chloride) and consequently their ability to modulate the spinal sensorimotor loop, although, paradoxically, motor deficits seemed relatively light. On the contrary, results indicate that, after the total knee arthroplasty, the afferent responses and the sensorimotor function were slightly altered but that motor deficits were more severe. We conclude that neural changes attested by the recovery of the metabosensitive afferent activity and the sensorimotor loop were induced when a total knee replacement was performed and that these changes may disrupt or delay the locomotor recovery.

Probing the Cellular Size Distribution in Cell Samples Undergoing Cell Death.

A polydisperse scattering model adapted for concentrated medium, namely the polydisperse structure factor model, was examined to explain the backscatter coefficients (BSCs) measured from packed cell samples undergoing cell death. Cell samples were scanned using high-frequency ultrasound in the 10-42 MHz bandwidth. A parameter estimation procedure was proposed to estimate the volume fraction and the relative impedance contrast that could explain the changes in BSC pattern by considering the actual change in cellular size distribution. Quantitative ultrasound parameters were estimated and related to the percentage of dead cells determined by flow cytometry. The standard deviation of scatterer size distribution extracted from the polydisperse structure factor model and the spectral intercept were found to be strongly correlated to the percentage of dead cells (r2 = 0.79 and r2 = 0.72, respectively). This study contributes to the understanding of ultrasonic scattering from cells undergoing cell death toward the monitoring of cancer therapy.

Nonlinear ultrasound parameter to monitor cell death in cancer cell samples.

A scaling subtraction method was proposed to analyze the radio frequency data from cancer cell samples exposed to an anti-cancer drug and to estimate a nonlinear parameter. The nonlinear parameter was found to be well correlated (R2 = 0.62) to the percentage of dead cells in apoptosis and necrosis. The origin of the nonlinearity may be related to a change in contacts between cells, since the nonlinear parameter was well correlated to the average total coordination number of binary packings (R2 ≥ 0.77). These results suggest that the scaling subtraction method may be used to early quantify chemotherapeutic treatment efficiency.

Granulocyte-colony stimulating factor enhances bone fracture healing.

BACKGROUND: Circulating mesenchymal stem cells contribute to bone repair. Their incorporation in fracture callus is correlated to their bioavailability. In addition, Granulocyte-colony stimulating factor induces the release of vascular and mesenchymal progenitors. We hypothesized that this glycoprotein stimulates fracture healing, and analyzed the effects of its administration at low doses on bone healing. METHODS: 27 adult male Sprague-Dawley rats underwent mid-femur osteotomy stabilized by centromedullar pinning. In a post (pre) operative group, rats were subcutaneously injected with 5 µg/kg per day of Granulocyte-colony stimulating factor for 5 days after (before) surgery. In a control group, rats were injected with saline solution for 5 days immediately after surgery. A radiographic consolidation score was calculated. At day 35, femurs were studied histologically and underwent biomechanical tests. FINDINGS: 5 weeks after surgery, mean radiographic scores were significantly higher in the Preop group 7.75 (SD 0.42) and in the Postop group 7.67 (SD 0.52) than in the control group 6.75 (SD 0.69). Biomechanical tests showed femur stiffness to be more than three times higher in both the Preop 109.24 N/mm (SD 51.86) and Postop groups 100.05 N/mm (SD 60.24) than in control 32.01 N/mm (SD 15.78). Mean maximal failure force was twice as high in the Preop group 68.66 N (SD 27.78) as in the control group 34.21 N (SD 11.79). Histological results indicated a later consolidation process in control than in treated groups. INTERPRETATION: Granulocyte-colony stimulating factor injections strongly stimulated early femur fracture healing, indicating its potential utility in human clinical situations such as programmed osteotomy and fracture.

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

Structure factor model for understanding the measured backscatter coefficients from concentrated cell pellet biophantoms.

Ultrasonic backscatter coefficient (BSC) measurements were performed on K562 cell pellet biophantoms with cell concentrations ranging from 0.006 to 0.30 in the 10-42 MHz frequency bandwidth. Three scattering models, namely, the fluid-filled sphere model (FFSM), the particle model (PM), and the structure factor model (SFM), were compared for modeling the scattering from an ensemble of concentrated cells. A parameter estimation procedure was developed in order to estimate the scatterer size and relative impedance contrast that could explain the measured BSCs from all the studied cell concentrations. This procedure was applied to the BSC data from K562 cell pellet biophantoms in the 10-42 MHz frequency bandwidth and to the BSC data from Chinese hamster ovary cell pellet biophantoms in the 26-105 MHz frequency bandwidth given in Han, Abuhabsah, Blue, Sarwate, and O'Brien [J. Acoust. Soc. Am. 130, 4139-4147 (2011)]. The data fitting quality and the scatterer size estimates show that the SFM was more suitable than the PM and the FFSM for modeling the responses from concentrated cell pellet biophantoms.

Structural remodeling of unweighted soleus myotendinous junction in monkey.

This study describes the morphology of the soleus myotendinous junction (MTJ) in the Rhesus monkey. Ultrastructural observations revealed a structural complexity that probably reflects functional adaptations. We also studied ultrastructural modifications of the MTJ in response to 14 days of hypokinesia and microgravity (Bion 11 mission). The reduced limb mobility of the animals, placed in a safety seat aboard the satellite, induced a sarcolemmal remodeling that was enhanced by the microgravity conditions. Signs of MTJ remodeling such as alterations of contractile apparatus and myofilament-anchoring structures, T-tubule dilation, and autophagic vacuoles could be ascribed to the microgravity.