The Use of Proton and Carbon Ion Radiation Therapy for Sarcomas.

The unique physical and biological characteristics of proton and carbon ions allow for improved sparing of normal tissues, decreased integral dose to the body, and increased biological effect through high linear energy transfer. These properties are particularly useful for sarcomas given their histology, wide array of locations, and age of diagnosis. This review summarizes the literature and describes the clinical situations in which these heavy particles have advantages for treating sarcomas.

One Year of Clinic-Wide Cherenkov Imaging for Discovery of Quality Improvement Opportunities in Radiation Therapy.

PURPOSE: Cherenkov imaging is clinically available as a radiation therapy treatment verification tool. The aim of this work was to discover the benefits of always-on Cherenkov imaging as a novel incident detection and quality improvement system through review of all imaging at our center. METHODS AND MATERIALS: Multicamera Cherenkov imaging systems were permanently installed in 3 treatment bunkers, imaging continuously over a year. Images were acquired as part of normal treatment procedures and reviewed for potential treatment delivery anomalies. RESULTS: In total, 622 unique patients were evaluated for this study. We identified 9 patients with treatment anomalies occurring over their course of treatment, which were only detected with Cherenkov imaging. Categorizing each event indicated issues arising in simulation, planning, pretreatment review, and treatment delivery, and none of the incidents were detected before this review by conventional measures. The incidents identified in this study included dose to unintended areas in planning, dose to unintended areas due to positioning at treatment, and nonideal bolus placement during setup. CONCLUSIONS: Cherenkov imaging was shown to provide a unique method of detecting radiation therapy incidents that would have otherwise gone undetected. Although none of the events detected in this study reached the threshold of reporting, they identified opportunities for practice improvement and demonstrated added value of Cherenkov imaging in quality assurance programs.

The influence of CO2 on spatiotemporal features of mechanically induced cough in anesthetized cats.

Effective cough requires a significant increase in lung volume used to produce the shear forces on the airway to clear aspirated material. This increase in tidal volume during cough, along with an increase in tidal frequency during bouts of paroxysmal cough produces profound hyperventilation and thus reduces arterial CO2. While there are several reports in the literature regarding the effects of hypercapnia, hyperoxia, and hypoxia on cough, there is little research quantifying the effects of hypocapnia on the cough reflex. We hypothesized that decreased CO2 would enhance coughing. In 12 spontaneously breathing adult male cats, we compared bouts of prolonged mechanically stimulated cough, in which cough induced hyperventilation (CHV) was allowed to occur, with isocapnic cough trials where we maintained eupneic end-tidal CO2 by adding CO2 to the inspired gas. Isocapnia slightly increased cough number and decreased esophageal pressures with no change in EMG magnitudes or phase durations. The cough-to-eupnea transition was also analyzed between CHV, isocapnia, and a third group of animals that were mechanically hyperventilated to apnea. The transition to eupnea was highly sensitive to added CO2, and CHV apneas were much shorter than those produced by mechanical hyperventilation. We suggest that the cough pattern generator is relatively insensitive to CHV. In the immediate post-cough period, the appearance of breathing while CO2 is very low suggests a transient reduction in apneic threshold following a paroxysmal cough bout.

The Antibody Dependant Neurite Outgrowth Modulation Response Involvement in Spinal Cord Injury.

Spinal cord injury (SCI) represents a major medical challenge. At present, there is still no cure to treat it efficiently and enable functional recovery below the injury site. Previously, we demonstrated that inflammation determines the fate of the physiopathology. To decipher the molecular mechanisms involved in this process, we performed a meta-analysis of our spatio-temporal proteomic studies in the time course of SCI. This highlighted the presence of IgG isotypes in both spinal cord explants and their secretomes. These IgGs were detected in the spinal cord even if no SCI occurred. However, during the time course following SCI, abundance of IgG1 and IgG2 subclasses (a, b, c) varied according to the spatial repartition. IgG1 was clearly mostly abundant at 12 h, and a switch to IgG2a was observed after 24 h. This IgG stayed predominant 3, 7, and 10 days after SCI. A protein related to IgM as well as a variable heavy chain were only detected 12 h after lesion. Interestingly, treatment with RhoA inhibitor influenced the abundance of the various IgG isotypes and a preferential switch to IgG2c was observed. By data reuse of rat dorsal root ganglion (DRG) neurons RNAseq datasets and RT-PCR experiments performed on cDNA from DRG sensory neurons ND7/23 and N27 dopaminergic neural cell lines, we confirmed expression of immunoglobulin heavy and light chains (constant and variable) encoding genes in neurons. We then identified CD16 and CD32b as their specific receptors in sensory neuron cell line ND7/23 and their activation regulated neurites outgrowth. These results suggest that during SCI, neuronal IgG isotypes are released to modulate neurites outgrowth. Therefore, we propose a new view of the SCI response involving an antibody dependent neurite outgrowth modulation (ADNM) which could be a precursor to the neuroinflammatory response in pathological conditions.

Sirt3 Pharmacologically Promotes Insulin Sensitivity through PI3/AKT/mTOR and Their Downstream Pathway in Adipocytes.

Sirtuin-3 (Sirt3) is a major mitochondrial deacetylase enzyme that regulates multiple metabolic pathways, and its expression is decreased in diabetes type 1 and type 2 diabetes. This study aimed to elucidate Sirt3's molecular mechanism in regulating insulin sensitivity in adipocytes that can contribute to the effort of targeting Sirt3 for the treatment of obesity and type 2 diabetes. We found that the Sirt3 activator honokiol (HNK) induced adipogenesis compared to the control, in contrast to Sirt3 inhibitor, 3-TYP. Accordingly, HNK increased expression of adipocyte gene markers, gene-involved lipolysis and glucose transport (GLUT4), while 3-TYP reduced expression of those genes. Interestingly, 3-TYP caused an increase in gene expression of adipocyte-specific cytokines including IL6, resistin, and TNF-α. However, changes in adipocyte-specific cytokines in HNK treated cells were not significant. In addition, HNK stimulated insulin pathway by promoting insulin receptor beta (IRß) and PI3K/AKT/mTOR pathways, resulting in an increase in phosphorylation of the forkhead family FoxO1/FoxO3a/FoxO4 and glycogen synthase kinase-3 (GSK-3ß), opposing 3-TYP. In line with these findings, HNK increased free fatty acid and glucose uptake, contrary to 3-TYP. In conclusion, Sirt3 activator-HNK induced adipogenesis and lipolysis reduced adipocytes specific cytokines. Intriguingly, HNK activated insulin signaling pathway and increased free fatty acid as well as glucose uptake and transport, in sharp contrast to 3-TYP. These results indicate that, via insulin signaling regulation, Sirt3 activation by HNK improves insulin resistance, while Sirt3 inhibition by 3-TYP might precipitate insulin resistance.

Importance of Cobalt-60 Dose Rate and Biologically Effective Dose on Local Control for Intracranial Meningiomas Treated With Stereotactic Radiosurgery.

BACKGROUND: Radiosurgery dose rate and biologically effective dose (BED) are associated with outcomes after stereotactic radiosurgery (SRS) for functional neurosurgical conditions and some benign tumors. It is not known if these factors affect the efficacy of SRS for meningioma. OBJECTIVE: To determine the association between cobalt-60 dose rate and BED on outcomes in patients with meningioma treated with SRS. METHODS: A single-institution cohort of 336 patients treated between 2005 and 2018 with cobalt-based SRS for 414 separate meningioma lesions was assembled. BED was calculated using an SRS-specific monoexponential model accounting for treatment time per lesion, assuming α/ß = 2.47 Gy. Cumulative incidences of local failure (LF) were reported after considering the competing risk of death, on a per-lesion basis. Multivariable analysis of LF was performed using a proportional hazards model. RESULTS: The most common SRS dose was 12 Gy (n = 227); 140 lesions received 14 Gy. Five-year LF was 15.6% (95% confidence interval 10.4-21.9) and 4.3% (1.4-9.8) in patients who had a dose rate of <2.95 and ≥2.95 Gy/min, respectively (P = .0375). Among 354 grade I or unresected lesions treated with SRS, BED >50 Gy2.47 was associated with a lower incidence of LF (P = .0030). Each 1 Gy/min increase in dose rate was associated with an adjusted hazard ratio of 0.53 (95% confidence interval, 0.29-0.97, P = .041) for LF. Prescription dose >12 Gy was not associated with a lower incidence of LF. CONCLUSION: Patients with meningiomas treated with lower dose rates experienced a higher incidence of LF than those treated with higher dose rates.

Therapeutic anti-glioma effect of the combined action of PCSK inhibitor with the anti-tumoral factors secreted by Poly (I:C)-stimulated macrophages.

Macrophages plasticity is a key feature in cancer progression. Neoplastic cells can alter their immune functions and orient them into a pro-tumoral phenotype. In this context, we developed a new therapeutic strategy to switch macrophages phenotype and reactivate their anti-tumoral functions. We showed a dual activity of a proprotein convertases inhibitor as anti-glioma drug and anti-tumoral macrophages' reactivation drug. Proprotein convertases are proteases that cleave proteins into functional proteins. Several of their substrates are involved in tumorigenesis and immunosuppression. We combine here proprotein convertases inhibitor with Poly (I:C), a TLR3 ligand, to increase the anti-tumoral activity of macrophages. With mass spectrometry-based proteomics, system biology, combined with biological assays, we established that a stimulation of macrophages with Poly (I:C) increased their secretion of pro-inflammatory cytokines and anti-tumoral factors. 3D invasion assay showed the efficacy of these anti-tumoral factors against mixed glioma cells and macrophages spheroids. Besides, immunofluorescence and proliferation assays showed an additive effect of the proprotein convertases inhibitor and the anti-tumoral factors secreted by Poly (I:C)-treated macrophages on both anti-glioma activity and macrophages anti-tumoral orientation directly in tumor microenvironment, leading to an innovative glioma therapy.

Influence of intrathoracic vagotomy on the cough reflex in the anesthetized cat.

Recurrent laryngeal afferent fibers are primarily responsible for cough in response to mechanical or chemical stimulation of the upper trachea and larynx in the guinea pig. Lower airway slowly adapting receptors have been proposed to have a permissive effect on the cough reflex. We hypothesized that vagotomy below the recurrent laryngeal nerve branch would depress mechanically or chemically induced cough. In anesthetized, bilaterally thoracotomized, artificially ventilated cats, thoracic vagotomy nearly eliminated cough induced by mechanical stimulation of the intrathoracic airway, significantly depressed mechanically stimulated laryngeal cough, and eliminated capsaicin-induced cough. These results support an important role of lower airway sensory feedback in the production of tracheobronchial and laryngeal cough in the cat. Further, at least some of this feedback is due to excitation from pulmonary volume-sensitive sensory receptors.

The role of neuronal excitation and inhibition in the pre-Bötzinger complex on the cough reflex in the cat.

Brainstem respiratory neuronal network significantly contributes to cough motor pattern generation. Neuronal populations in the pre-Bötzinger complex (PreBötC) represent a substantial component for respiratory rhythmogenesis. We studied the role of PreBötC neuronal excitation and inhibition on mechanically induced tracheobronchial cough in 15 spontaneously breathing, pentobarbital anesthetized adult cats (35 mg/kg, iv initially). Neuronal excitation by unilateral microinjection of glutamate analog d,l-homocysteic acid resulted in mild reduction of cough abdominal electromyogram (EMG) amplitudes and very limited temporal changes of cough compared with effects on breathing (very high respiratory rate, high amplitude inspiratory bursts with a short inspiratory phase, and tonic inspiratory motor component). Mean arterial blood pressure temporarily decreased. Blocking glutamate-related neuronal excitation by bilateral microinjections of nonspecific glutamate receptor antagonist kynurenic acid reduced cough inspiratory and expiratory EMG amplitude and shortened most cough temporal characteristics similarly to breathing temporal characteristics. Respiratory rate decreased and blood pressure temporarily increased. Limiting active neuronal inhibition by unilateral and bilateral microinjections of GABAA receptor antagonist gabazine resulted in lower cough number, reduced expiratory cough efforts, and prolongation of cough temporal features and breathing phases (with lower respiratory rate). The PreBötC is important for cough motor pattern generation. Excitatory glutamatergic neurotransmission in the PreBötC is involved in control of cough intensity and patterning. GABAA receptor-related inhibition in the PreBötC strongly affects breathing and coughing phase durations in the same manner, as well as cough expiratory efforts. In conclusion, differences in effects on cough and breathing are consistent with separate control of these behaviors.NEW & NOTEWORTHY This study is the first to explore the role of the inspiratory rhythm and pattern generator, the pre-Bötzinger complex (PreBötC), in cough motor pattern formation. In the PreBötC, excitatory glutamatergic neurotransmission affects cough intensity and patterning but not rhythm, and GABAA receptor-related inhibition affects coughing and breathing phase durations similarly to each other. Our data show that the PreBötC is important for cough motor pattern generation, but cough rhythmogenesis appears to be controlled elsewhere.

Surfaceome Proteomic of Glioblastoma Revealed Potential Targets for Immunotherapy.

Glioblastoma (GBM) is the most common and devastating malignant brain tumor in adults. The mortality rate is very high despite different treatments. New therapeutic targets are therefore highly needed. Cell-surface proteins represent attractive targets due to their accessibility, their involvement in essential signaling pathways, and their dysregulated expression in cancer. Moreover, they are potential targets for CAR-based immunotherapy or mRNA vaccine strategies. In this context, we investigated the GBM-associated surfaceome by comparing it to astrocytes cell line surfaceome to identify new specific targets for GBM. For this purpose, biotinylation of cell surface proteins has been carried out in GBM and astrocytes cell lines. Biotinylated proteins were purified on streptavidin beads and analyzed by shotgun proteomics. Cell surface proteins were identified with Cell Surface Proteins Atlas (CSPA) and Gene Ontology enrichment. Among all the surface proteins identified in the different cell lines we have confirmed the expression of 66 of these in patient's glioblastoma using spatial proteomic guided by MALDI-mass spectrometry. Moreover, 87 surface proteins overexpressed or exclusive in GBM cell lines have been identified. Among these, we found 11 specific potential targets for GBM including 5 mutated proteins such as RELL1, CYBA, EGFR, and MHC I proteins. Matching with drugs and clinical trials databases revealed that 7 proteins were druggable and under evaluation, 3 proteins have no known drug interaction yet and none of them are the mutated form of the identified proteins. Taken together, we discovered potential targets for immune therapy strategies in GBM.

Differential effects of acute cerebellectomy on cough in spontaneously breathing cats.

The role of the cerebellum in controlling the cough motor pattern is not well understood. We hypothesized that cerebellectomy would disinhibit motor drive to respiratory muscles during cough. Cough was induced by mechanical stimulation of the tracheobronchial airways in anesthetized, spontaneously breathing adult cats (8 male, 1 female), and electromyograms (EMGs) were recorded from upper airway, chest wall, and abdominal respiratory muscles. Cough trials were performed before and at two time points after total cerebellectomy (10 minutes and >1 hour). Unlike a prior report in paralyzed, decerebrated, and artificially ventilated animals, we observed that cerebellectomy had no effect on cough frequency. After cerebellectomy, thoracic inspiratory muscle EMG magnitudes increased during cough (diaphragm EMG increased by 14% at 10 minutes, p = 0.04; parasternal by 34% at 10 minutes and by 32% at >1 hour, p = 0.001 and 0.03 respectively). During cough at 10 minutes after cerebellectomy, inspiratory esophageal pressure was increased by 44% (p = 0.004), thyroarytenoid (laryngeal adductor) muscle EMG amplitude increased 13% (p = 0.04), and no change was observed in the posterior cricoarytenoid (laryngeal abductor) EMG. Cough phase durations did not change. Blood pressure and heart rate were reduced after cerebellectomy, and respiratory rate also decreased due to an increase in duration of the expiratory phase of breathing. Changes in cough-related EMG magnitudes of respiratory muscles suggest that the cerebellum exerts inhibitory control of cough motor drive, but not cough number or phase timing in response to mechanical stimuli in this model early after cerebellectomy. However, results varied widely at >1 hour after cerebellectomy, with some animals exhibiting enhancement or suppression of one or more components of the cough motor behavior. These results suggest that, while the cerebellum and behavior-related sensory feedback regulate cough, it may be difficult to predict the nature of the modulation based on total cerebellectomy.

The Role of Proprotein Convertases in the Regulation of the Function of Immune Cells in the Oncoimmune Response.

Proprotein convertases (PC) are a family of 9 serine proteases involved in the processing of cellular pro-proteins. They trigger the activation, inactivation or functional changes of many hormones, neuropeptides, growth factors and receptors. Therefore, these enzymes are essential for cellular homeostasis in health and disease. Nine PC subtilisin/kexin genes (PCSK1 to PCSK9) encoding for PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P and PCSK9 are known. The expression of PC1/3, PC2, PC5/6, Furin and PC7 in lymphoid organs such as lymph nodes, thymus and spleen has suggested a role for these enzymes in immunity. In fact, knock-out of Furin in T cells was associated with high secretion of pro-inflammatory cytokines and autoantibody production in mice. This suggested a key role for this enzyme in immune tolerance. Moreover, Furin through its proteolytic activity, regulates the suppressive functions of Treg and thus prevents chronic inflammation and autoimmune diseases. In macrophages, Furin is also involved in the regulation of their inflammatory phenotype. Similarly, PC1/3 inhibition combined with TLR4 stimulation triggers the activation of the NF-κB signaling pathway with an increased secretion of pro-inflammatory cytokines. Factors secreted by PC1/3 KD macrophages stimulated with LPS exert a chemoattractive effect on naive auxiliary T lymphocytes (Th0) and anti-tumoral activities. The link between TLR and PCs is thus very important in inflammatory response regulation. Furin regulates TL7 and TLR8 processing and trafficking whereas PC1/3 controls TLR4 and TLR9 trafficking. Since PC1/3 and Furin are key regulators of both the innate and adaptive immune responses their inhibition may play a major role in oncoimmune therapy. The role of PCs in the oncoimmune response and therapeutic strategies based on PCs inhibition are proposed in the present review.

Intra-Arterial, but Not Intrathecal, Baclofen and Codeine Attenuates Cough in the Cat.

Centrally-acting antitussive drugs are thought to act solely in the brainstem. However, the role of the spinal cord in the mechanism of action of these drugs is unknown. The purpose of this study was to determine if antitussive drugs act in the spinal cord to reduce the magnitude of tracheobronchial (TB) cough-related expiratory activity. Experiments were conducted in anesthetized, spontaneously breathing cats (n = 22). Electromyograms (EMG) were recorded from the parasternal (PS) and transversus abdominis (TA) or rectus abdominis muscles. Mechanical stimulation of the trachea or larynx was used to elicit TB cough. Baclofen (10 and 100 µg/kg, GABA-B receptor agonist) or codeine (30 µg/kg, opioid receptor agonist) was administered into the intrathecal (i.t.) space and also into brainstem circulation via the vertebral artery. Cumulative doses of i.t. baclofen or codeine had no effect on PS, abdominal muscle EMGs or cough number during the TB cough. Subsequent intra-arterial (i.a.) administration of baclofen or codeine significantly reduced magnitude of abdominal and PS muscles during TB cough. Furthermore, TB cough number was significantly suppressed by i.a. baclofen. The influence of these drugs on other behaviors that activate abdominal motor pathways was also assessed. The abdominal EMG response to noxious pinch of the tail was suppressed by i.t. baclofen, suggesting that the doses of baclofen that were employed were sufficient to affect spinal pathways. However, the abdominal EMG response to expiratory threshold loading was unaffected by i.t. administration of either baclofen or codeine. These results indicate that neither baclofen nor codeine suppress cough via a spinal action and support the concept that the antitussive effect of these drugs is restricted to the brainstem.

Hydroxyl functionalized multi-walled carbon nanotubes modulate immune responses without increasing 2009 pandemic influenza A/H1N1 virus titers in infected mice.

Growing use of carbon nanotubes (CNTs) have garnered concerns regarding their association with adverse health effects. Few studies have probed how CNTs affect a host's susceptibility to pathogens, particularly respiratory viruses. We reported that exposure of lung cells and mice to pristine single-walled CNTs (SWCNTs) leads to significantly increased influenza virus H1N1 strain A/Mexico/4108/2009 (IAV) titers in concert with repressed antiviral immune responses. In the present study, we investigated if hydroxylated multi-walled CNTs (MWCNTs), would result in similar outcomes. C57BL/6 mice were exposed to 20 µg MWCNTs on day 0 and IAV on day 3 and samples were collected on day 7. We investigated pathological changes, viral titers, immune-related gene expression in lung tissue, and quantified differential cell counts and cytokine and chemokine levels in bronchoalveolar lavage fluid. MWCNTs alone caused mild inflammation with no apparent changes in immune markers whereas IAV alone presented typical infection-associated inflammation, pathology, and titers. The co-exposure (MWCNTs + IAV) did not alter titers or immune cell profiles compared to the IAV only but increased concentrations of IL-1ß, TNFα, GM-CSF, KC, MIPs, and RANTES and inhibited mRNA expression of Tlr3, Rig-i, Mda5, and Ifit2. Our findings suggest MWCNTs modulate immune responses to IAV with no effect on the viral titer and modest pulmonary injury, a result different from those reported for SWCNT exposures. This is the first study to show that MWCNTs modify cytokine and chemokine responses that control aspects of host defenses which may play a greater role in mitigating IAV infections.

The Role of a Proprotein Convertase Inhibitor in Reactivation of Tumor-Associated Macrophages and Inhibition of Glioma Growth.

Tumors are characterized by the presence of malignant and non-malignant cells, such as immune cells including macrophages, which are preponderant. Macrophages impact the efficacy of chemotherapy and may lead to drug resistance. In this context and based on our previous work, we investigated the ability to reactivate macrophages by using a proprotein convertases inhibitor. Proprotein convertases process immature proteins into functional proteins, with several of them having a role in immune cell activation and tumorigenesis. Macrophages were treated with a peptidomimetic inhibitor targeting furin, PC1/3, PC4, PACE4, and PC5/6. Their anti-glioma activity was analyzed by mass spectrometry-based proteomics and viability assays in 2D and 3D in vitro cultures. Comparison with temozolomide, the drug used for glioma therapy, established that the inhibitor was more efficient for the reduction of cancer cell density. The inhibitor was also able to reactivate macrophages through the secretion of several immune factors with antitumor properties. Moreover, two proteins considered as good glioma patient survival indicators were also identified in 3D cultures treated with the inhibitor. Finally, we established that the proprotein convertases inhibitor has a dual role as an anti-glioma drug and anti-tumoral macrophage reactivation drug. This strategy could be used together with chemotherapy to increase therapy efficacy in glioma.

Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.

Hemophilia A (HA) is a bleeding disorder characterized by spontaneous and prolonged hemorrhage. The disease is caused by mutations in the coagulation factor 8 gene (F8) leading to factor VIII (FVIII) deficiency. Since FVIII is primarily produced in endothelial cells (ECs) in a non-diseased human being, ECs hold great potential for development as a cell therapy for HA. We showed that HA patient-specific induced pluripotent stem cells (HA-iPSCs) could provide a renewable supply of ECs. The HA-iPSC-derived ECs were transduced with lentiviral vectors to stably express the functional B domain deleted F8 gene, the luciferase gene, and the enhanced green fluorescent protein gene (GFP). When transplanted intramuscularly into neonatal and adult immune deficient mice, the HA-iPSC-derived ECs were retained in the animals for at least 10-16 weeks and maintained their expression of FVIII, GFP, and the endothelial marker CD31, as demonstrated by bioluminescence imaging and immunostaining, respectively. When transplanted into HA mice, these transduced HA-iPSC-derived ECs significantly reduced blood loss in a tail-clip bleeding test and produced therapeutic plasma levels (11.2%-369.2%) of FVIII. Thus, our studies provide proof-of-concept that HA-iPSC-derived ECs can serve as a factory to deliver FVIII for the treatment of HA not only in adults but also in newborns.

Compressive fatigue and endurance of juvenile bovine articular cartilage explants.

Articular cartilage is an enduring tissue. For most individuals, articular cartilage facilitates a lifetime of pain-free ambulation, supporting millions of loading cycles from activities of daily living. Although early studies into osteoarthritis focused on the role of mechanical fatigue in articular cartilage degeneration, much is still unknown regarding its strength and endurance characteristics. The compressive strength of juvenile, bovine articular cartilage explants was determined to be loading rate-dependent, reaching a maximum strength of 29.5 ±â€¯4.8 MPa at a strain rate of 0.10 %/sec. The fatigue and endurance properties of articular cartilage were characterized utilizing a material testing system, as well as a custom, validated instrument termed the two degrees-of-freedom endurance meter (endurometer). These instruments characterized fatigue in articular cartilage explants at loading levels ranging from 10 to 80 % strength (%S), up to 100,000 cycles. Cartilage explants displayed characteristics of fatigue - fatigue life increased as the loading magnitude decreased. All explants failed within 14,000 cycles at loading levels between 50 and 80 %S. At 10 and 20 %S, all explants endured 100,000 loading cycles. There was no significant difference in equilibrium compressive modulus between run-out explants and unloaded controls, although the pooled modulus increased in response to testing. Histological staining and biochemical assays revealed no material changes in collagen, sulfated glycosaminoglycan, or hydration content between unloaded controls and explants cyclically loaded to run-out. These results suggest articular cartilage may have a putative endurance limit of 20 %S (5.86 MPa), with implications for articular cartilage biomechanics and mechanobiology.

Potential long-term treatment of hemophilia A by neonatal co-transplantation of cord blood-derived endothelial colony-forming cells and placental mesenchymal stromal cells.

BACKGROUND: Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cell-based gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. METHODS: In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. RESULTS: In vivo bioluminescence imaging showed that the ECFC only and the co-transplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks post-transplantation. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype, and generated functional vasculature. Next, co-transplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13 ± 19.84 µl to 155.78 ± 44.93 µl in a tail-clip assay. CONCLUSIONS: This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA.

Enhancing Retention of Human Bone Marrow Mesenchymal Stem Cells with Prosurvival Factors Promotes Angiogenesis in a Mouse Model of Limb Ischemia.

Mesenchymal stem/stromal cells (MSCs) offer great promise in the treatment of ischemic injuries, including stroke, heart infarction, and limb ischemia. However, poor cell survival after transplantation remains a major obstacle to achieve effective MSC therapies. To improve cell survival and retention, we transplanted human bone marrow MSCs with or without a specific prosurvival factor (PSF) cocktail consisting of IGF1, Bcl-XL, a caspase inhibitor, a mitochondrial pathway inhibitor, and Matrigel into the limbs of immune deficient mice, after induction of hindlimb ischemia. The PSF markedly prolonged the retention of the MSCs in the ischemic limb muscles as demonstrated by bioluminescence imaging. Using microcomputed tomography to image the limb muscle vasculature in the mice 9 weeks after the transplantation, we found that the mice transplanted with MSCs without PSF did not show a significant increase in the blood vessels in the ischemic limb compared with the nontransplanted control mice. In contrast, the mice transplanted with MSCs plus PSF showed a significant increase in the blood vessels, especially the larger and branching vessels, in the ischemic limb compared with the control mice that did not receive MSCs. Thus, we demonstrated that prolonged retention of MSCs using PSF effectively promoted angiogenesis in ischemic animal limbs. This study highlights the importance of enhancing cell survival in the development of effective MSC therapies to treat vascular diseases.

3D MALDI mass spectrometry imaging reveals specific localization of long-chain acylcarnitines within a 10-day time window of spinal cord injury.

We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.