Targeting the PI3K/mTOR pathway in murine endocrine cell lines: in vitro and in vivo effects on tumor cell growth

The mammalian target of rapamycin (mTOR) inhibitors, such as rapalogues, are a promising new tool for the treatment of metastatic gastroenteropancreatic endocrine tumors. However, their mechanisms of action remain to be established. We used two murine intestinal endocrine tumoral cell lines, STC-1 and GLUTag, to evaluate the antitumor effects of rapamycin in vitro and in vivo in a preclinical model of liver endocrine metastases. In vitro, rapamycin inhibited the proliferation of cells in the basal state and after stimulation by insulin-like growth factor-1. Simultaneously, p70S6 kinase and 4EBP1 phosphorylation was inhibited. In vivo, rapamycin substantially inhibited the intrahepatic growth of STC-1 cells, irrespectively of the timing of its administration and even when the treatment was administered after cell intrahepatic engraftment. In addition, treated animals had significantly prolonged survival (mean survival time: 47.7 days in treated animals versus 31.8 days in controls) and better clinical status. Rapamycin treatment was associated with a significant decrease in mitotic index and in intratumoral vascular density within STC-1 tumors. Furthermore, the antitumoral effect obtained after treatment with a combination of rapamycin and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 was more significant than with rapamycin alone in both cell lines. Our results suggest that the antitumor efficacy of rapamycin in neuroendocrine tumors results from a combination of antiproliferative and antiangiogenic effects. Interestingly, a more potent antitumor efficiency could be obtained by simultaneously targeting several levels of the PI3K/mTOR pathway.     

Interleukin-1β inhibition prevents choroidal neovascularization and does not exacerbate photoreceptor degeneration

The pro-inflammatory cytokine IL-1β has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1β in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1β expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1β was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1β significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1β on choroidal endothelial cell proliferation. Inhibition of IL-1β in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1β inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.     

Multisensory adaptation of spatial-to-motor transformations in children with developmental coordination disorder

Recent research has demonstrated that adaptation to a visuomotor distortion systematically influenced movements to auditory targets in adults and typically developing (TD) children, suggesting that the adaptation of spatial-to-motor transformations for reaching movements is multisensory (i.e., generalizable across sensory modalities). The multisensory characteristics of these transformations in children with developmental coordination disorder (DCD) have not been examined. Given that previous research has demonstrated that children with DCD have deficits in sensorimotor integration, these children may also have impairments in the formation of multisensory spatial-to-motor transformations for target-directed arm movements. To investigate this hypothesis, children with and without DCD executed discrete arm movements to visual and acoustic targets prior to and following exposure to an abrupt visual feedback rotation. Results demonstrated that the magnitudes of the visual aftereffects were equivalent in the TD children and the children with DCD, indicating that both groups of children adapted similarly to the visuomotor perturbation. Moreover, the influence of visuomotor adaptation on auditory-motor performance was similar in the two groups of children. This suggests that the multisensory processes underlying adaptation of spatial-to-motor transformations are similar in children with DCD and TD children.     

Gastric and pyloric motor pattern control by a modulatory projection neuron in the intact crab Cancer pagurus

Neuronal release of modulatory substances provides motor pattern generating circuits with a high degree of flexibility. In vitro studies have characterized the actions of modulatory projection neurons in great detail in the stomatogastric nervous system, a model system for neuromodulatory influences on central pattern generators. Less is known about the activities and actions of modulatory neurons in fully functional and richly modulated network settings, i.e., in intact animals. It is also unknown whether their activities contribute to the motor patterns in different behavioral conditions. Here, we show for the first time the activity and effects of the well-characterized modulatory projection neuron 1 (MCN1) in vivo and compare them to in vitro conditions. MCN1 was always spontaneously active, typically in a rhythmic fashion with its firing being interrupted by ascending inhibitions from the pyloric motor circuit. Its activity contributed to pyloric motor activity, because 1) the cycle period of the motor pattern correlated with MCN1 firing frequency and 2) stimulating MCN1 shortened the cycle period while 3) lesioning of the MCN1 axon reduced motor activity. In addition, gastric mill motor activity was elicited for the duration of the stimulation. Chemosensory stimulation of the antennae moved MCN1 away from baseline activity by increasing its firing frequency. Following this increase, a gastric mill rhythm was elicited and the pyloric cycle period decreased. Lesioning the MCN1 axon prevented these effects. Thus modulatory projection neurons such as MCN1 can control the motor output in vivo, and they participate in the processing of exteroceptive sensory information in behaviorally relevant conditions.     

Enhanced catecholamine release in mice expressing PKB/SGK-resistant GSK3

Glycogen synthase kinase 3 (GSK3) plays a decisive role in the regulation of multiple functions. GSK3 is phosphorylated and its activity inhibited by protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which are in turn activated by growth factors through phosphoinositide (PI) 3 kinase signaling. PI3/PKB/Akt/SGK-dependent inhibition of GSK3 is disrupted in gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3α,? (gsk3 ^KI ) where the serine of the PKB/SGK phosphorylation site has been replaced by alanine. Recent experiments revealed that blood pressure is significantly higher in those mice than in wild type mice (gsk3 ^WT ). The present study was performed to elucidate the underlying cause. Blood pressure was determined with the tail cuff method, heart rate by ECG measurements, catecholamine concentrations by ELISA, and vanillylmandelic acid by high pressure liquid chromatography. As a result, blood pressure and heart rate were significantly higher in gsk3 ^KI than in gsk3 ^WT mice. The α-adrenergic blocker prazosin (1 μg/g body weight, b.w.) and the ganglion blocker hexamethonium (40 μg/g b.w.) decreased blood pressure to a larger extent in gsk3 ^KI than in gsk3 ^WT mice and virtually abrogated the difference between genotypes. Similarly, the β-adrenergic blocker atenolol (5 μg/g b.w.) decreased the heart rate to a larger extent in gsk3 ^KI than in gsk3 ^WT mice and again dissipated the difference of heart rate between genotypes. Plasma epinephrine and norepinephrine concentrations, as well as urinary excretion of vanillylmandelic acid, were significantly higher in gsk3 ^KI than in gsk3 ^WT mice. The observations reveal a completely novel function of PKB/Akt/SGK-dependent GSK3 signaling, i.e., regulation of catecholamine release.     

Xpert MTB/RIF assay for diagnosis of pleural tuberculosis

We prospectively investigated the diagnostic utility of the Xpert MTB/RIF (Mycobacterium tuberculosis/rifampin [RIF] resistance) assay in 20 cases with confirmed tuberculous pleural effusion. The sensitivity and specificity of the Xpert assay in pleural fluid were 25% and 100%, respectively. All cases positive by the Xpert assay were also positive by pleural fluid culture.     

Comparison of ventral corpectomy and plate-screw-instrumented fusion with dorsal laminectomy and rod-screw-instrumented fusion for treatment of at least two vertebral-level spondylotic cervical myelopathy

The objective of the article is to verify the hypothesis that the dorsal multilevel laminectomy and rod-screw-instrumented fusion (DLF) for multilevel spondylotic cervical myelopathy (MSCM) is less strenuous for patients, and less prone to perioperative complications, than ventral multilevel corpectomy and plate-screw-instrumented fusion (VCF), while clinical outcome is comparable. One hundred and three successive patients were treated for at least two vertebral-level MSCM, 42 of them by VCF and 61 by DLF. The two patients groups were retrospectively compared. VCF patients were slightly younger than DLF patients (62.5 ± 10.61 years versus 66 ± 12.4 years, P = 0.012). In VCF patients, a median of 2 (2–3) corpectomies and in DLF patients a median of 3 (2–5) laminectomies were performed. In VCF patients, surgery lasted longer than in DLF patients (229 ± 60 min versus 183 ± 46 min, P ≤ 0.001). Between the VCF and the DLF patients groups, no significant difference was found in perioperative complications (e.g. hardware failure rates of 16.7% in VCF and of 6.6% in the DLF patients) and mortality rates. The postoperative outcome, as assessed by the postoperative change of the Nurick scores, the change of neck pain, the patients’ satisfaction, and the change of the subaxial Cobb angle of the spine did not differ between the two patients groups. However, when comparing the postoperative Nurick scores directly, VCF patients fared somewhat better than DLF patients [median of 2 (0–5) versus 3 (1–5), P = 0.003]. The hypothesized advantages of DLF over VCF in the surgical treatment of at least two vertebral-level MSCM could not be confirmed in this retrospective study. A prospective randomized study is warranted to clarify this issue.     

Hemodynamic Changes in a Model of Chronic Heart Failure Induced by Multiple Sequential Coronary Microembolization in Sheep

Although a large variety of animal models for acute ischemia and acute heart failure exist, valuable models for studies on the effect of ventricular assist devices in chronic heart failure are scarce. We established a stable and reproducible animal model of chronic heart failure in sheep and aimed to investigate the hemodynamic changes of this animal model of chronic heart failure in sheep. In five sheep (n = 5, 77 ± 2 kg), chronic heart failure was induced under flouroscopic guidance by multiple sequential microembolization through bolus injection of polysterol microspheres (90 µm, n = 25.000) into the left main coronary artery. Coronary microembolization (CME) was repeated up to three times in 2 to 3‐week intervals until animals started to develop stable signs of heart failure. During each operation, hemodynamic monitoring was performed through implantation of central venous catheter (central venous pressure [CVP]), arterial pressure line (mean arterial pressure [MAP]), implantation of a right heart catheter {Swan‐Ganz catheter (mean pulmonary arterial pressure [PAPmean])}, pulmonary capillary wedge pressure (PCWP), and cardiac output [CO]) as well as pre‐ and postoperative clinical investigations. All animals were followed for 3 months after first microembolization and then sacrificed for histological examination. All animals developed clinical signs of heart failure as indicated by increased heart rate (HR) at rest (68 ± 4 bpm [base] to 93 ± 5 bpm [3 mo][P < 0.05]), increased respiratory rate (RR) at rest (28 ± 5 [base] to 38 ± 7 [3 mo][P < 0.05]), and increased body weight 77 ± 2 kg to 81 ± 2 kg (P < 0.05) due to pleural effusion, peripheral edema, and ascites. Hemodynamic signs of heart failure were revealed as indicated by increase of HR, RR, CVP, PAP, and PCWP as well as a decrease of CO, stroke volume, and MAP 3 months after the first CME. Multiple sequential intracoronary microembolization can effectively induce myocardial dysfunction with clinical and hemodynamic signs of chronic ischemic cardiomyopathy. The present model may be suitable in experimental work on heart failure and left ventricular assist devices, for example, for studying the impact of mechanical unloading, mechanisms of recovery, and reverse remodeling.     

Fat extravasation due to unreamed and experimentally reamed intramedullary nailing of the sheep femur

Aim

To compare systemic fat extravasation in unreamed and experimentally reamed nailing.

Methods

An osteotomy was created in the proximal third of the femoral shaft in 16 sheep, and intramedullary pressure increase and fat extravasation were monitored for the two nailing techniques.

Results

The highest intramedullary pressures, median 2700 mmHg, and highest percentages of fat extravasation, peaking at almost 90% of fat, were found for the unreamed nailing technique. The values for the reamed group were significantly lower.

Conclusions

The extravasation of intramedullary fat can be attributed to the great increase in intramedullary pressure that occurs during unreamed nailing. Correctly performed intramedullary reaming with the new reaming system produces lower pressures and much less systemic fat extravasation, reducing the risk for fat embolism.