Downregulation of inflammatory markers by conjugated linoleic acid isomers in human cultured astrocytes

Objectives: Conjugated linoleic acid (CLA) isomers have been shown to possess anti-inflammatory activity in the central nervous system. In this study, we aimed to evaluate whether modulation of the fatty acid profile by the CLA isomers c9,t11 or t10,c12CLA was associated with changes in the expression of pro-inflammatory molecules in human astrocytes.

Methods: Cultured astrocytes were treated for 6 days with 100?µM fatty acids (c9,t11CLA or t10,c12CLA or oleic acid). Following the treatment, the fatty acid profile of the cell and pro-inflammatory molecule expression were assessed.

Results: Only the t10,c12CLA isomer induced a significant decrease in arachidonic acid and increased the ratio of docosahexaenoic acid/eicosapentaenoic acid, which constitutes indirect evidence of peroxisome proliferator-activated receptor alpha activation. Inhibition of tumour necrosis factor-α, interleukin-1β, and RANTES expression was observed in astrocytes treated with c9,t11CLA and t10,c12CLA.

Discussion: Current data demonstrate that CLA isomers, particularly t10,c12, may affect neuroinflammation by reducing the pro-inflammatory molecules in cultured astrocytes, suggesting a potential nutritional role of CLA isomers in modulating the astrocyte inflammatory response.     

Platelet-rich plasma injections for the treatment of refractory Achilles tendinopathy: results at 4 years

Background

Chronic Achilles tendinopathy is responsible for a severe reduction in physical performance and persistent pain. There is currently a number of therapeutic options and the local administration of growth factors is an emerging treatment strategy. In particular, platelet-rich plasma (PRP) is a widely used way to provide a local regenerative stimulus for tendon healing. The aim of this study was to document the mid-term results obtained after treating recalcitrant Achilles tendinopathy with injections of high concentrate, leucocyte-rich PRP.

Materials and methods

Twenty-seven patients (mean age: 44.6 years; 22 men and 5 women) affected by chronic mid-portion Achilles tendinopathy (7 bilateral, for a total of 34 tendons), refractory to previous treatments, were enrolled. Patients were treated with three ultrasound-guided intra-tendinous injections of PRP at 2-week intervals. Patients were prospectively evaluated at baseline, and then at 2, 6, and up to a mean of 54.1 months of follow-up (minimum 30 months), using the following tools: Blanzina, VISA-A, EQ-VAS for general health, and Tegner scores.

Results

The VISA-A score showed a significant improvement: the baseline score of 49.9±18.1 increased to 62.9±19.8 at 2 months (p=0.002), with a further improvement at 6 months (84.3±17.1, p<0.0005), and stable results at 4.5 years (90.0±13.9). The EQ-VAS score also showed a similar positive trend. An evaluation of the activity level confirmed these findings, showing a significant improvement in the Tegner score over time (p=0.017 for the final evaluation). The longer duration of symptoms before treatment was associated with a slower return to sport (p=0.041).

Discussion

PRP injections produced good overall results for the treatment of chronic recalcitrant Achilles tendinopathy with a stable outcome up to a medium-term follow-up. Longer symptom duration was related with a more difficult return to sporting activity.     

A Protein Kinase A–Independent Pathway Controlling Aquaporin 2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis Associated with Polycystic Kidney Disease 1 Haploinsufficiency

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1^+/− mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1^+/− mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca²⁺ levels, promotes AQP2 trafficking independent of the AVP–PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.In most mammals, regulation of water balance is critically dependent on water intake and excretion, which is under control of the antidiuretic hormone arginine vasopressin (AVP). In the kidney, AVP binds to the V2 vasopressin (V2R) receptor, activating the cAMP/protein kinase A (PKA) signal transduction cascade, promoting the fusion of intracellular vesicles containing aquaporin 2 (AQP2) to the apical plasma membrane, and increasing luminal permeability.^1–3 This translocation is accompanied by AVP-dependent phosphorylation of AQP2 at serine-256 (pS256).Mice in which S256 could not be phosphorylated (AQP2-S256L) develop polyuria and hydronephrosis because of a defect in AQP2 trafficking to the plasma membrane.⁴ Interestingly, it connects to polycystic kidney disease (PKD). Mutations in polycystin-1 (Pkd1^+/−) gene cause PKD, whereas PKD1 haplo-insufficient mice (Pkd1^+/−), showing an inappropriate antidiuresis, display significantly higher levels of pS256 compared with wild-type (WT) littermates; the prominent expression at the apical plasma membrane of collecting duct principal cells, despite normal V2R expression and normal cAMP levels, is associated with unchanged AVP expression in the brain, despite chronic hypo-osmolality.⁵These observations underscore the crucial role of AQP2 phosphorylation at S256 in controlling the cellular distribution and fate of AQP2.^1,6,7 As for many proteins, the function and the trafficking of AQP2 are modulated by a balance of reversible phosphorylation and dephosphorylation. Preventing dephosphorylation of AQP2 with okadaic acid, inhibitor of phosphatase 1 (PP1), inhibitor of phosphatase 2A (PP2A), and inhibitor of phosphatase 2B (PP2B) significantly increased AQP2-pS256.⁸ Proteomic analysis of inner medulla collecting duct identified PP2A as a phosphoprotein isolated from inner medullary collecting duct samples treated with either calyculin-A, a specific PP2A inhibitor, or vasopressin,⁹ suggesting the possible participation of this phosphatase in cellular events triggered by physiologic stimulus, such as vasopressin in renal collecting duct cells.The complexity of AQP2 regulation was further increased by phosphoproteomics studies showing that, other than S256, vasopressin modulates the phosphorylation status of three other sites within the C terminus (S261, S264, and S269). Although vasopressin increases S264 and S269 phosphorylation, it decreases S261 phosphorylation.^9–12 Regarding the potential kinases responsible for the phosphorylation of these sites, c-Jun N-terminal kinase, p38, and cyclin-dependent kinases (cdks) cdk1 and cdk5 can phosphorylate AQP2 peptides at S261 in vitro.^13,14 Here, in the attempt to investigate the potential involvement of cdks in AQP2 regulation, we discovered a new PKA-independent signal transduction pathway regulating AQP2 phosphorylation and localization. We found that selective inhibition of cdks with R-roscovitine is associated with a decrease of intracellular Ca²⁺ levels and a significant downregulation of the phosphatase PP2A activity, resulting in an increase of AQP2 phosphorylation at S256 and targeting to the apical membrane. Physiologically, this novel regulatory mechanism might be of clinical interest, because it better elucidates the molecular bases of pathologic states characterized by disturbances in water balance.     

Spontaneous clostridial myonecrosis after pregnancy – emergency treatment to the limb salvage and functional recovery: a case report

Clostridial myonecrosis (CM) is a rare, life threatening necrotizing infection of a skeletal muscle caused by Clostridium perfringens in the majority of cases. The diagnosis may be difficult because of few diagnostic and cutaneous signs early in its course. Standard therapy involves surgical debridements of a devitalized tissue and high‐dose organism‐specific antibiotic therapy. The hyperbaric oxygen has also showed its usefulness in the treatment of these infections. Autograft systems as tissue replacement, based on bioengineered materials, have been demonstrated to be safe and effective treatments for chronic wounds and a suitable physiotherapy is recommended for the recovery of functional impairments of upper extremities. We present a rare case of CM of right upper limb treated with a combination of standard treatments and new techniques.     

New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK

Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients.We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models.X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner.In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents.Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors.     

Cognitive rehabilitation for early post-surgery inpatients affected by primary brain tumor: a randomized, controlled trial

Cognitive impairment is one of the most common neurological disorders in neuro-oncological patients and exerts a deep negative impact on quality of life interfering with familiar, social and career-related activities. To test the effectiveness of early cognitive rehabilitation treatment for inpatients affected by primary brain tumors. Out of 109 consecutive patients enrolled in the study, 58 patients were randomly assigned to a rehabilitation group or to a control group. The rehabilitation consisted of 16 one-hour individual sessions of therapist-guided cognitive training, spread over 4 weeks, combining computer exercises and metacognitive training. Patients in the control group received usual care without cognitive training. All patients were evaluated by means of a comprehensive neuropsychological battery at the admission (T0) and after 4 weeks (T1). Patients in the rehabilitation group showed a significant improvement of cognitive functions. In particular, the domains that benefited most from the training were visual attention and verbal memory. The control group exhibited only a slightly, not statistically relevant, enhancement of cognitive performances. Cognitive rehabilitation for neuro-oncological inpatients resulted in a significant enhancement of cognitive performances after the training, also providing a foundation for early administration. Future research should be aimed to clarify the patients’ characteristics that predict neuropsychological improvement, to identify the most effective elements in rehabilitative programs and to study the effects of treatment extension to everyday life.