As the effects of ultrasound on human brain functions might bear therapeutic potential, in this study, we examined the effects of diagnostic, i.e. non-thermal, ultrasound, on morphology, networking, and metabolic activity of SH- SY5Y human neurons in culture, as well as on the expression of GAP-43, Hsp90 and VEGF proteins, with and without selenium in the culture medium. The rationale for studying selenium lays in the observation that selenium improves functional neurologic outcome in traumatic brain injury and, therefore, analysis of the interactions between ultrasound and selenium may be of clinical interest. In the presence of selenium, ultrasound increased the overall number and length of elongations arising from the neuron bodies, thus reflecting an increase in the complexity of neuronal networks and circuits. The expression of GAP-43, Hsp90 and VEGF and metabolic activity of SH-SY5Y neurons, studied as markers of cell damage, were not affected by ultrasound or selenium. This study suggests that ultrasound may modulate neuronal networking in vitro without inducing cellular or molecular damage and highlights the potential role of selenium in the ultrasound-elicited cellular responses.

In this study we report the results of a protocol for improving brain lymphatic flow in autism through lymphatic drainage massage, a technique successfully used in a variety of conditions where intracranial lymphatic circulation is hampered by obstacles at the level of deep cervical nodes. At the end of May 2018, the Biomedical Centre for Autism Research and Treatment started implementing a protocol of manual lymphatic drainage of the deep cervical nodes on autistic subjects. By October 2018, several scores of patients had been treated with this protocol. In this report, we describe the cases of three autistic patients for whom manual lymphatic massage was remarkably effective. To our knowledge, this is the first report of lymphatic drainage massage at the level of the deep cervical nodes in autism. Symptomatic improvement was robust and we attribute these results to the effects of the massage on the intracranial lymph or sometimes referred to as the glymphatic circulation with improvement of brain lymphatic drainage believed leading to a decrease of neuroinflammation. In addition to stimulating lymphatic drainage, we postulate that the protocol may serve also as vagus nerve stimulation. The protocol also targets the larynx in a manner similar as described for laryngeal manual therapy for the treatment of dysphonia, and this factor may be contributing to the overall improvement of symptoms, with particular reference to speech. Based on the cases described in this report and on our ongoing research, we are convinced that this type of inexpensive, harmless and easy-to-implement approach of manual lymphatic drainage can be beneficial to autistic patients and represents a new and promising treatment. We expect that the described protocol will play a central role in future treatments for autism, both alone and in combination with other therapies such as behavioral therapies or nutritional interventions.

Cognitive behavioral therapy (CBT) is a well established first line treatment for obsessive compulsive disorder (OCD). It consists of a cognitive and a behavioral treatment (exposure and response prevention therapy (ERP)) [1]. The goal of the cognitive treatment is the cognitive restructuring of meta-cognitions while the goal of exposure therapy is enhancing and facilitating patients’ fear extinction processes in front of the feared object, situation or thought/obsession. Several studies have tried to enhance fear extinction through pharmacological agents with mixed results [2]. High-frequency repetitive transcranial magnetic stimulation (HF-rTMS), through its capacity to induce long-term potentiation, is able to modulate learning processes and hippocampal plasticity [3]. Moreover, several studies showed that HF-rTMS over the dosrsolateral prefrontal cortex (DLPFC) is able to induce better working memory performance after active stimulation [4]. Thus, rTMS could represent a promising tool to enhance cognitive restructuring and fear extinction learning processes during the cognitive-behavioral treatment sessions. Recently, we published a case report of a patient treated with HF-rTMS over the left DLPFC before each ERP session [5]. Here we adopted a new protocol in order to potentiate both the cognitive and the behavioral part of the treatment. The aim of this study was to evaluate the potential CBT enhancing effect of HF-rTMS over the left DLPFC in OCD patients who failed to respond to a previous CBT trial. Each patient underwent a 16 sessions CBT trial (1 session per week, 50/60 minutes per session). The first six sessions were dedicated to establishing an individual hierarchy of obsessions and compulsions and to a cognitive restructuring of meta-cognitions. The following 10 sessions focused on in vivo ERP exercises. For the first six sessions patients underwent a HF-rTMS session over the left DLPFC immediately before the session in order to enhance working memory processes during cognitive restructuring of metacognitions (6 stimulation sessions). Subsequently, each exposure session was immediately followed by an HF-rTMS session over the left DLPFC (10 stimulation sessions). During the treatment, 32 trains of 5 seconds has been applied at 10 Hz and at 110% of the left motor trheshold over the left DLPFC, with a 25 seconds inter-train interval (a total of 1600 stimuli per session). No pharmacological treatment changes were allowed during the CBT trail and patients were stable on their undergoing medications since 4 weeks before starting the CBT sessions. We enrolled two OCD patients (with prominent checking and symmetry/ordering symptoms respectively) who failed a previous CBT trial and showed only slight depressive symptoms. At the end of the 16 CBT sessions patients showed a 35% and 30% symptoms reduction on the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) respectively. HF-rTMS was well tolerated. rTMS could represent e tolerable tool in order to enhance the effect of CBT with exposure and response prevention techniques in patients with obsessive-compulsive disorder.

Our study is focused on the development of a new method for the automatic analysis of cell images. We focused on neurons (cells line SH-SY5Y) treated/untreated with ultrasound and stained with Haematoxylin-Eosin. The aim of the algorithm is the automatic detection of the cell body as well as the determination of the number and the length of neuron elongations. Starting point of the algorithm was the convolution of an image with a bank of rotating Gaussian kernels and the construction of a module map. Then several strategies were implemented to detect cell bodies and to detect and extract data about cell elongations. We have also realized a graphical user interface allowing the loading, saving and processing of images. Results show that this method is able to properly and efficiently detect cell contours and elongations. The automated evaluation is in strong agreement with manual evaluation performed by an expert operator, with an average error of 11% with most parameter combinations. This tool constitutes an important support in biological research activities, where operators need to analyze a large number of images to investigate about cell morphology before and after a treatment.

Aim. Aim of the present study was to prevent overtraining and to ensure that the athletic training program will result in performance improvements, regular performance tests as well as heart rate evaluation and questionnaires are necessary. However, none of them are routinely performed on regular basis mainly for their interference with the session training, thus leading to the risk of an over-reaching which in turn may result in an overtraining. In this study we evaluate the possibility to monitor the development of the training program of high level rugby players by a simple, non-invasive and fast salivary testosterone and cortisol measure.Methods. Testosterone and cortisol samplings were performed in several moments of the preseason training program. Measures were correlated with anthropometric evaluations performed by bioelectrical impedance analysis before and after the training program. To assess a potential role of genetic, athletes were also genotyped for ACTN3 and ACE gene polymorphisms.Results. Data from this study show that hormone salivary measures correlate with changes in anthropometric values during the training program. No correlations were observed between genotypes and variations in hormone levels; however, ACE genotype correlates with the different team position of rugby players.Conclusion. Hormone salivary measures represent a valid, fast, non-invasive and stress-free system of monitoring the athletic preparation.

Deglycosylated vitamin D-binding protein-derived macrophage-activating factor (GcMAF) is known to be a strong immune stimulatory natural molecule. Data in literature demonstrate that GcMAF has a direct role in decreasing cell proliferation of different cancer cell lines. In this study we evaluate the direct effect of GcMAF complexed with oleic acid (OA-GcMAF) on human multiple myeloma cells (KMS-12BM), as well as the effect on the same cell line of human macrophages (CRL9853) previously activated by OA-GcMAF. Cell viability and living cell number were evaluated respectively by tetrazolium dye cell viability assay and by Trypan blue staining. Interactions between activated macrophages and myeloma cells were studied by time lapse photography. Our results show that OA-GcMAF decreases the cell viability of KMS-12-BM with a dose-dependent pathway. Furthermore OA-GcMAF activates human macrophages, which in turn phagocytise myeloma cancer cells. OA-GcMAF confirms its double effect on cancer cells: a direct inhibition of their viability and, at the same time, an efficient macrophage activation leading to a significant depletion of cancer cell population. Introduction: In recent years the interest of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) as a potent immunotherapeutic agent has increased. The GcMAF has been shown to be effective in stimulating murine macrophages in vitro to phagocytose human breast carcinoma cultures (1, 2), as well as inhibiting the growth of prostate cancer cells (3). It has also been the agent that has been referenced as reducing tumour burden in several clinical approaches (4, 5). In previous studies GcMAF has been used to stimulate Raw 264.7 cells (murine macrophage cell line) that were observed in vitro to phagacytose MCF-7 cells (human breast carcinoma). In this study we demonstrate the effect of GcMAF stabilized with oleic acid (OA-GcMAF) directly on KMS-12-BM multiple myeloma cells and on co-culture of stimulated human macrophages (CRL9853) and KMS-12-BM. Materials and Methods: Cell lines: KMS-12-BM: human multiple myeloma cell line was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultured in RPMI 1640 supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies, Paisley, UK). Cultures were passaged every 3-4 days. CRL9853: human spleen macrophage was purchased from ATCC (American Type Culture Collection, Teddington, UK) and routinely cultured in IMDM supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies). Cultures were passaged every 3-4 days. Prior to assay, CRL9853 cells were activated culturing them for 72h in the presence of OAGcMAF at a concentration 100ng/ml in complete medium. Stimuli: OA-GcMAF, commercially available, was prepared at Immuno Biotech Ltd. (Guernsey, Channel Island) with a proprietary procedure previously described (6). Cell viability Abstracts of the 9th International Conference of Anticancer Research, 6-10 October 2014, Porto Carras, Sithonia, Greece 6175 Figure 1. KMS-12-BM cell viability assay and Trypan Blue viable cell counts. (A) Results for (8 pM) demonstrate a slight decrease on cell proliferation which becomes significantly greater at (800 pM) indicating a direct effect on cell viability. (B) The decrease in viable cell number as the OA-GcMAF concentration increases corroborating the observed cell viability assay data. Results are expressed as mean value±S.E.M. for five replicates. (*p<0.01). assay: Cell viability was evaluated by the reduction of a tetrazolium salt (WST-8) as an index of cell dehydrogenases’ activity. KMS-12-BM cells were seeded into a 96-well plate at a density of 3x104 cells/well in their appropriate starvation medium (without FBS). After incubation for 24h the cell line was treated for 24h with the following different concentrations of OA-GcMAF ([8-80-800 pM). At the end of the treatment, the medium was replaced with 100μl of fresh starvation medium plus 10μl of WST-8. The 96-well plate was incubated for 3h at 37 C and the optical density (O.D.) was directly measured at A450nm by Multiscan FC photometer (ThermoScientific, Milano, Italy). Cell counting – Trypan blue assay: To corroborate the results obtained by cell viability assay, a viable cell count was performed. Briefly, KMS-12-BM cells were plated into a 6-well plate at a density of 2x105 cells/well in starvation medium. After 24h incubation, human multiple myeloma cells were treated with OA-GcMAF at the increasing concentrations (8-80-800 pM) for 24h. At the end of the treatment, a volume of cell suspension was collected and the viable cell number was counted by Trypan Blue staining. Video-time lapse photography: KMS-12-BM cells were seeded into a 24-well plate at a density of 1x106 cells/well with a 1ml volume. The cells were allowed to settle for a minimum of 2h prior to the addition of the OA-GcMAF-activated CRL9853 macrophages. The staging mat was set at a temperature of 37 C and allowed to equilibrate prior to placement of the 24-well plate. The OA-GcMAF-activated CRL9853 macrophages were added to a final concentration of 5x105 cells per well in 1ml. HEPES (Fisher Scientific, Loughborough, UK) was added to each well to provide a final concentration of 25mM to stabilize the culture pH. Once activated macrophages and the HEPES were added, the 24-well plate was observed microscopically and an image selected. An initial frame was taken and a timelapse film initiated. A frame was taken every 3 minutes until filming was stopped. Results: Cell viability assay: Cell viability (Figure 1), evaluated both by tetrazolium dye cell assay (A) and by Trypan blue staining (B), decreased when KMS-12-BM cells were treated with increasing concentrations of OAGcMAF. In particular, when cells were treated with OAGcMAF (800 pM) a significant reduction (p<0.01) in cell ANTICANCER RESEARCH 34: 5761-6258 (2014) 6176 Figure 2. Selected frame shots over time of KMS-12-BM cells in the presence of OA-GCMAF-stimulated CRL9853 human macrophages. (A) KMS-12-BM suspension culture typical growth pattern. (B) KMS-12-BM cells 24h post addition of OA-GcMAF activated human macrophages. CRL9853 cultures as large masses of suspension cells and the dense collection of cells can be observed sitting on the KMS-12-BM suspension cells. The OA-GcMAF-activated macrophages are extremely motile under observation and move around clearing the KMS-12-BM cells. During the phagocytosis smaller dense “clusters” of cells break off and scavenge the KMS-12-BM cells quite rapidly. (C) KMS-12-BM cells 48h post addition of OA-GcMAF-activated human macrophages. CRL9853 cells now have a few large cell mass “clusters” and many smaller “clusters” as can be observed (red arrows). The number of KMS-12-BM cells has been decreased and the clearance of cells can be easily observed. (D) KMS-12BM cells 72h post addition of OA-GcMAF-activated human macrophages CRL9853. OA-GcMAF-activated CRL9853 cells are still extremely active with the phagocytotic activity and are clearing the KMS-12-BM cells from the culture. This image shows the smaller clusters of cells as indicated by the red arrows. The clusters have become smaller and rapid moving seeking out and destroying the remaining KMS-12-BM cells. viability was observed in comparison to the untreated control cells. Discussion: It has been shown that Gc-MAF activates Raw 264.7 murine macrophages to phagocytose and destroy MCF-7 human breast carcinoma cells (1). An identical effect has been recorded for the first time with the stimulation of human macrophages CRL9853 on KMS-12-BM cell line . The OA-GcMAF-stimulated human macrophages seek out surround and phagocytose the myeloma cell lines destroying them. This demonstrates that the CRL9853 behave as postulated against KMS-12BM cell line. In addition the effect of increased cell death in the presence of OA-GcMAF alone as indicated by direct evaluation of viable cell counts and viability assay also provides supports to the previous data generated (1). This study provides some inferred evidence to support the in vivo clinical data that has recently been published (6) providing some insight into the method of potential tumour removal by stimulated macrophages. In conclusion OA-GcMAF has demonstrated two major effects: a direct decrease of KMS-12BM cell viability and an efficient activation of human macrophages, which become able to phagocytose and destroy the myeloma cells. The studies will be expanded further to encompass additional cancer cell lines. 1 Pacini S et al: Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells. Anticancer Res, 2012; 32(1):45-52. 2 Thyer L et al: A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages. Nutrients 2013; 5(7):2577-2589. 3 Gregory KJ et al: Vitamin D binding protein-macrophage activating factor directly inhibits proliferation, migration, and uPAR expression of prostate cancer cells. PLoS One 2010; 5(10):e13428. 4 Thyer L et al: Therapeutic effects of highly purified deglycosylated GcMAF in the immunotherapy of patients with chronic diseases. Am J Immunol 2013; 9(3):78-84. 5 Yamamoto Net al: Immunotherapy for prostate cancer with gc protein derived macrophage-activating factor OAGcMAF. Trans Oncol 2008; 1:65-72 6 Ruggerio M et al: Oleic acid, deglycosylated vitamin Dbinding protein, nitric oxide: a molecular triad made lethal to cancer. Anticancer Res 2014; 34: 3569-3578. 605 SENESCENCE LINKED INFLAMMATION, REPLICATION STRESS, FRAGILE SITES AND DNA METHYLATION CHANGES IN SPORADIC BREAST AND PROSTATE CANCER.

Background: Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). Materials and Methods: The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrst microscopy, immunohistochemestry and western blotting, and chorioallantoic membrane (CAM) assay. Results: DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. Conclusion: These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential.

This Article-in-Press has been permanently withdrawn. The editorial policy of Medical Hypotheses makes it clear that the journal considers “radical, speculative, and non-mainstream scientific ideas”, and articles will only be acceptable if they are “coherent and clearly expressed.” However, we received serious expressions of concern about the quality of this article. Given these important signals of concern, we commissioned an external expert panel to investigate the circumstances in which this article came to be published online. The panel recommended that the article should be externally peer-reviewed. Following a peer-review process managed by The Lancet editorial team, all five external reviewers recommended rejection, as a result of which the expert panel recommended permanent withdrawal. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Initially identified as components of the signaling pathways triggered by receptor tyrosine kinases and leading to Ras activation, Shc proteins have been more recently implicated in the regulation of signals controlling not only cell proliferation, but also cell survival and apoptosis. Here we briefly review the current understanding of Shc proteins as promoters of apoptosis. Specifically, we focus on the 66 kDa isoform of ShcA, whose paramount importance in the regulation of oxidative stress responses leading to cell apoptosis and ageing has been by now firmly established.

Human endogenous plasma heparin associates with proteins that mask its anticoagulant activity. This association persists after exhaustive proteolysis of plasma, and resulting peptide/heparin complexes have no anticoagulant activity. Looking for functions other than inhibition of coagulation, we considered that commercial preparations of heparin from bovine or porcine sources show alternative effects on angiogenesis, either stimulating or inhibiting the process. However, the effects of endogenous human heparin on angiogenesis are unknown. In this study, the fraction of plasma containing endogenous heparin was prepared by means of exhaustive proteolysis, either in the presence or in the absence of 35S-labeled heparin. Plasma from healthy donors was digested and the supernatant was precipitated with 66% ethanol, dialyzed, and submitted to basic and acidic ion-exchange chromatography. 35S-Labeled heparin as well as endogenous heparin bound plasmatic peptides, forming acidic, basic, and neutral complexes. Binding of peptides, eluting from both resins, impaired migration of heparin on cellulose acetate electrophoresis. Endogenous neutral complexes (i.e., those formed by human endogenous plasma heparin and peptides) were tested for angiogenic activity in chick embryo chorioallantoic membrane assay. Bovine heparin induced a moderate angiogenic response. Neutral complexes of human endogenous plasma heparin and basic plasma peptides induced a very strong angiogenic response. Treatment of neutral complexes with nitrous acid, which degrades heparin, abolished the angiogenic effect, thus demonstrating that it was due to the presence of heparin. These results demonstrate that proteolysis of human plasma generates angiogenic peptide/heparin complexes.

Interstitial cells of Cajal (ICC) are distributed throughout the gastrointestinal muscle coat with a region-specific location, and are considered to be pace-maker and/or mediators of neurotransmission. Little is known about their shape, size, distribution and relationships with excitatory and inhibitory nerves in human stomach. With this aim, we labeled the ICC, using c-Kit immunohistochemistry, followed by a quantitative analysis to evaluate the distribution and area occupied by these cells in the circular and longitudinal muscle layers and at the myenteric plexus level in the human fundus, corpus and antrum. Furthermore, by NADPH-d histochemistry and substance P (SP) immunohistochemistry, we labeled and quantified nitric oxide (NO)-producing and SP-containing nerves and evidenced their relationships with the ICC in these three gastric regions. In the fundus, the ICC appeared as bipolar cells and in the corpus and antrum they mainly appeared as multipolar cells, with highly ramified processes. The networks formed by ICC differed in the three gastric regions. The ICC number was significantly higher and cell area smaller in the fundus compared to the corpus and antrum. The area occupied by the ICC was significantly higher at the myenteric plexus level compared with circular and longitudinal muscle layers. Everywhere, NADPH-d-positive nerves were more numerous than SP-positive ones. Both kinds of fibers were closely apposed to the ICC in the corpus and antrum. In conclusion, in the human stomach, the ICC have region-specific shape, size and distribution and in the corpus and antrum have close contact with both inhibitory and excitatory nerves. Presumably, as suggested for laboratory mammals, these differences are in relationship with the motor activities peculiar to each gastric area.

Of the three She isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.

Background:It is well documented that exercise reduces the risk of thromboembolic disease, possibly by increasing the plasma concentration of anticoagulant-antithrombotic compounds.Objectives:As plasma glycosaminoglycans (GAGs) play a role in the anticoagulant-antithrombotic potential of plasma, to examine the concentration and profile of these compounds in well trained, long distance runners and sedentary subjects.Methods:Plasma GAGs were measured in 10 male, long distance runners and 10 sedentary counterparts before and after ergometric tests. GAGs were extracted, purified, and identified by electrophoretic and enzymatic methods, and measured as hexosamine.Results:Plasma GAGs found in sedentary subjects were slow migrating heparan sulphates I and II, keratan sulphate I, and chondroitin 4–6-sulphate. Those found in trained athletes were slow migrating heparan sulphate I, chondroitin 4–6-sulphate (or keratan sulphate I), and fast migrating heparan sulphate. Total plasma concentrations of GAGs were higher in athletes than in sedentary subjects at rest. In sedentary subjects, plasma GAGs did not change after cycle ergometric exercise at 80% of their anaerobic threshold. However, the appearance of a novel band of heparan sulphate migrating faster than fast migrating heparan sulphate was observed in athletes after exercise.Conclusions:Exercise changes the amount and profile of plasma GAGs; these changes may play a role in protecting subjects who practise aerobic sports against developing cardiovascular disease.

Psoriasis is a chronic inflammatory skin disorder associated with both genetic, immunologic, and environmental factors. Changes in proliferation and metabolism of keratinocytes, together with neo-angiogenesis and neutrophilic infiltration into the dermis lead to a broad spectrum of clinical manifestations, so that therapeutic strategy has to be adapted in each patient [ [1] Camp RDR. (1992) Psoriasis. In: Champion RH, Burton JL., Ebling FJG, editors. Textbook of Dermatology Rook/Wilkinson/Ebling. Oxford: Blackwell Scientific Publications. Google Scholar ].