Hexosamine Biosynthetic Pathway and Glycosylation Regulate Cell Migration in Melanoma Cells.

The Hexosamine Biosynthetic Pathway (HBP) is a branch of glycolysis responsible for the production of a key substrate for protein glycosylation, UDP-GlcNAc. Cancer cells present altered glucose metabolism and aberrant glycosylation, pointing to alterations on HBP. Recently it was demonstrated that HBP influences many aspects of tumor biology, including the development of metastasis. In this work we characterize HBP in melanoma cells and analyze its importance to cellular processes related to the metastatic phenotype. We demonstrate that an increase in HBP flux, as well as increased O-GlcNAcylation, leads to decreased cell motility and migration in melanoma cells. In addition, inhibition of N- and O-glycosylation glycosylation reduces cell migration. High HBP flux and inhibition of N-glycosylation decrease the activity of metalloproteases 2 and 9. Our data demonstrates that modulation of HBP and different types of glycosylation impact cell migration.

Hypertonic stress induces VEGF production in human colon cancer cell line Caco-2: inhibitory role of autocrine PGE2.

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E2 are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE2 generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE2 generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE2 production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE2 on VEGF production, Caco-2 cells were treated with cPLA2 (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE2 generation. The Caco-2 cells were also treated with a non selective PGE2 receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE2 or selective EP2 receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE2 appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl2 was decreased by inhibition of concomitant PGE2 generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE2 plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP2 activation.

Hypertonic environment elicits cyclooxygenase-2-driven prostaglandin E2 generation by colon cancer cells: role of cytosolic phospholipase A2-alpha and kinase signaling pathways.

Cyclooxygenase (COX)-2-derived prostaglandin (PG)E(2) controls many aspects of colon cancer development, modulating from apoptosis resistance and cell proliferation to angiogenesis, invasion, and metastasis. Here, we investigated the role of different phospholipases (PL)A(2) in supplying arachidonic acid (AA) for COX-2-dependent PGE(2) generation and signaling pathways involved in activation of colon cancer cells by a physiologically relevant stimulus. To emulate the hypertonic environment found physiologically in colon, the human colon cancer cell line Caco-2 was maintained in hypertonic complete DMEM medium. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked AA release, COX-2 induction and PGE(2) generation. Selective secretory (s)PLA(2) and calcium-independent (i)PLA(2) inhibitors did not modify PGE(2) generation, while either COX-2 or cytosolic (c)PLA(2) inhibitors completely inhibited PGE(2) generation. cPLA(2)-alpha was responsible for AA supply for PGE(2) generation, but had no role in COX-2 induction. Mitogen-activated protein (MAP) kinases, ERK 1/2, p38, and JNK, participated in the signaling events that lead to PGE(2) generation by modulating AA release, but only ERK 1/2 was involved in COX-2 upregulation. Our results indicate that hypertonic stress activates PGE(2) generation by Caco-2 cells through a mechanism dependent on MAP kinase-regulated AA mobilization, increased cPLA(2)-alpha activity, and COX-2 induction.

Leukotriene B4 mediates gammadelta T lymphocyte migration in response to diverse stimuli.

Herein, we investigated the involvement of the 5-LO-derived lipid mediator LTB(4) in gammadelta T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB(4) triggered gammadelta T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli endotoxin (LPS) triggered increased levels of LTB(4) in pleural cavities. The in vivo inhibition of LTB(4) biosynthesis by the 5-LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS-induced gammadelta T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit gammadelta T cells into the inflammatory site after i.pl. LPS. Antagonists of the high-affinity LTB(4) receptor BLT1, CP105,696, and LY292476 also attenuated LPS-induced gammadelta T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS-simulated mice. LTB(4)/BLT1 also accounted for gammadelta T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS-recruited gammadelta T cells. Isolated gammadelta T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB(4) in vitro, confirming that gammadelta T lymphocytes can respond directly to LTB(4). In addition to its direct effect on gammadelta T cells, LTB(4) triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that gammadelta T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB(4)/BLT1.

[Cervical lymphoadenopathy due to Pseudomonas aeruginosa following mesotherapy]. / Linfoadenopatia cervicale da Pseudomonas aeruginosa dopo trattamento con mesoterapia.

Mesotherapy is a treatment method devised for controlling several diseases by means of subcutaneous microinjections given at or around the affected areas at short time intervals. It is used to treat a variety of medical conditions, amongst which all orthopaedic diseases and rheumatic pain. Mesotherapy is especially indicated for neck pain. The mechanism of action is twofold: a pharmacological effect due to the drug administered, and a reflexogenic effect, the skin containing many nerve endings that are sensitive to the mechanical action of the needle. Although this therapy is safe, like any other medical intervention it cannot be considered free of complications that may occur, such as allergies, haematomas, bruising, wheals, granulomas and telangiectasias. Infective complications are also possible, due to pathogenic bacteria that are inoculated through contamination of products, of the materials used for the procedure or even from germs on the skin. We present the case of a patient who had cervical lymphadenopathy due to Pseudomonas aeruginosa after mesotherapy treatment for neck pain.

[Post-surgical neuropathic pain]. / Dolore neuropatico post-chirurgico.

The traumatic lesions during surgical interventions often turn into a persistent pain. Pain persists in the location of surgical intervention for a long time, beyond the usual course of natural healing of an acute pain and it is different from that suffered preoperatively. It is usually a chronic pain and it is associated to lesions of the central or peripheral nervous system. Pain is usually described as burning or tingling, or electric shock-like; it can be continuous or parossistic, often associated to paraesthesia, iperalgesia and allodinya. If circumstances preclude the surgical revision, the treatment of post-surgical neuropathic pain is based on drugs, according to the guidelines. The drugs of choice are the tricyclic antidepressants, the serotonin and adrenaline re-uptake selective inhibitors (SSRI), local antiepileptics of new generation (gabapentin, pregabalin) and topical anaesthetics. Drugs of second line are: opioid analgesics, tramadol; drugs of third line are: mexiletine, antagonist of NMDA receptor and capsaicine. The post-surgical neuropathic pain is often resistant to the pharmacologic treatment; for this reason the spinal cord neuromodulation can be applied only after careful selection of the patients according to the international guidelines. The incidence of post-surgical neuropathic pain in the Pain Units is approximately 20% of the patients admitted to hospital. Therefore it is necessary a greater attention for the post-surgical analgesia, adopting appropriate surgical techniques in order to avoid the onset of the post-surgical neuropathic pain.

Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells.

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE(2), but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3 K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A(2)-alpha, while facilitated AA mobilization and synthesis of PGE(2) within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA (2)-alpha-driven enhanced AA mobilization and PGE(2)production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.

[Percutaneous electrical nerve stimulation of peripheral nerve for the intractable occipital neuralgia]. / Neurostimolazione elettrica subcutanea del nervo periferico utilizzata nel controllo della nevralgia occipitale refrattaria.

UNLABELLED: Occipital neuralgia is characterized by pain paroxysm occurring within distribution of the greater or lesser occipital nerves. The pain may radiates from the rear head toward the ipso-lateral frontal or retro-orbital regions of head. Though known causes include head injuries, direct occipital nerve trauma, neuroma formation or upper cervical root compression, most people have no demonstrable lesion. METHOD AND MATERIALS: A sample of 8 patients (5 females, 3 males) aging 63,5 years on the average with occipital neuralgia has been recruited. The occipital neuralgic pain had presented since 4, 6 years and they had been treated by pharmacological therapy without benefit. Some result has been obtained by blocking of the grand occipital nerve so that the patients seemed to be suitable for subcutaneous peripheral neurostimulation. The pain was evaluated by VAS and SVR scales before treatment (TO) and after three and twelve months (T1, T2). RESULTS: During the follow up period 7 patients have been monitored for a whole year while one patient was followed only for 3 months in that some complications have presented. In the other 7 patients pain paroxysms have interrupted and trigger point disappeared with a VAS and SVR reduction of about 71% and 60%, respectively. CONCLUSIONS: Our experience demonstrates a sound efficacy of such a technique for patients having occipital neuralgia resistant to pharmacological therapies even if action mechanisms have not yet clearly explained. Some hypothesis exist and we think it might negatively affect the neurogenic inflammation that surely acts in pain maintaining.

[Cannabinoids in the control of pain]. / I cannabinoidi nella terapia del dolore.

Hemp (Cannabis sativa L.) has been used since remotes ages as a herbal remedy. Only recently the medical community highlighted the pharmacological scientific bases of its effects. The most important active principle, Delta-9-tetrahydrocannabinol, was identified in the second half of the last century, and subsequently two receptors were identified and cloned: CB1 that is primarily present in the central nervous system, and CB2 that is present on the cells of the immune system. Endogenous ligands, called endocannabinoids, were characterized. The anandamide was the first one to be discovered. The effectiveness of the cannabinoids in the treatment of nausea and vomit due to anti-neoplastic chemotherapy and in the wasting-syndrome during AIDS is recognized. Moreover, the cannabinoids are analgesic, and their activity is comparable to the weak opioids. Furthermore, parallels exist between opioid and cannabinoid receptors, and evidence is accumulating that the two systems sometimes may operate synergistically. The interest of the pharmaceutical companies led to the production of various drugs, whether synthetic or natural derived. The good ratio between the polyunsatured fatty acids omega-3 and omega-6 of the oil of Cannabis seeds led to reduction of the phlogosis and an improvement of the pain symptoms in patients with chronic musculo-skeletal inflammation.

Continuous intrathecal morphine infusion in patients with vertebral fractures due to osteoporosis.

OBJECTIVES: Vertebral fractures are the most common consequences of severe osteoporosis. The chronic pain from collapse of osteoporotic vertebrae affects quality of life (QOL) and autonomy of patients. The management of pain with oral or transdermal opiates can cause severe side effects. Continuous intrathecal administration of morphine via an implantable pump might represent an alternative therapy to conventional oral or transdermal administration of opioids and has some advantages and disadvantages for pain relief and improvement in QOL when compared with conventional opioid delivery. It is our objective to report our experience using intrathecal delivery of analgesics in a population of patients with refractory pain due to vertebral fractures. MATERIALS AND METHODS: In 24 patients, refractory to conventional delivery of opioids, we used intrathecal analgesic therapy. To test for efficacy and improvement in QOL, we administered the visual analog scale for pain and the Questionnaire of the European Foundation of Osteoporosis (QUALEFFO). Before patients were selected for pump implantation, an intraspinal drug delivery trial was performed to monitor side effects and responses to intrathecal therapy. RESULTS: Significant pain relief was obtained in all implanted patients. Using the QUALEFFO, we observed significant improvement of all variables such as quality of daily life, domestic work, ambulation, and perception of health status, before and after 1 year after pump implantation. With intrathecal morphine infusion, none of the 24 patients required additional systemic analgesic medication. The mean morphine dose during the spinal trial was 11.28 mg/d, 7.92 mg/d at pump implantation, and 16.32 mg/d at 1-year follow-up. CONCLUSIONS: Our results show that intrathecal administration of morphine efficiently relieves the symptoms of pain and improves QOL. Continuous intrathecal administration of morphine appears to be an alternative therapy to conventional analgesic drug delivery and has advantages in those patients who have severe side effects with systemic administration of analgesics.

[Management of pain from osteoporotic vertebral fractures with continuous intrathecal administration of morphine]. / Trattamento del dolore cronico benigno da crollo vertebrale osteoporotico con infusione continua intratecale di morfina.

UNLABELLED: Vertebral fractures are the most common consequences of severe osteoporosis. The chronic pain from collapse of osteoporotic vertebrae affects quality of life and autonomy of patients. The management of pain with oral or transdermal opiates can cause severe side-effects. Continuous intrathecal administration of morphine with an implantable pump represents a good alternative therapy and has advantages in pain relief and quality of life. MATERIALS AND METHODS: We report our experience in the treatment of refractory chronic pain due to vertebral fractures using pumps for intrathecal infusion of morphine in 24 patients. We administered the Visual Analogue Scale (VAS) and the Quality of Life Questionnaire of the European Foundation of Osteoporosis (QUALEFFO). RESULTS: A significant pain relief was obtained in all implanted patients. Using the QUALEFFO we observed significant improvements of all variables such as QDL (quality of life), DW (domestic work), ambulation and PHS (perception of health status), before and after 1 year from pump implantation. With intrathecal morphine infusion none of the patients required additional systemic analgesics. CONCLUSIONS: Our results show that intrathecal administration of morphine efficiently relieves the symptoms of pain and improve the quality of life. Continuous intrathecal administration of morphine represents a good alternative therapy and has advantages in those patients who suffer from severe side-effects with systemic administration.

The management of pain from collapse of osteoporotic vertebrae with continuous intrathecal morphine infusion.

Objectives. Vertebral fractures are the most common consequences of severe osteoporosis. The chronic pain from collapse of osteoporotic vertebrae affects quality of life (QoL) and autonomy of patients. The management of pain with oral or transdermal opiates can cause severe side-effects. Continuous intrathecal administration of morphine through an implantable pump might represent an alternative therapy to conventional oral or transdermal administration of opioids and has some advantages and disadvantages for pain relief and improvement in QoL when compared to conventional opioid delivery. It is our objective to report our experience using intrathecal delivery of analgesics in a population of patients with refractory pain due to vertebral fractures. Materials and Methods. In 24 patients, refractory to conventional delivery of opioids, we used intrathecal analgesic therapy. To test for efficacy and improvement in QoL, we administered the visual analog scale (VAS) for pain and the Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO). Before patients were selected for pump implantation, an intraspinal drug delivery trial was performed to monitor side-effects and responses to intrathecal therapy. Results. Significant pain relief was obtained in all implanted patients. Using the QUALEFFO, we observed significant improvement of all variables such as QDL (quality of daily life), DW (domestic work), ambulation, and PHS (perception of health status), before and after one year after pump implantation. With intrathecal morphine infusion, none of the 24 patients required additional systemic analgesic medication. The mean morphine dose during the spinal trial was 11.28 mg/day, 7.92 mg/day at pump implantation, and 16.32 mg/day at one-year follow-up. Conclusions. Our results show that intrathecal administration of morphine efficiently relieves the symptoms of pain and improves QoL. Continuous intrathecal administration of morphine appears to be an alternative therapy to conventional analgesic drug delivery and has advantages in those patients who have severe side-effects with systemic administration of analgesics.

The median body of Giardia lamblia: an ultrastructural study.

Giardia lamblia is an intestinal parasite of several mammals. The most striking feature of Giardia is the presence of a complex and unique cytoskeleton, and among its components the median body (MB) is the least defined microtubular structure. In the present study, we used a technique that allowed the removal of the plasma membrane and observation of cytoskeletal structures by both routine scanning electron microscopy (SEM) and field emission high resolution SEM. This technique permitted new observations such as details and insights of the median bodies, not previously described or controversial in the literature. Light microscopy after Panotic staining, immunofluorescence microscopy using several antibodies, and thin sections were also used to better characterized the Giardia MB. The new observations concerning the median bodies were : (1) they are not one or two structures, but varied in number, shape and position ; (2) they were found in mitotic and interphasic trophozoites, in disagreement with previous works ; (3) they were present in about 80 % of the cells, and not in 50 % of the cells, as previously described ; (4) they could be connected either to the plasma membrane, to the adhesive disc, and caudal flagella, and thus they are not completely free in the cells, as published before ; (5) they can protrude the cell surface ; (6) their microtubules react with several anti-tubulin and -beta giardin antibodies. These observations add new data on the scarce literature and to this largely understudied cell structure.

Visualization of the funis of Giardia lamblia by high-resolution field emission scanning electron microscopy--new insights.

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a cytoskeleton made of several microtubular structures-an adhesive disc, four pairs of flagella, median body, and funis. This protozoan displays different types of movements, including a lateral and dorso-ventral dislocation of its posterior region, which has not been completely elucidated. In the present study, high-resolution field emission scanning electron microscopy was used to analyze the funis structure of G. lamblia trophozoites. It was shown that the funis is made of short arrays of microtubules emanating from the axonemes of the caudal flagella, which are anchored to dense rods that run parallel to the posterior-lateral flagella. After emergence of the posterior-lateral flagella, funis microtubules are anchored to the epiplasm, a fibrous layer that underlies the portion of membrane that presents tail contractility. Based on these observations a model for the tail flexion of G. lamblia is proposed.