Unexpected Absence of Skeletal Responses to Dietary Magnesium Depletion: Basis for Future Perspectives?

It's known that a magnesium (Mg)-deficient diet is associated with an increased risk of osteoporosis. The aim of this work is to investigate, by a histological approach, the effects of a Mg-deprived diet on the bone of 8-weeks-old C57BL/6J male mice. Treated and control mice were supplied with a Mg-deprived or normal diet for 8 weeks, respectively. Body weight, serum Mg concentration, expression of kidney magnesiotropic genes, and histomorphometry on L5 vertebrae, femurs, and tibiae were evaluated. Body weight gain and serum Mg concentration were significantly reduced, while a trend toward increase was found in gene expression in mice receiving the Mg-deficient diet, suggesting the onset of an adaptive response to Mg depletion. Histomorphometric parameters on the amount of trabecular and cortical bone, number of osteoclasts, and thickness of the growth plate in femoral distal and tibial proximal metaphyses did not differ between groups; these findings partially differ from most data present in the literature showing that animals fed a Mg-deprived diet develop bone loss and may be only in part explained by differences among the experimental protocols. However, the unexpected findings we recorded on bones could be attributed to genetic differences that may have developed after multiple generations of inbreeding.

Phosphoinositide-specific phospholipase C isoforms are conveyed by osteosarcoma-derived extracellular vesicles.

Cancer cells are able to release high amounts of extracellular vesicles, thereby conditioning the normal cells in the surrounding tissue and/or in distant target organs. In the context of bone cancers, previous studies suggested that osteosarcoma cancer cells produce transforming extracellular vesicles able to induce a tumour-like phenotype in normal recipient cells. Indeed, phosphoinositide-specific phospholipase C (PI-PLC) enzymes are differentially expressed in osteosarcoma cell lines with increasing aggressiveness, thus providing helpful insights to better define their role and functions in this bone tumour. By confocal microscopy analysis, we demonstrated that osteosarcoma-derived extracellular vesicles convey all the assessed PI-PLC isoforms, and that they localize into cell membrane bubble-like structures, resembling extracellular vesicles about to be released, as conveyed and/or membrane protein. Cytofluorimetric analysis confirmed the presence of PI-PLC isoforms in the extracellular vesicles collected from conditioned media of osteosarcoma cells. These findings suggest the feasibility to use circulating extracellular vesicles as biomarkers of osteosarcoma progression and/or the monitoring of this distressing disease.

Phosphoinositide-specific phospholipase C in normal human liver and in alcohol abuse.

The phosphoinositide (PI) signal transduction pathway participates in liver metabolism. Abnormal activity or expression of PI-specific phospholipase C (PLC) enzymes has been described in different liver diseases. We resume the role of the PI metabolism in liver and PLC abnormalities in different liver diseases. Moreover, we present the results of PLC analyses in a normal human liver and an alcohol-damaged liver. PLC enzymes and the expression of the corresponding genes in liver biopsies from individuals deceased for complications of the alcoholic liver disease (ALD) at different stages compared with normal controls (deceased individuals with histologically normal livers without alcohol addiction anamnesis) were analyzed by using immunohistochemistry and molecular biology techniques. The expression panel of PLCs was described in normal and alcohol abuse liver. Our observations suggest that the regulation of PLC expression might be due to posttranscriptional events and that alcohol affects the epigenetic control of PLC expression belonging to PI signaling. We also describe the alternate expression of PLCB1 and PLCH1 genes in liver. Our results corroborate literature data suggesting that PLC enzymes are differently expressed in normal versus pathological liver, playing a role in the histopathogenesis of liver tissue damage. The expression and/or localization of selected PLC isoforms is especially affected in alcohol-related liver tissue histopathology. Our present observations confirm that the modulation of protein synthesis plays a role in the regulation of PLC enzymes. We also suggest that this modulation might act at the transcription level. Further studies are required to investigate related epigenetic mechanisms.

The Phosphoinositide Signal Transduction Pathway in the Pathogenesis of Alzheimer's Disease.

BACKGROUND: During aging and in age-associated disorders, such as Alzheimer's Disease (AD), learning abilities decline. Probably, disturbances in signal transduction in brain cells underlie the cognitive decline. The phosphorylation/dephosphorylation imbalance occurring in degenerating neurons was recently related to abnormal activity of one or more signal transduction pathways. AD is known to be associated with altered neuronal Ca2+ homeostasis, as Ca2+ accumulates in affected neurons leading to functional impairment. It is becoming more and more evident the involvement of signal transduction pathways acting upon Ca2+ metabolism and phosphorylation regulation of proteins. A growing interest raised around the role of signal transduction systems in a number of human diseases including neurodegenerative diseases, with special regard to the systems related to the phosphoinositide (PI) pathway and AD. The PI signal transduction pathway plays a crucial role, being involved in a variety of cell functions, such as hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, cytoskeleton regulation, cell cycle control, apoptosis, cell and tissue polarity, and contributes to regulate the Ca2+ levels in the nervous tissue. CONCLUSION: A number of observations indicated that PI-specific phospholipase C (PLC) enzymes might be involved in the alteration of neurotransmission. To understand the role and the timing of action of the signalling pathways recruited during the brain morphology changes during the AD progression might help to elucidate the aetiopathogenesis of the disease, paving the way to prognosis refinement and/or novel molecular therapeutic strategies.

LPS, Oleuropein and Blueberry extracts affect the survival, morphology and Phosphoinositide signalling in stimulated human endothelial cells.

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes' expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.

Wounds Difficult to Heal: An Effective Treatment Strategy.

OBJECTIVE: Treatment of wounds difficult to heal concerns 50% of the elderly population in Italy and is therefore a relevant social burden. The present study shows how the treatment with autologous leuco-platelets reduces the healing time of wounds improving the functional recovery. PATIENTS AND METHODS: Patients (n=100) with ulcers of the legs were divided in two groups: 1) 50 patients treated with conventional therapies; 2) 50 patients treated with autologous leuco-platelet concentrate (LPC) and hyaluronic acid (HIAFF, Hyalofill-F® ) as a scaffold. RESULTS: After 2 months, a 49% reduction in wound area was observed in the second group and in about 65% wound reduction was achieved in 15 days (4 LPC dressings). In contrast, patients treated by conventional therapies, showed a longer healing time and a greater percentage of failures. Morphometric analysis of biopsy samples obtained from the edge as well as from the bottom of the lesions obtained from the LPC group, detected an abundant presence of neoformed capillaries, characterized by a cubic, "reactive endothelium", close to the site of LPC infiltration. CONCLUSION: These results suggest that healing was promoted not only by limiting bacterial infections but also by the release of chemotactic and proangiogenic factors from leukocytes and platelets, improving the neoformation of capillaries.

Different expression and subcellular localization of Phosphoinositide-specific Phospholipase C enzymes in differently polarized macrophages.

Macrophages' phenotypic and functional diversity depends on differentiating programs related to local environmental factors. Recent interest was deserved to the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. The expression panel of PLCs and the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), as well as in M1 and M2 macrophages to list the expressed isoforms and their subcellular localization. Furthermore, we investigated whether inflammatory stimulation modified the basal panel of PLCs' expression and subcellular localization. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes' mRNA expression and intracellular localization of PLC enzymes. The panel of PLC genes' expression and PLC proteins' presence slightly changes after inflammatory stimulation. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization.

U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving Phosphoinositide-specific Phospholipases C.

The definition of the number and nature of the signal transduction pathways involved in the pathogenesis and the identification of the molecules promoting metastasis spread might improve the knowledge of the natural history of osteosarcoma, also allowing refine the prognosis and opening the way to novel therapeutic strategies. Phosphatydil inositol (4,5) bisphosphate (PIP2), belonging to the Phosphoinositide (PI) signal transduction pathway, was related to the regulation of ezrin, an ezrin-radixin-moesin protein involved in metastatic osteosarcoma spread. The levels of PIP2 are regulated by means of the PI-specific Phospholipase C (PLC) enzymes. Recent literature data suggested that in osteosarcoma the panel of expression of PLC isoforms varies in a complex and unclear manner and is related to ezrin, probably networking with Ras GTPases, such as RhoA and Rac1. We analyzed the expression and the subcellular localization of PLC enzymes in cultured human osteosarcoma MG-63 cells, commonly used as an experimental model for human osteoblasts, using U-73122 PLC inhibitor, U-73343 inactive analogue, and by silencing ezrin. The treatment with U-73122 significantly reduces the number of MG-63 viable cells and contemporarily modifies the expression and the subcellular localization of selected PLC isoforms. U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving PI-specific Phospholipases C.

Relapsing polychondritis: A clinical update.

Relapsing polychondritis (RP) is a rare connective tissue disease in which recurrent bouts of inflammation, involve the cartilage of the ears, nose, larynx, tracheobronchial tree and cardiovascular system. RP is generally observed in the fourth and fifth decades of life and occurs with equal frequency in both sexes. The cause of RP is still unknown. It is considered an immune-mediated disease, as there is an overlap between well documented RP with other rheumatic and autoimmune diseases. There is a significant association of RP with the antigen HLA-DR4. RP includes loss of basophilic staining of cartilage matrix perichondral accompanied by inflammation of the cartilage. Cells are present perivascular mononuclear and polymorphonuclear cells infiltrated. The chondrocytes become vacuolated and necrotic and are replaced by fibrous tissue. Common symptoms are often absent in the early stages of the disease in almost half the cases, resulting in delay in diagnosis. The development of chondrite allows the diagnosis of RP in patients initially evaluated for joint abnormalities, ocular, cutaneous, or audio-vestibular. Diagnostic criteria for RP are based on characteristic clinical manifestations. According to Damiani and Levine, the diagnosis can be considered final when one or more of the clinical features are present in conjunction with biopsy confirmation. The course of symptoms for patients with relapsing polychondritis is often unpredictable. Patients with mild signs of acute inflammation are usually treated with non-steroidal anti-inflammatory drugs and small doses of prednisone. Patients with severe manifestations, such as airway compromise may require high doses of prednisone or even intravenous pulse methyl-prednisone.

Impairment and reorganization of the phosphoinositide-specific phospholipase C enzymes in suicide brains.

A number of studies suggested that suicide may be associated with specific neurobiological abnormalities. Neurobiology studies focused upon abnormalities of signalling mechanisms with special regard to the serotonin system and the related Phosphoinositide (PI) signalling system. Previous data suggested the involvement of the PI-specific phospholipase C (PLC) family in neuropsychiatric disorders. By using PCR and morphological microscopy observation we examined the whole panel of expression of PLC isoforms in the brains of 28 individuals who committed suicide and in normal controls in order to evaluate the involvement of specific PLC isoforms. The overall PLC expression was reduced and a complex reorganization of the isoforms was observed. The knowledge of the complex network of neurobiological molecules and interconnected signal transduction pathways in the brain of suicide victims might be helpful to understand the natural history and the pathogenesis of the suicidal behavior. That might lead to obtain prognostic suggestions in order to prevent suicide and to new therapeutic agents targeting specific sites in this signalling cascade.

Silencing of phosphoinositide-specific phospholipase C ε remodulates the expression of the phosphoinositide signal transduction pathway in human osteosarcoma cell lines.

BACKGROUND: Ezrin, a member of the ezrin-radixin-moesin family, is involved in the metastatic spread of osteosarcoma. Ezrin binds phosphatydil inositol-4,5-bisphosphate (PIP2), a crucial molecule of the phosphoinositide signal transduction pathway. PIP2 levels are regulated by phosphoinositide-specific phospholipase C (PI-PLC) enzymes. PI-PLCε isoform, a well-characterized direct effector of rat sarcoma (RAS), is at a unique convergence point for the broad range of signaling pathways that promote RAS GTPase-mediated signalling. MATERIALS AND METHODS: By using molecular biology methods and microscopic analyses, we analyzed the expression of ezrin and PLC genes after silencing of PLCE (OMIM *608414) in 143B and Hs888 cell lines. RESULTS: The growth rate of the cells was slowed, and the expression of ezrin, PLCB1, PLCG2 and PLCD4 was significantly modified. Ezrin displacement from the plasma membrane was observed. CONCLUSION: The present results corroborate the hypothesis that ezrin and the PI signal transduction system are involved in a common network.

Degradation of silicone rubber causes Provox 2 voice prosthesis malfunctioning.

OBJECTIVE/HYPOTHESIS: Provox 2 voice prosthesis requires periodic replacement due to biofilm proliferation which causes malfunctioning of the valve. The aim of this study was to show that Provox 2 voice prosthesis malfunctioning is due not only to valve obstruction caused by biofilm but also to the silicone variations. DESIGN METHODS: Prospective study on the malfunction of Provox2 voice prostheses. METHODS: Through photographic and electron microscopic assessment, the authors studied nine Provox 2 voice prostheses, which were removed due to malfunctioning. RESULTS: Findings revealed that the silicone undergoes a degenerative process, thus causing the surface to become rough, deformed, swollen, and translucent. Furthermore, electron microscopy confirmed the presence of immune system cells and biofilm on the prosthesis surface and their role in creating a structural nonhomogenous structure in the silicone, which is deformed due to the presence of "crests" caused by material degeneration. CONCLUSION: The degenerative process of the silicone seems to be related to the oxygen present in the trachea and esophagus and to the production of oxygen-free radicals on the biofilm's part and the immune system.

Fibroblast growth factor acts upon the transcription of phospholipase C genes in human umbilical vein endothelial cells.

Besides the control of calcium levels, the phosphoinositide-specific phospholipases C (PI-PLCs), the main players in the phosphoinositide signalling pathway, contribute to a number of cell activities. The expression of PI-PLCs is strictly tissue specific and evidence suggests that it varies under different conditions, such as tumour progression or cell activation. In previous studies, we obtained a complete panel of expression of PI-PLC isoforms in human umbilical vein endothelial cells (HUVEC), a widely used experimental model for endothelial cells (EC), and demonstrated that the expression of the PLC genes varies under inflammatory stimulation. The fibroblast growth factor (FGF) activates the PI-PLC γ1 isoform. In the present study, PI-PLC expression in FGF-treated HUVEC was performed using RT-PCR, observed 24 h after stimulation. The expression of selected genes after stimulation was perturbed, suggesting that FGF affects gene transcription in PI signalling as a possible mechanism of regulation of its activity upon the AkT-PLC pathway. The most efficient effects of FGF were recorded in the 3-6-h interval. To understand the complex events progressing in EC might provide useful insights for potential therapeutic strategies. The opportunity to manipulate the EC might offer a powerful tool of considerable practical and clinical importance.

Is Montgomery tracheal Safe-T-Tube clinical failure induced by biofilm?

OBJECTIVES: Montgomery Safe-T-Tube deterioration and early biofilm colonization may explain the discomfort claimed by many patients and clinical failures. The aim of the study was to analyze the deterioration of Montgomery Safe-T-Tube morphological and mechanical properties in vivo in 16 patients by using microbiological methods, optical and electron microscopy, and engineering tests. STUDY DESIGN: Prospective controlled study at a single medical center. SETTING: University hospital. SUBJECTS AND METHODS: The study, conducted from April 2007 to February 2012 at the "Sapienza" University of Rome, was designed to collect 2 Montgomery Safe-T-Tubes from each patient. The first was removed 3 to 15 days after insertion (group A) and the second at least 90 days after (group B). Specimens underwent microbiologic assays, electron microscopic analysis, immunocytologic analysis, and mechanical tests. RESULTS: Microorganisms were not isolated in 2 group A cases (12%), whereas they were in all group B cases. Biofilm was identified in 11 of 16 (69%) group A samples and in 16 of 16 (100%) group B samples (P = .0149) using scanning electron microscopy. Immunohistochemistry showed monocyte-granulocyte line cells producing interleukin-1ß on the external surfaces of Montgomery Safe-T-Tubes. The tensile test showed that the wear related to the longer period of use makes Montgomery Safe-T-Tubes more rigid than newer ones. CONCLUSION: Early biofilm colonization takes place in Montgomery Safe-T-Tubes in most cases. The mechanical decay could be justified in part by the destructive biofilm activity and by the release of inflammatory effectors and enzymes.

Expression of Phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines.

The definition of the number and nature of signal transduction pathways networking in the pathogenesis of osteosarcoma raised great interest. Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration is regulated by signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol (4,5) bisphosphate (PIP2) is critical for many cellular activities. The levels of PIP2 are regulated by means of Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes. We delineated the panel of expression of PI-PLC enzymes in four human osteosarcoma cell lines. In MG-63 cell line, PI-PLC ß1, ß2, ß3, ß4, γ1, γ2, δ1, δ3 and ε resulted expressed. In 143B cell line, PI-PLC ß1, ß2, ß3, ß4, γ1, γ2, δ1, δ3 and ε were expressed. In SaOS-2 cell line, PI-PLC ß1, ß3, ß4, γ1, γ2, δ1, δ3, ε and η1. In Hs888 cell line, PI-PLC ß1, ß3, ß4, γ1, δ1, δ3, δ4, ε and η1 the administration of U-73122 to cultures briefly modifies the levels of PI-PLC transcripts. The obtained complete expression panel of PI-PLC isoforms will be a useful tool for further functional studies about the role of the PI signal transduction pathway in osteosarcoma.

Phosphoinositide-specific Phospholipase C ß1 gene deletion in bipolar disorder affected patient.

The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC ß1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.

Phosphoinositide pathway and the signal transduction network in neural development.

The development of the nervous system is under the strict control of a number of signal transduction pathways, often interconnected. Among them, the phosphoinositide (PI) pathway and the related phospholipase C (PI-PLC) family of enzymes have been attracting much attention. Besides their well-known role in the regulation of intracellular calcium levels, PI-PLC enzymes interact with a number of molecules belonging to further signal transduction pathways, contributing to a specific and complex network in the developing nervous system. In this review, the connections of PI signalling with further transduction pathways acting during neural development are discussed, with special regard to the role of the PI-PLC family of enzymes.

Expression of phosphoinositide-specific phospholipase C enzymes in normal endometrium and in endometriosis.

OBJECTIVE: To delineate the panel of expression of phosphoinositide-specific phospholipase C (PI-PLC) signaling enzymes in normal endometrium and in endometriosis. DESIGN: Clinical/experimental study. SETTING: University. PATIENT(S): Healthy donor woman and endometriosis-affected woman. INTERVENTION(S): Normal endometrium and endometriosis surgical biopsies were analyzed using gene expression analyses methodology (reverse transcriptase-polymerase chain reaction [PCR], bioanalyses). MAIN OUTCOME MEASURE(S): Gene expression (messenger RNA concentration) measures of 12 PI-PLC enzymes: PI-PLC ß1, PI-PLC ß2, PI-PLC ß3, PI-PLC ß4, PI-PLC γ1, PI-PLC γ2, PI-PLC δ1, PI-PLC δ3, PI-PLC δ4, PI-PLC ε, PI-PLC η1, and PI-PLC η2. RESULT(S): PI-PLC ß1, PI-PLC ß3, PI-PLC δ1, and PI-PLC δ3 enzymes were detected, although differently expressed in normal and endometriosis tissues. CONCLUSION(S): The involvement of PI-PLC enzymes in inflammation and the consistency of susceptible endometriosis loci with PI-PLC genes mapping corroborate the hypothesis that PI signaling might be involved in the pathogenesis of endometriosis.

Deletion of PLCB1 gene in schizophrenia-affected patients.

A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC ß1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients.

IL-1ß and IL-23 in amniotic fluids of ultrasound-detected aortic intima/media thickness and growth retardation.

Intrauterine growth restriction (IUGR) and/or neonatal low birth weight are often associated with increased intima/media thickness of the abdominal aortic wall (aIMT). Several studies in children suggested that aIMT might be related to inflammation, probably indicating an early stage of adulthood diseases, such as atherosclerosis. Our previous study performed on the abdominal aortic wall of a stillbirth presenting with IUGR and aIMT suggested an association among IUGR, aIMT, and inflammation, also highlighting the presence of fibroblastoid cells, which are thought to represent peculiar elements of the pre-atherosclerotic lesions. These observations led us to analyze two cytokines involved in the inflammation cascade, IL-1ß and IL-23, in amniotic fluid samples of IUGR fetuses and small-for-gestational-age newborns presenting with aIMT and in normal controls. Our results indicate that IL-23, but not IL-1ß, concentrations differed in the groups analyzed. Therefore, IL-23, a regulatory element that bridges the innate and adaptive arms of the immune system, might be involved in the inflammatory process observed in fetal aIMT.