Does the purinergic system affect extracellular matrix functions in the central nervous system?

Exracellular matrix (ECM) consists of a plethora of proteins and polysaccharides, which aggregate into an organized network connected to the surface of the producing cells. It is structurally and functionally present in all components of tissues and organs and represents the substrate on which cells adhere, migrate, proliferate and differentiate, influencing their survival, shape and function. In response to acute (trauma) or chronic (degenerative) insults, brain ECM modifies its composition and function, actively contributing to "scar forming" gliosis or tissue degeneration/remodelling. Moreover, morphological changes in dendritic spines associated with extracellular matrix remodeling play key roles in rewiring synaptic circuitry pertinent to memory formation. In the present report, we collected the main acquisitions on the functional interplay between ECM alterations and the adenine-/guaninebased purine system with particular regard on how purine compounds and their respective receptors may affect and be affected by changes of the cerebral ECM.

Cyanidin reduces preadipocyte differentiation and relative ChREBP expression.

Adipogenesis is a continuous process even in adult adipose tissue for the presence of preadipocytes that, when subjected to appropriate stimuli can proliferate and differentiate. ChREBP, the essential transcription factor for lipogenesis, is expressed in all tissues, but mainly in lipogenic organs. In this study, we focused on ChREBP expression during preadipocytes differentiation. Since it was found that cyanidin-3 reduces body weight in mice even in the presence of a high-fat diet, by decreasing levels of blood glucose and by improving insulin sensitivity, we studied the effect of this substance on adipogenic differentiation. For this purpose we used preadipocytes obtained from subcutaneous and visceral human adipose explant tissue, characterized and stimulated to differentiate in selective media. On cytofluorimetric analysis these cells showed mesenchymal markers (CD29, CD90, CD44), whereas they were negative for hematopoietic markers (CD45, CD10, CD117,CD31). ChREBP expression levels were quantified by immunoelectron-microscopy and western blotting analysis. In this report we show that ChREBP is expressed in preadipocytes (both nuclear and cytoplasmic compartments); the cytoplasmic level of ChREBP increased by 50 percent on day seven of differentiation into mature adipocytes. Cyanidin reduced differentiation by 20 percent (as evaluated by red oil O staining) and the expression of ChREBP. In addition, cyanidin-treated cells showed abnormal morphology, a square shape with irregular size, probably due to the fact that cyanidin may interfere with the extracellular matrix. These findings suggest that dietary cyanidin, may have inhibitory effects on adipogenesis.

Tissue distribution and metabolism of guanosine in rats following intraperitoneal injection.

Guanosine has long been known as an endogenous purine nucleoside deeply involved in the modulation of several intracellular processes, especially G-protein activity. More recently, it has been reported to act as an extracellular signaling molecule released from neurons and, more markedly, from astrocytes either in basal conditions or after different kinds of stimulation including hypoxia. Moreover, in vivo studies have shown that guanosine plays an important role as both a neuroprotective and neurotrophic agent in the central nervous system. Specific high-affinity binding sites for this nucleoside have been found on membrane preparations from rat brain. The present study was undertaken to investigate the distribution and metabolic profiles of guanosine after administering the nucleoside to gain a better understanding of the biological effects of this potential drug candidate. Rats were given an intraperitonal (i.p.) injection of 2, 4, 8 or 16 mg/kg of guanosine combined with 0.05% of [3H]guanosine. Plasma samples were collected 7.5, 15, 30, 60 and 90 min after the guanosine-mixture administration and analyzed by either a liquid scintillation counter or by HPLC connected to a UV and to an on-line radiochemical detector to measure the levels of guanosine and its metabolic products guanine, xanthine and uric acid. The levels of guanosine, guanine and xanthine were also measured in brain, lung, heart, kidney and liver tissue homogenates at the defined time points after the injection of 8 mg/kg of the guanosine-mixture. We found that the levels of radioactivity in plasma increased linearly in a dose- and time-dependent manner. Guanosine was widely distributed in all tissues examined in the present study, at almost twice its usual levels. In addition, guanine levels dramatically increased in all the organs. Interestingly, enzymatic analysis of the plasma samples showed the presence of a soluble purine nucleoside phosphorylase, a key enzyme in the purine salvage pathway and nucleoside catabolism. Since guanosine has been shown to be neuroprotective and astrocytes have been reported to play critical roles in mediating neuronal survival and functions in different neurodegenerative disorders, we also performed uptake and release.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

Vascular endothelial growth factor enhances in vitro proliferation and osteogenic differentiation of human dental pulp stem cells.

Mesenchymal stem cells (MSC), isolated from dental tissues, are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from human dental pulp (DPSC) of normal impacted third molars that, when cultured in lineage-specific inducing media, differentiate into osteoblasts and adipocytes (evaluated by Alizarin Red S and Red Oil O stainings, respectively), thus showing a multipotency. We confirmed that DPSC, grown under undifferentiating conditions, are negative for hematopoietic (CD45, CD31, CD34, CD144) and positive for mesenchymal (CD29, CD90, CD105, CD166, CD146, STRO-1) markers, that underwent down-regulation when cells were grown in osteogenic medium for 3 weeks. In this condition, they also exhibit an increase in the expression of osteogenic markers (RUNX-2, alkaline phosphatase) and extracellular calcium deposition, whereas the expression of receptors (VEGFR-1 and -2) for vascular endothelial growth factors (VEGF) and related VEGF binding proteins was similar to that found in undifferentiated DPSC. Exposure of DPSC growing under undifferentiating or osteogenic conditions to VEGF-A165 peptide (10-40 ng/ml) for 8 days dose- and time-dependently increased the number of proliferating cells without inducing differentiation towards endothelial lineage, as evaluated by the lack of expression of specific markers (CD31, CD34, CD144). Additionally, exposure of DPSC cultured in osteogenic medium to VEGF-A165 for a similar period enhanced cell differentiation towards osteoblasts as evaluated after 14 and 21 days by Alizarin Red S staining and alkaline phosphatase activity quantification. These findings may have clinical implications possibly facilitating tissue repair and remodeling.

Guanosine protects human neuroblastoma cells from oxidative stress and toxicity induced by Amyloid-beta peptide oligomers.

Amyloid-beta (Abeta) peptide aggregation forms such as soluble oligomers (O) have a causal role in neuronal dysfunction and death associated with Alzheimer?s Disease (AD). The main efforts for the development of neuroprotective drugs are therefore focused on preventing Abeta production, aggregation or downstream neurotoxic events. We therefore investigated the effect of guanosine (GUO), a guanine based purine, that exerts neurotrophic and neuroprotective effects. The GUO showed the ability to reduce neuronal death in terms of apoptosis, but not necrosis, elicited by Abeta1-42O in human neuroblastoma SH-SY5Y cells. The neuroprotective effect was recorded only when the GUO was added simultaneously to treatment of the SH-SY5Y cells with Abeta1-42O. By contrast, the GUO treatment of SH-SY5Y cells before and after the appearance of beta1-42O toxicity had no neuroprotective effects. The employment of specific inhibitors showed the involvement of neuronal survival pathways, such as PI3K?Akt and MAPK-ERK for the GUO anti-apoptotic effects observed. In parallel, the SH-SY5Y cells treated with GUO, in experimental conditions similar to those adopted to evaluate neuronal death, showed a marked decrease of the early reactive oxygen species formation induced by Abeta1-42O and pro-oxidant H2O2. In the same neuronal model, GUO was also shown to inhibit the extra- and intra-cellular Abeta1-42 release as well as the beta-secretase activity evoked by H2O2 pro-oxidant action. Based on these findings, GUO and other guanine based purines appear to be a promising class of compounds with neuroprotective properties that may play an important role in the therapy of AD.

Changes in matrix extracellular phosphoglycoprotein expression before and during in vitro osteogenic differentiation of human dental papilla mesenchymal cells.

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing beta-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration.

Analysis of the occlusal stress transmitted to the inferior alveolar nerve by an osseointegrated threaded fixture.

BACKGROUND: Altered sensation can occur after the placement or loading of mandibular implants. Limited evidence exists with regard to the proper distance between the implant and the mandibular nerve to ensure the nerve's integrity and physiologic activity. The proper distance should come from evaluation of clinical data as well as from biomechanical analyses. METHODS: A numeric mandibular model based on the boundary element method was created to simulate a mandibular segment containing a threaded fixture so that the pressure on the trigeminal nerve, as induced by the occlusal loads, could be assessed. Such pressure distributions were evaluated with different distances of the fixture from the mandibular canal and considering different bone densities. Although all simulations considered a canal that was orthogonal to the implant axis, in one case, the effects of an inclined canal were analyzed. RESULTS: The nerve pressure increased rapidly with a bone density decrease. A low mandibular cortical bone density caused a major nerve pressure increase. CONCLUSION: Our study suggested a distance of 1.5 mm to prevent implant damage to the underlying inferior alveolar nerve when biomechanical loading was taken into consideration.

Upper airway obstructive disease in mucopolysaccharidoses: polysomnography, computed tomography and nasal endoscopy findings.

In mucopolysaccharidoses, upper airway obstruction has multiple causative factors and progressive respiratory disease may severely affect morbidity and mortality. In a cross-sectional study over 2 years we evaluated upper airway obstructive disease through overnight polysomnography, upper airway computed tomography and nasal endoscopy in 5 children and 6 adults with mucopolysaccharidoses of various types. Measurements of apnoea and apnoea-hypopnoea index, arousal index, and sleep efficiency were obtained through polysomnography. Retropalatal and retroglossal spaces were calculated through computed tomography, and the degree of adenoid hypertrophy was assessed through endoscopy. Apnoea index and apnoea-hypopnoea index were significantly higher in children than in adults with mucopolysaccharidoses (p = 0.03 and p = 0.03, respectively). Compared to healthy controls, retropalatal and retroglossal spaces were significantly smaller in children (p = 0.03 and p = 0.004, respectively) or adults with mucopolysaccharidoses (p = 0.004 and p = 0.004, respectively). All subjects had adenoid hypertrophy causing first-degree (36%) or second-degree (64%) obstruction at endoscopy. Overnight polysomnography, upper airway computed tomography and nasal endoscopy are useful tools for diagnosing obstructive sleep apnoea syndrome in mucopolysaccharidoses, and identifying the site and severity of airway obstruction.

Activation of P2X(7) receptors stimulates the expression of P2Y(2) receptor mRNA in astrocytes cultured from rat brain.

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X(7) ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X(7) receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y(2) in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y(2) expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y(2) sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y(2) mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X(7)-antagonists oATP and BBG. The BzATP-evoked P2Y(2) receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y(2)siRNA, thus supporting evidence of increased P2Y(2) activity. To further investigate the mechanisms by which P2X(7) receptors mediated the P2Y(2) mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y(2) mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X(7)-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X(7) receptors in astrocytes enhances P2Y(2) mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways.

Morphostructural analysis of human follicular stem cells on highly porous bone hydroxyapatite scaffold.

In this study we investigated the in vitro behaviour, morphostructure and extracellular matrix synthesis of human dental follicular stem cells (hDFSCs) isolated from human dental bud, which resulted to be positive for mesenchymal markers (CD29, CD90, CD146 and CD166) by FACS analysis. Cells were analysed by light and electronic microscopy to evaluate their biological response either at week 1, that is before differentiation, or at weeks 3-6, when they had been cultured in osteogenic medium onto a highly porous natural scaffold material (Bio-Oss). Microscopy analysis of primary culture cells showed they had a mesenchymal stem cell-like morphostructure, spindle shaped, similar to the culture of mesenchymal stem cells derived from adult bone marrow. Also, after osteogenic differentiation, these analyses indicate typical osteoblast morphostructure and reveale a tri-dimensional organization of the cells and deposition of extracellular matrix (ECM) in close contact with biomaterial. This approach would allow to personalize the scaffold for bone tissue engineering in order to accelerate the process of osteogenesis.

Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes.

Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes. In contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

P2Y2 receptor up-regulation induced by guanosine or UTP in rat brain cultured astrocytes.

Among P2 metabotropic ATP receptors, P2Y2 subtype seems to be peculiar as its upregulation triggers important biological events in different cells types. In non-stimulated cells including astrocytes, P2Y2 receptors are usually expressed at levels lower than P2Y1 sites, however the promoter region of the P2Y2 receptors has not yet been studied and little is known about the mechanisms underlying the regulation of the expression of this ATP receptor. We showed that not only UTP and ATP are the most potent and naturally occurring agonist for P2Y2 sites, but also guanosine induced an up-regulation of astrocyte P2Y2 receptor mRNA evaluated by Northern blot analysis. We also focused our attention on this nucleoside since in our previous studies it was reported to be released by cultured astrocytes and to exert different neuroprotective effects. UTP and guanosine-evoked P2Y2 receptor up-regulation in rat brain cultured astrocytes was linked to an increased P2Y2-mediated intracellular calcium response, thus suggesting an increased P2Y2 activity. Actinomycin D, a RNA polymerase inhibitor, abrogated both UTP and guanosine-mediated P2Y2 up-regulation, thus indicating that de novo transcription was required. The effect of UTP and guanosine was also evaluated in astrocytes pretreated with different inhibitors of signal transduction pathways including ERK, PKC and PKA reported to be involved in the regulation of other cell surface receptor mRNAs. The results show that ERK1-2/MAPK pathway play a key role in the P2Y2 receptor up-regulation mediated by either UTP or guanosine. Moreover, our data suggest that PKA is also involved in guanosine-induced transcriptional activation of P2Y2 mRNA and that increased intracellular calcium levels and PKC activation may also mediate P2Y2 receptor up-regulation triggered by UTP. The extracellular release of ATP under physiological and pathological conditions has been widely studied. On the contrary, little is known about the release of pyrimidines and in particular of UTP. Here we show that astrocytes are able to release UTP, either at rest or during and following hypoxia/hypoglycemia obtained by submitting the cells to glucose-oxygen deprivation (OGD). Interestingly, also P2Y2 receptor mRNA increased by about two-fold the control values when the cultures were submitted to OGD. It has been recently reported that P2Y2 receptors can play a protective role in astrocytes, thus either guanosine administration or increased extracellular concentrations of guanosine and UTP reached locally following CNS injury may increase P2Y2-mediated biological events aimed at promoting a protective astrocyte response.

Trophic effects of purines in neurons and glial cells.

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.

Nickel distribution and DNA lesions induced in rat tissues by the carcinogen nickel carbonate.

DNA lesions in nuclei isolated from rat tissues following i.p. injection of the carcinogen nickel carbonate were measured by the alkaline elution technique. Kidney, liver, lung, and thymus gland nuclei were examined at 3 and 20 hr after treatment for the presence of DNA single-strand breaks and cross-links. Single-strand breaks were detectable in lung and kidney nuclei, and both DNA-protein and DNA interstrand cross-links were detectable in kidney nuclei. No DNA damage was observed in liver or thymus gland nuclei. A dose response to both single-strand breaks and cross-links was observed in kidney nuclei. Time course studies revealed that maximum DNA damage in kidney nuclei occurred at 2 to 4 hr following injection and also revealed the presence of an active repair process in these nuclei. Repair-resistant DNA-protein cross-links were observed to persist through 48 hr. Tissue and intracellular nickel concentrations as measured by electrothermal atomic absorption spectroscopy were observed to correlate with the levels of DNA damage and repair. A dose response to the concentration of nickel in tissues and nuclei was observed. These results are discussed relative to the solubilization, toxicity, and carcinogenicity of nickel compounds.

Nickel-bound chromatin, nucleic acids, and nuclear proteins from kidney and liver of rats treated with nickel carbonate in vivo.

The in vivo binding of nickel to chromatin, nucleic acids, and nuclear proteins from rat kidney and liver was investigated. Evidence is presented for the direct interaction of nickel with chromatin from rat tissues following i.p. injection of nickel carbonate. A gentle DNA isolation procedure was developed and used to isolate nickel-bound whole chromatin, DNA + histone octamer complex (polynucleosomes), and deproteinized DNA from kidney and liver nuclei. A similar procedure was developed for isolating nickel-bound RNA from the cytoplasm. The level of nickel bound to whole chromatin from kidney was higher than that from liver, and these levels could be related to the nuclear concentration of nickel. Much higher levels of nickel were bound to the DNA + histone octamer complex and purified, deproteinized DNA from kidney as compared to liver. Substantial levels of nickel were bound to nonhistone proteins and/or chromatin-associated RNA from kidney and liver nuclei. Nickel was also associated with histone octamer proteins from kidney; however, little or no nickel was associated with histone octamer proteins from liver. 63Ni binding to nuclear proteins in rats given injections of 63Ni(II) was investigated by using two different gel electrophoretic systems. Electrophoretic conditions in both systems were found to remove protein-bound 63Ni. These results are discussed relative to the molecular mechanism of nickel carcinogenesis.

Synergic development of pharmacokinetics and bioanalytical methods as support of pharmaceutical research.

The development of pharmacokinetics led this science to achieve a relevant role in the investigation of new chemical entities for therapeutic application, and has allowed a series of new useful realizations of out of patent drugs like prolonged release and delayed release formulations, therapeutic delivery system (TDS) for drugs to be active in systemic circulation avoiding the first pass effect, orodispersible and effervescent formulations, intramuscular and subcutaneous depot formulations acting over a long period, oral inhalatory systems, and drug association at fixed dose. The above applications had pharmacokinetics as protagonist and have required the support from bioanalytical methods to assay drug concentrations, even in pg·mL(-1) of plasma, that really have paralleled the synergic development of pharmacokinetics.The complexity of the above realizations required specific guidelines from the regulatory authorities, mainly the US FDA and EU EMA, which have normalized and, in most cases, simplified the above applications admitting some waivers of in vivo bioequivalence.However, this review highlights some critical points, not yet focused on by operating guidelines, which need to be clarified by regulatory authorities. One of the most relevant issues is about the planning and conducting bioavailability and bioequivalence trials with endogenous substances, that possess own homeostatic equilibria with fluctuations, in some cases with specific rhythms, like melatonin and female sex hormones. The baseline subtraction required by guidelines to define the net contribute to the exogenous absorbed drug in most cases is a non-solvable problem.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Treatment of Gorham's disease with zoledronic acid.

Gorham's disease (GD) is a rare disorder characterized by spontaneous and progressive osteolysis of one or more bones and thought to belong to lymphangiomatoses spectrum of diseases. Surgical, radiation and medical therapies have been performed with variable and often discouraging outcomes and currently there is no recognized effective treatment. In this paper we describe a 24-year-old girl with GD localized to mandible who was effectively managed with zoledronic acid, a nitrogen-containing high-potency bisphosphonate.

P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes.

Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured astrocytes P2X7ATP receptors may participate in the control of CysLT release thus further supporting a role for extracellular ATP as an integral component of the inflammatory brain response.